bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of PI3‐k/Akt signaling pathway in sublytic C5b‐9 complexes‐induced synthesis of thrombospondin‐1 (Tsp‐1), transforming growth factor‐β1 (TGF‐β1) and proliferation of glomerular mesangial cells (GMCs) including secretion of extracelluar matrix (ECM)
Authors: Wang Yingwei
Published: June 2021

Sublytic complement C5b‐9 complexes can cause many forms of immune‐mediated diseases. But the role and relative signal pathway of sublytic C5b‐9 on the pathologic changes of glomerular mesangial cells (GMCs) has not been demonstrated. In this study, we first explored the effects of sublytic C5b‐9 on the synthesis of thrombospondin‐1 (Tsp‐1) and transforming growth factor‐β1 (TGF‐β1) including proliferation of GMCs and secretion of extracelluar matrix (ECM) i.e. fibronectin (FN), and then ascertained the roles of phosphatidylinositol 3‐kinase (PI3‐k)/Akt signal pathway in these processes by using small hairpin RNAs (shRNA). The results showed that the expression of Tsp‐1, TGF‐β1, cyclin D2 and 3H‐thymidine into DNA as well as FN in the GMCs stimulated by sublytic C5b‐9 complexes could be increased, and the elevation of these parameters mentioned‐above was also markedly reduced in the GMCs transfected with PI3‐k and Akt shRNA expression vectors or LY294002 (PI3‐k inhibitor) respectively. These data indicate that sublytic C5b‐9 can induce the sythesis of Tsp‐1, TGF‐β1 and promote the proliferation of GMCs and secretion of FN, and PI3‐k/Akt signal pathway plays a partial role in these reactions mediated by sublytic C5b‐9 complexes at least.
Plasmacytoid Dendritic Cells both produce interferon‐λ and respond to interferon‐λ stimulation
Authors: Yin Zhiwei , Amrute Sheela , Lewis‐antes Anita , Donnelly Raymond P. , Kotenko Serguei , Bocarsly Patricia F.
Published: June 2021

PDC are considered to be “professional” type I IFN producing cells in that they produce 10–100‐fold more IFN‐α in response to enveloped viruses than other cell types. We investigated the ability of human PDC to produce and respond to IFN‐λ (IL‐28/‐29). We developed an intracellular flow cytometry assay to assess the expression of IFN‐λ protein. A subset of IFN‐α+ PDC was found to express IFN‐λ after stimulation with HSV, flu, Sendai virus, as well as with TLR9 and ‐7 agonists, with IFN‐λ expressed by those cells with the highest IFN‐α expression. As we previously reported for IFN‐α, cross‐linking of CD4 on PDC inhibited HSV‐induced IFN‐λ production. Also as with IFN‐α, HSV‐induced IFN‐λ production in PDC was mediated through TLR9. By ELISA, PDC produced ~10‐fold more IFN‐α than –λ. Both unstimulated and stimulated PDC were found to express high levels of IFN‐λ receptor mRNA and stimulated PDC expressed both IFN‐λ1 (IL‐29) and –λ2 (IL‐28) mRNA. IFN‐λ treatment of PDC led to upregulation of the expression of both IFN‐α and IFN‐λ protein. In addition, exogenous IFN‐λ inhibited apoptosis of PDC. We conclude that PDC are major producers of IFN‐λ in response to viral stimulation and also express receptors for this cytokine. Moreover, these receptors are functional in that IFN‐λ has autocrine effects that strengthen the antiviral activity of the PDC by increasing both IFN‐α and IFN‐λ production and promoting PDC survival. (AI26806).
The immunosuppressive tumor microenvironment in hepatocellular carcinoma and the identification of a new type of Foxp3‐expressing cell
Authors: Pang Yanli , Zhang Huagang , Zhang Yu , Chen Weifeng
Published: June 2021

Increasing evidence supports the existence of many immunosuppressive factors in tumor microenvironment. To investigate the immune status in hepatocellular carcinoma tissues, we isolated tumor infiltrating lymphocytes (TILs) and non‐tumor infiltrating lymphocytes (NILs) from fresh tissue. In comparison to NILs, CD4+ T cells in TILs were elevated, whereas CD8+ T cells, NK cells, CD56+ T cells, γδT cells, and CCR7−CD45RA+CD8+ cells were reduced. Moreover, CD8+ cells in TILs showed impaired capacity for the production of IFN‐γ and perforin. Reduced IFN‐γ production was also observed for CD4+ cells. On the other hand, these cells secreted more IL‐10. Subsequently, we examined the representation of cells with suppressive functions. TILs were found to contain a markedly increased number of CD4+CD25+Foxp3+, CD3+CD56+Foxp3+, and CD3−CD56+Foxp3+ cells. Further analysis of the CD3+CD56+Foxp3+ cells demonstrated that they were mainly CD8−CD4+Valpha24− cells, which produced significant amount of IL‐4 and IL‐10, but rarely IFN‐γ or perforin. In conclusion, the hepatocellular carcinoma microenvironment appears to be dominated by negative immune regulators, and the CD3+CD56+Foxp3+ cells may represent a new type of regulatory cells.
The CD8 T cell‐derived lipase, pancreatic lipase related protein 2, mediates tumor killing through an indirect mechanism.
Authors: Alves Bryce N. , Tamang David Lee , Leong Jeff , Redelman Doug , Lowe Mark E. , Hudig Dorothy
Published: June 2021

What purpose could a pancreatic lipase possibly serve in cytotoxic T lymphocytes (CTLs)? Pancreatic lipase related protein 2 (PLRP2) was first identified as an IL‐4 inducible T cell protein in the early 90s. Later studies using PLRP2 knockout mice showed that PLRP2 deficient T cells have reduced lytic capacity when compared to wild type T cells, suggesting that PLRP2 may function in T cell mediated killing. Our lab has focused on the characterization of PLRP2 expression in CTLs and to determine if PLRP2 is directly or indirectly toxic to tumor cells. Expression of PLRP2 was determined over a 5 day period in the presence of IL‐4. PLRP2 mRNA expression first appeared on days 4, with increased expression by day 5. T cells grown in increasing concentrations of IL‐4 displayed a dose response in the induced expression of PLRP2 up to 250 units/ml IL‐4 (7.15x10−10M). Enrichment and depletion of both the CD4+ and CD8+ T cell populations indicated CD8+ T cells as the PLRP2 expressing subpopulation when grown in IL‐4. We tested if PLRP2 could mediate direct or indirect killing of targets using recombinant PLRP2. When rPLRP2 at 5–10ug/ml was added to tumor cells with physiological triglycerides levels, tumor viability was reduced. The tumors were refractory to PLRP2 without the triglyceride substrate. These results suggest PLRP2 may use an indirect method to mediate its toxic effect on tumors.
In vivo evaluation of the immunomodulatory activity of the polysaccharides purified from Salicornia herbacea
Authors: Im Sun‐A , Lee Young‐Ran , Lee Young‐Hee , Kim Hyunjin , Chang Pan‐Young , Jung Ki‐Sung , Lee Chong‐Kil
Published: June 2021

Immunomodulatory polysaccharides isolated from plants or mushrooms have been shown to activate diverse types of cells of the immune system in vitro. However, the in vivo efficacy of the immunomodulation has been a matter of question due to the high molecular weight nature of the polysaccharides. In the present study, we examined the in vivo immunomodulatory activity of the polysaccharides isolated from Salicornia herbacea. The immunomodulatory polysaccharides were purified from the hot water extract of Salicornia herbacea by passage through a DEAE‐Sephacel column, and then the gel filtration chromatography. The polysaccharides efficiently induced cytokine production, nitric oxide release, expression of surface molecules and phagocytic activity, in a dose dependent manner, when added to cultures of a mouse monocytic cell line, RAW 264.7 cells. In vivo efficacy was examined in mice implanted with tumor cells, and also in mice immunized with ovalbumin (OVA). We found that the polysaccharides enhance antitumor immune responses in mice implanted with Sarcoma‐180 cells. In addition, we found that the polysaccharides induce strong OVA‐specific CTL responses in mice. These results confirm that the polysaccharide purified from Salicornia herbacea exerts immunostimulatory activity in vivo.
Different Immunologic Microenvironments in Halo Nevi and Melanomas
Authors: Yu Ping , Liu Wenhua , Tretiakova Maria , Mentzel Thomas , Hui Shiu‐Ki , Shea Christopher , Petronic‐Rosic Vesna , Fu Yangxin , Krausz Thomas
Published: June 2021

In halo nevi, the nevus cells get destroyed with the passage of time. A complete regression is rare even with brisk lymphocytic infiltrate in melanomas. We compared the immunologic microenvironments in these entities in order to understand better the regression mechanism in these lesions. We found that the ratio of CD8+/CD4+ cells was comparable in nevi and melanomas, Foxp3+/CD4+ was 1/2 and 1/15, respectively, which was much higher in halo nevi. Perforin+ T cells counted 60% of the CD8+ cells in halo nevi, while they were less than 5% in melanomas. We concluded that the majority of tumor‐infiltrating CD8+ T cells express perforin, suggesting that perforin‐mediated cytotoxicity may be the key mechanism involving the early regression of halo nevi. Increased Foxp3+ Treg most likely reflect the negative feedback mechanisms to prevent tissue damage from severe immunoreaction in halo nevi. Although CD8+ T cells significantly increase in melanomas similarly to in halo nevi, these T cells lack perforin, suggesting defective CTL around tumor, which may contribute to the mechanisms of immune evasion leading to the aggressive growth of the tumor. The failed immunoreaction in melanomas does not cause tissue damage, which may reflect to the paucity of Foxp3+ T cells in melanomas. In an attempt to study what mechanisms inactivate effector CTL independent of Treg, we found many TGF‐β‐secreting cells in melanomas but none in halo nevi. Indeed, melanoma regressed upon combination treatment of TGF‐β blockade and anti‐41BB in a murine model. Our study suggested that immune suppressive cytokines in the tumor environment may be important factors independent of Treg to suppress anti‐tumor immunity.
OX40‐mediated tumor protection is decreased in aging mice: the role of CD4+CD25+FoxP3+ T cells and IL‐12
Authors: Ruby Carl E , Weinberg Andrew D
Published: June 2021

OX40 agonists have potent immunotherapeutic effects against a variety of tumors, yet it is unclear to what extent age‐related immune senescence influences the activities of these agonists. We found that treating aged tumor‐bearing mice (12 and 20 months old) with anti‐OX40 showed less tumor free survival compared to treated younger mice (2 and 6 months old). This effect was observed in both male and female mice, and was not due to changes in the expression of OX40 on T cells. Despite the absence of a productive anti‐tumor response in anti‐OX40‐treated aging mice, anti‐OX40 equally enhanced the activation of young and aging T cells, as measured by increases in CD8 T cell IFN gamma and CD4 T cell IL‐2 compared to controls. To better understand the mechanisms of these age‐related differences, we measured the accumulation of CD4+CD25+FoxP3+ T cells within the tumors of treated mice. Anti‐OX40 treated young mice showed a significant reduction in the accumulation of CD4+CD25+FoxP3+ T cells compared to controls, however anti‐OX40 failed to reduce the accumulation of these cells in the tumors of aging mice. Finally, IL‐12 in concert with anti‐OX40 partially restored the defective anti‐tumor responses in aging mice, coinciding with a significant decrease in the accumulation of CD4+CD25+FoxP3+ T cells. Our data suggest that anti‐OX40‐mediated anti‐tumor responses in aging mice are influenced by IL‐12 and CD4+CD25+FoxP3+ T cells.
SNP in exon 2 of the MUC1 gene generates a candidate minor histocompatibility antigen through alternative splicing
Authors: Zhang Lixin , Finn Olivera J
Published: June 2021

Minor histocompatibility antigens (mHAg's) are allelic peptides encoded by polymorphic genes that can bind MHCI or II molecules and initiate an immune response in allele‐negative hosts. MUC1 is a type I transmembrane glycoprotein with variable numbers of tandem repeats (VNTRs) and aberrant expression in many tumors makes it an ideal target for cancer therapy. MUC1 is polymorphic not only in the VNTR region but also in sequences outside of the VNTR. At least 33 single nucleotide polymorphisms (SNPs) in the MUC1 gene have been reported, one of which is a 3506 G/A SNP located in the exon 2. In the 54 MUC1 isoforms we recently isolated, 27 isoforms have 3506 G while the other half have 3506 A SNPs. The 3506 G allele encodes a 27‐bp additional sequence from 3' end of intron 1 that encodes a 9 a.a. peptide, which theoretically affects the cleavage site selection of the signal peptide by adding 10 a.a. to the N‐terminus of the mature proteins. Three peptides were synthesized according to the epitope prediction results and MHCI stabilization assay showed that they could bind HLA‐A0201 as well as H‐2Kb . In vivo assay are being done now in MUC1.Tg mice. This MUC1 minor antigen may serve as a new target for tumor immunotherapy. Supported by NIH grant RO1CA56103.
Designer glycopeptides for cytotoxic T cell‐based elimination of carcinomas
Authors: Franco Alessandra , Sidney John , Sette Alessandro , Maewal Ajesh
Published: June 2021

Our laboratory has elucidated the immunochemical requirements for recognition of tumor associated carbohydrate antigens (TACA) by mouse and human CD8+ cytotoxic T cells. These results proved that TcRs accommodate small TACA as the Thomsen Freidenreich antigen (TF) and its monomer Tn, conventionally presented to T cells in the context of peptides linked to MHC molecules.

TF‐ and Tn‐containing glycopeptides with high affinity for class I MHC molecules were capable of priming Th‐independent primary and memory TACA‐specific CTL responses in vivo in mice and normal human donors. These CTL could kill a variety of TACA‐expressing tumors in vitro.

We are currently performing pre‐clinical studies with a variety of Tn‐containing glycopeptides derived from the MUC1 glycoprotein (MUC1 contains Tn and TF), aiming at the prevention/treatment of carcinomas. These glycopeptides contain the “PDTR” fragment of the variable number of tandem repeat (VNTR) domains of MUC1. “PDTR”‐containing glycopeptides bind with very high affinity A2 class I MHC molecules since have been designed to incorporate primary and secondary anchor residues for optimal binding.
Consumption of the slow digesting starch waxy maize leads to blunted and sustained carbohydrate utilization but does not influence energy expenditure or appetite
Authors: Sands Amanda L. , Leidy Heather J. , Hamaker Bruce R. , Maguire Paul , Campbell Wayne W.
Published: June 2021

Background: The postprandial metabolic and appetitive responses of waxy maize, a slow‐digesting carbohydrate (CHO), are undocumented in humans and were examined in this study.

Methods: 12 young, physically fit men and women (age: 23 ± 1 y; BMI: 22.2 ± 0.7 kg/m2; physical activity: 556 ± 120 min/wk) consumed, on separate testing days, 50 g of available CHO as either fast‐digesting maltodextrin (M) or waxy‐maize starch (WM). Plasma glucose and insulin concentrations, whole body energy expenditure and appetite (hunger, fullness, and desire to eat) were measured over 4 h.

Results: Compared to M, WM led to lower postprandial glucose (1082 ± 151 vs. 2186 ± 262 mg/dL•240 min; p<0.001) and insulin (833 ±164 vs. 1628 ± 165 uU/mL•240 min; p<0.005) area under the curve during the 4 h period. These differences were driven by the blunted glucose response during the first hour (p<0.001) and the blunted insulin response during the first two hours (p<0.005) in WM. CHO utilization was lower after 1 h but higher after 4 h following WM vs. M. Composite CHO utilization over the entire 4 h period was not different between WM and M. No differences in energy expenditure or appetite were observed between WM vs. M.

Conclusion: These results support that waxy maize provides sustained glucose availability in young, physically fit adults.

Support: US Army, Natick Soldier RDECOM, Combat Feeding Directorate; Ingestive Behavior Res. Ctr., Purdue University
Dietary Modulation of Plasma and Adipose Tissue n‐3:n‐6 PUFA Ratio has no Effect on Insulin Resistance and Inflammation in Obese Gambian Women
Authors: Savy Mathilde , Nyan Ousman , Browning Lucy M. , Chatfield Mark , Prentice Andrew M.
Published: June 2021

This study examined whether increasing long‐chain n‐3 polyunsaturated fatty acids (LC n‐3 PUFA) intake have benefits on glucose tolerance in obese Gambian women and on their fatty acid and lipid profiles, anthropometrics, blood pressure and inflammation status. Fifty four obese women with impaired glucose tolerance entered a 24‐week randomised intervention study. Women received LC n‐3 PUFA capsules and high n‐3 cooking oil (n = 30) or placebo capsules and high n‐6 cooking oil (n = 24). Measurements were taken at baseline, 12 and 24 weeks intervention and expressed as percentage of baseline values. In the intervention group, LC n‐3 PUFA increased in plasma (208%, p<0.0001) and adipose tissue (168%, p<0.0001), leading to an increase in n‐3:n‐6 PUFA ratio. There was no effect of high n‐3 PUFAs on fasting glucose, 120‐min glucose or fasting insulin, although insulin sensitivity assessed by the homeostasis model assessment improved modestly in the intervention group (p = 119%, p = 0.04 at 24 weeks). No differences were observed in weight, BMI and fat mass between the two groups, or in inflammatory markers, blood pressure or triglycerides. We conclude that although LC n‐3 PUFA supplementation modifies the fatty acid profile in obese Gambian women, there was no evidence that it had benefits on their glucose tolerance, triglycerides or inflammatory status.

This work was supported by UK MRC.
Assessment of methylsulfonylmethane (MSM) on the development of osteoarthritis (OA): An animal study
Authors: Amiel David , Healey Robert M , Oshima Yasushi
Published: June 2021

OA is characterized as articular cartilage degeneration, & a recent study shows that MSM supplementation is clinically effective (1). The mechanism, however, is still unclear. We examined the effect of MSM in OA.

The ACL was transected in the right knee joints of mature NZW rabbits (n=10) (2). 5 wks after ACLT, MSM constant delivery system to the joint was created by implanting an Alzet osmotic pump. In the control group no add’l treatment was done. The rabbits were sac’d 9 wks postop, & OA grades of the femoral surface were: (Grade I intact surface; Grade II minimal fibrillation; Grade III overt fibrillation; & Grade IV erosion of the articular cartilage surface) (2).

Results showed 2 Grade III & 1 Grade IV (avg 3.3) in the control group (ACLT). The MSM‐treated group showed 1 Grade I, 3 Grade II, 1 Grade III & 2 Grade IV (avg 2.6). Expression of type II collagen and aggrecan on cartilage showed no difference between control (ACLT) & experimental groups, however expression of TNF‐α in both cartilage and synovial tissue was decreased (p<0.01).

MSM has preserved the articular cartilage surface during development of OA & relieved the inflammation level (i.e. TNF‐α) in both cartilage & synovium statistically. MSM therapy is a potential application for non‐invasive treatment of OA joints.

Support from Bergstrom Nutrition and UCSD Ortho Connective Tissue Rsrch.
Garlic Therapy retards coronary artery calcification
Authors: Budoff Matthew Jay , Ahmadi Naser , Gul Khawar M , Liu Sandy T , Tiano Jima , Flores Ferdinand , Takasu Junichiro
Published: June 2021

We sought to assess whether a garlic plus homocysteine‐lowering formulation can change the rate of atherosclerosis progression as compared to placebo.

The study is a placebo‐controlled, double‐blind, randomized trial to determine whether Aged Garlic Extract (AGE) plus B‐vitamins, L‐Arginine and Folate can change the rate of atherosclerotic plaque burden (CAC) compared to placebo in 65 intermediate risk patients. Four capsules daily each containing either placebo or AGE + vitamins for the duration of 1 year. Patients’ were followed and repeat CAC measure were performed at 1 year.

Results: The mean age was 60±9 and 79% of participants were male. AGE Group had less CAC progression (22 ± 18 Agatston Units) than placebo group (62±43) over 1 year(p=0.03). Multivariate regression analysis demonstrated garlic group had less CAC progression (Odds Ratio −0.24, 95% CI 0.06–0.9,P=0.01), lower total cholesterol (0.97, 95% CI 0.92–0.99,P=0.05), LDL‐C(0.96, 95% CI 0.91–0.98,P=0.04), and homocysteine (0.77, 95% CI 0.06–0.9,P=0.03) upon followup than placebo group. Also garlic therapy was associated with a significant increase in HDL‐C compared to the placebo group (1.10 (95% CI 1.02–1.21, p=0.04). This double‐blind, placebo‐controlled study demonstrates the ability of garlic plus vitamins to retard progression of CAC, and biomarkers of atherosclerosis independent of age, gender and conventional risk factors.
BOLD (Beef in an Optimal Lean Diet) Effects on Established and Emerging Cardiovascular Disease (CVD) Risk Factors: Study Design and Planning
Authors: Roussell Michael Antonio , Cifelli Amy , Bagshaw Deborah , West Sheila , Heuvel John Vanden , Ulbrecht Jan , Kris‐Etherton Penny
Published: June 2021

Several clinical trials have shown that the incorporation of lean red meat in a Step I or AHA diet does not attenuate the reduction of traditional risk factors for cardiovascular disease (CVD). However, epidemiologic data consistently correlates increased red meat consumption with increased incidence of CVD. Recent trials also have shown beneficial effects of a diet containing moderate protein (PRO) levels (mainly from plant sources). The BOLD (Beef in an Optimal Lean Diet) Study is a randomized, four period, crossover controlled feeding study designed to compare the effects of the average American diet with the DASH diet (50% carbohydrates [CHO], 18% PRO, 38% fat, 2–3oz lean red meat), a heart healthy diet rich in lean red meat (BOLD; 50% CHO, 18% PRO, 28% fat, 5–7oz lean red meat), and a moderate PRO diet also rich in lean red meat (BOLD+; 44% CHO, 28% PRO, 28% fat, 5–7oz lean red meat) on blood lipids, lipoproteins, and emerging risk factors for CVD. Eighty men and women (BMI 18.5–37m/kg2) will be split into two distinct populations, moderately hypercholesterolemic (LDL‐C between the 50th and 90th percentile) and moderately hypertriglyceridemic (TG>150 mg/dL). We hypothesize that the BOLD and BOLD+ diets will yield changes in LDL‐C similar to that seen during the DASH diet period. We also hypothesize that the BOLD and BOLD+ diet periods will produce better effects on HDL‐C, TG and glucose homeostasis.
Crack Cocaine Use Accelerates CD4 decline and is Associated With Poorer Nutritional Status in HIV+ Drug Users
Authors: Rafie Carlin , Campa Adriana , Sales Sabrina , Baum Marianna K
Published: June 2021

Objective:

To evaluate the effect of crack cocaine use on HIV disease progression and nutritional status.

Methods:

After consenting, 222 HIV+ drug users were followed for 30 mo. CD4 count, HIV viral load, anthropometries, 24‐hour intake recall, antiretroviral (ARV) treatment, and drug use was assessed every 6 months. Percent of adequate calorie and protein was determined using 3 dietary recalls taken over 12 months. Cox Regression was used to evaluate drug use and time to decline of CD4 to <200cells/μL.

Results:

Crack use was associated with a faster decline of CD4 to <200cells/μL (RR=2.027, CI: 1.024–4.013), after controlling for baseline CD4 and ARV use. Crack users had lower body mass index (logBMI: 1.38±0.08 vs. 1.40±0.08, p=0.044), greater likelihood of an albumin<4.0g/dL (p=0.032), and higher viral load (logVL: 4.21±1.02 vs 3.80±1.06, p=0.004). BMI and albumin were significantly correlated independent of viral load (r=0.142, p=0.035). Adequacy of energy and protein intake, monthly income, homelessness, food security, and ARVs were not different between crack users and non‐users.

Conclusions:

Crack use is associated with faster decline of CD4 count, and poorer nutritional status, despite similar energy and protein intake. Crack use may increase nutritional requirements, suggesting a need for specific nutritional intervention in crack users.

Support: Funded by NIDA
Chronic and portable fluorescence microendoscopy in freely moving mice
Authors: Schnitzer Mark
Published: June 2021

Fluorescence microendoscopy (FME) is emerging as a promising modality for cellular level imaging in live mammalian subjects. We have developed novel techniques for imaging the living nervous system using both one‐ and two‐photon fluorescence excitation forms of FME. Our microendoscope probes are based on minimally invasive compound gradient refractive index (GRIN) lenses (350–1000 μ m diameter) that provide micron‐scale resolution. Such probes have allowed us to visualize cellular details in regions of the nervous system inaccessible to conventional microscopy. However, the use of FME to date has been limited to acute studies of anesthetized subjects. We present two developments that extend FME usage to chronic imaging in anesthetized subjects over prolonged time periods, as well as to cellular level brain imaging in freely moving subjects. First, we present techniques for long‐term FME studies that have enabled imaging of CA1 hippocampal pyramidal neurons in live mice months after an initial surgery. Second, we present several forms of compact fluorescence microendoscopy instrumentation intended for brain imaging in freely behaving adult mice. Although further engineering remains, we expect that fluorescence imaging in freely moving mice will become increasingly feasible, allowing detailed comparisons of animal behavior, physiological dynamics, and cellular properties between normal and transgenic mouse subjects.
Dietary bovine lactoferrin does not alter intestinal lymphocyte apoptosis but decreases TNF‐α production during exercise induced intestinal inflammation
Authors: Spagnuolo Paul Anthony , Hoffman‐Goetz Laurie
Published: June 2021

Strenuous exercise can induce physiological alterations (e.g., ischemia‐reperfusion injury, generation of free radicals, increased stress hormones, apoptosis) which increase the likelihood of intestinal inflammation. Bovine lactoferrin (bLf), a common dietary protein which affects cytokine expression and lymphocyte apoptosis, is a potential therapeutic tool against the development of intestinal inflammation. The purpose of this study was to determine the effects of bLf on intestinal lymphocyte (IL) apoptosis and cytokine production following strenuous exercise. C57BL/6 female mice (n=89) were given three repeated bouts of exhaustive exercise, plasma collected and IL isolated. Exercise resulted in IL loss (p<0.05) attributed to increased expression of pro‐apoptotic caspase‐3 (p<0.001) and decreased expression of antiapoptotic Bcl‐2 (p<0.05) in IL. Elevations in corticosterone (p<0.05) and 8‐isoprostane F2α (p<0.05) in plasma and HSP 70 (p<0.01) in IL were also observed following exercise. Dietary bLf did not alter these exercise induced effects but did decrease TNF‐α expression (p<0.05), possibly through reduced levels of NFκB (p<0.05) in IL; there was no change in IL‐10. Thus, dietary bLf does not appear to alter exercise induced IL cell loss and apoptosis but may confer some intestinal protection by decreasing the TNF‐α (inflammatory) cytokine response. Research supported by NSERC of Canada.
Effect of the nutritional recovery on serum concentration and intracellular expression in T lymphocytes of Interleukin‐2 in severely malnourished children
Authors: Vasquez‐Garibay Edgar Manuel , Stein Katja , Méndez‐Estrada Concepción , Pérez‐Becerra Elías , Romero‐Velarde Enrique , Gutiérrez Isabel Ibarra , Ortega Miguel Angel Ortiz , Iglesias Trinidad García
Published: June 2021

With the purpose to demonstrate the effect of intensive nutritional support on the cellular and serum concentration of IL‐2 in children with severe protein energy malnutrition a clinical assay was carried out in a tertiary care hospital with 10 severely malnourished children < 48 months of age who received formula without lactose via enteral feeding for two weeks and ad libitum for an additional two weeks were included. Cellular and serum concentrations of IL‐2 and the subpopulation of CD4+ and CD8+ were obtained. A control group (n = 13) was included. A paired student t test for initial‐final determinations and the Mann‐Whitney Test for comparison with control group were used, and null hypothesis was rejected with a p value < 0.05. There was a noteworthy increase in the comparison between the initial vs. final percentage of the cellular expression of IL‐2 (p < 0.001) and in the serum concentration of IL‐2 (p = 0.001). However, at the end of the study the serum concentration of IL‐2 was still lower than in the control group (p = 0.013). Four weeks of nutritional recovery significantly restored the production of IL‐2, although, the total restoration may take longer and probably related with the severity of the PEM. Funded by CONACYT.
Stability of biochemical analytes in blood specimens subjected to delayed processing
Authors: Hitchcox Kristen , Wolf Megan , Zwart Sara R. , Smith Scott M.
Published: June 2021

Blood is nominally centrifuged within 45 min of phlebotomy and the plasma or serum is stored at −80°C until it is analyzed, but in some off‐nominal cases, whole blood may be held at 20–25°C for an extended period after phlebotomy. The standard recommendation for the processing time was designed to maximize analyte stability. We investigated the stability of several analytes related to bone metabolism and clinical chemistry. Blood was collected from 10 subjects and was maintained at ambient temperature and protected from light for 45 min and 2.5, 5, and 24 h after phlebotomy before it was centrifuged to collect serum. Significant changes were observed for parathyroid hormone (PTH, decreased at 24 h, P < 0.05) and osteocalcin (decreased at 24 h, P < 0.001). Homocysteine was significantly elevated at 24 h (P < 0.001). No changes were observed for the remaining analytes, including calcium, cortisol, methylmalonic acid, retinol‐binding protein, total antioxidant capacity, transferrin and transferrin receptors, 25‐hydroxyvitamin D, cholesterol, triglycerides, alkaline phosphatase, total protein, sodium, albumin, creatinine, aspartate aminotransferase, and alanine aminotransferase. These studies indicate that some blood components related to bone and iron metabolism, including PTH, osteocalcin, and homocysteine, are not stable for up to 24 h when stored at 25°C. This supported by the NASA Human Research Program.
Effects of folic acid on the myelin basic protein expression in the maternal brain
Authors: Lee Yo A , Lee Ji Hye , Oh Kyung Hee , Chang Namsoo
Published: June 2021

It is well known that folate is essential for fetal brain development, but the effect of folate on maternal brain is unknown. Myelin basic protein (MBP), a major structural protein of myelin, is thought to be important for the maintenance of myelin in the central nervous system (CNS). The synthesis of MBP requires a universal methyl donor, S‐adenosylmethionine, whose synthesis is dependent on folate and its involvement in one‐carbon metabolism. We investigated the effects of folic acid on the expression of MBP in the maternal CNS during pregnancy and lactation period. Female Sprague‐Dawley rats were fed either folic acid sufficient (8mg/kg diet) or deficient (0mg/kg diet) diet from 2 weeks prior to the mating throughout the entire pregnancy and lactation period. The MBP expression in the cortex, hippocampus and spinal cord was observed by immunohistochemical study and western blot analysis. At gestational day 20, the MBP expression in the spinal cord was increased in both groups, but the increase was greater in animals on a folic acid sufficient diet than those on a deficient diet. In conclusion, the maternal folic acid deficiency might inhibit the MBP synthesis in the spinal cord during pregnancy, and this might affect the maternal neurological function.

This work was supported by the second stage of Brain Korea 21 project in 2007
Comparison of Different Protein Diets on Renal Function and Bone Health in Normal Growing Female Rats
Authors: Gigliotti Joseph Christopher , Jaczynski Jacek , Tou Janet
Published: June 2021

Feeding animal proteins but not milk proteins, short‐term at conventional amounts, has been reported to alter renal function. The objective of this study was to investigate effects of different protein diets on renal function and bone health. Female rats (n=10/group) were individually housed in metabolic cages and fed ad libitum isonitrogenous and isocaloric diets consisting of either 10% casein or 10% krill protein concentrate (KPC) isolated from Antarctic krill (Euphausia superba)for 4 weeks. Results showed that rats fed KPC had lower (P<0.001) relative kidney weights compared to the rats fed casein. Rats fed KPC had lower kidney calcium (Ca) (P=0.008) and phosphorus (P) (P<0.001) levels compared to rats fed casein. Urinary output was higher (P=0.03) in the rats fed KPC than casein. However there were no changes in renal function indicated by absence of differences in serum levels of creatinine, blood urea nitrogen, protein, Ca, and P between the groups. There were no difference (P=0.49) in dietary Ca or P intake between the treatment groups. Urinary Ca (P=0.06) and P (P=0.69) excretion was not significantly different. There were no differences in femur total bone mineral content or femoral Ca and P. Femur strength measured by three‐point bending test was similar between rats fed KPC and rats fed casein. Based on the study results, short‐term consumption of KPC appeared comparable to casein for renal and bone health.
Investigation of the Role of Glypican 3 in Rat Hepatocyte Growth and Liver Regeneration
Authors: Liu Bowen , Paranjpe Shirish , Bowen William , Bell Aaron , Mars Wendy , Michalopoulos George
Published: June 2021

Glypicans are heparan sulfate proteoglycans (HSPGs) that are bound to the cell surface through a glycosylphosphatidylinositol (GPI) anchor. Six members of glypican family have been identified in mammals. Our study focused on glypican 3 (GPC3) which is mutated in the Simpson‐Golabi‐Behmel (SGB) syndrome, an X‐linked disorder characterized by pre‐ and postnatal overgrowth. GPC3 is reported to be over‐expressed in human hepatocellular carcinoma (HCC). Whether GPC3 is involved in liver regeneration and hepatocyte proliferation is still unknown. We investigated its role in regenerative liver using the 2/3rd partial hepatectomy (PHx) model in rats. Results showed that GPC3 is up‐regulated in liver starting from day 2 after PHx. GPC3 is also up‐regulated from day 7 in primary hepatocyte cultures, when the process of proliferation slows down. Yeast two hybrid assay revealed that GPC3 might interact with CD81, a member of tetraspanin and reported to involve in HCV infection and cell proliferation. Our study showed that CD81 level increased from day 2 after PHx, corresponding to the changes of GPC3. Immunofluorescence showed that CD81 and GPC3 co‐localize at day 2 and day 6 after PHx. Further study will include GPC3 transgenic mice screening and study, and function and growth study by silencing GPC3 using Lentivirus. We hypothesize that GPC3 negatively regulate liver regeneration and may interact with CD81.
Ethanol feeding enhances the hypotensive action of enalapril in telemetered female rats: Role of bradykinin B2 receptors
Authors: El‐Mas Mahmoud M , Abdel‐Rahman Abdel A
Published: June 2021

We investigsated the effect of chronic ethanol feeding on acute hemodynamic actions of enalapril in telemetered female rats and the role of bradykinin B2 receptors in the interaction. Changes evoked by enalapril (10 mg/kg i.p.) in blood pressure (BP), heart rate (HR), myocardial contractility (dP/dtmax), and power spectral indices of hemodynamic variability were evaluated in female rats receiving liquid diet with or without ethanol (5%, w/v, 8 weeks). Ethanol feeding reduced BP and dP/dtmax versus no effect on HR or hemodynamic variability. Enalapril decreased BP in control rats for at least 5 hrs and increased the low‐frequency (LF, 0.25–0.75 Hz), but not high‐frequency (HF, 0.75–3 Hz), spectral power of interbeat intervals (IBI) and IBILF/HF ratio, suggesting cardiac sympathetic dominance. In ethanol‐fed rats, enalapril elicited significantly greater hypotension and abolished the increase in cardiac sympathetic activity. Blockade of bradykinin B2 receptors by bradyzide (2 mg/kg i.p.) abolished the augmented hypotensive effect of enalapril in ethanol‐fed rats. In control rats, bradyzide eliminated the increase in cardiac sympathetic activity but had no effect on enalapril hypotension. These findings suggest that chronic ethanol enhances enalapril‐evoked hypotension in female rats, at least partly, via facilitation of bradykinin B2 receptor signaling.

Supported by Grant R01 AA014441 from NIAAA.
Cytochrome P450 3A4 regiospecific oxygenation of raloxifene: Evidence for the formation of the di‐quinone methide
Authors: Moore Chad Douglas , Yost Garold S
Published: June 2021

While the bioactivation of xenobiotics to toxic intermediates through cytochrome P450 (CYP)‐catalyzed oxygenation reactions is a well recognized process, the production of reactive electrophilic intermediates by P450s through dehydrogenation reactions is not well characterized. Raloxifene is a second generation selective estrogen receptor modulator used for the treatment and prevention of osteoporosis in postmenopausal women. Previous studies have shown CYP3A4‐mediated metabolism of raloxifene produces two hydroxylated metabolites and several glutathione (GSH) adducts. The hydroxylated metabolites and one of the GSH adducts could theoretically be derived from the same dehydrogenated intermediate. It is postulated that raloxifene is dehydrogenated to a reactive di‐quinone methide. To evaluate the formation of the di‐quinone methide, raloxifene was incubated with CYP3A4 in the presence of H218O or 18O2. LC/MS analysis showed that 3′‐hydroxyraloxifene incorporated oxygen only from 18O2, while the source of the oxygen for the 7‐hydroxyraloxifene was inconclusive. These results demonstrate CYP3A4 selectively oxygenates raloxifene from 18O2 at the 3′‐position, but not at the 7‐position. Furthermore, the lack of CYP3A4‐mediated oxygenation at the 7‐position suggests that the di‐quinone methide is hydrated by active‐site H2O, but labeled H218O could not serve as the source of hydration. Supported by NIH grant # GM074249 form the National Institute of General Medical Sciences.
Investigating naturally occurring variations in the Acetylcholinesterase gene of a human population
Authors: Valle Anne Marie , Radic Zoran , Ho Kwok‐Yiu , Mahboubi Vafa , Wessel Jennifer , Rao Fangwen , Rana Brinda K. , O’Connor Daniel T. , Taylor Palmer
Published: June 2021

We are studying single nucleotide polymorphisms (SNPs) in the acetylcholinesterase (AChE) gene of a human population in relation to catalytic properties and cardiovascular (CV) function. Cholinergic proteins play an important regulatory role in homeostatic maintenance of CV function. AChE found in whole blood enabled a biochemical characterization in addition to correlating genotype with phenotypic physiologic responses. Enzymatic activity determined in whole blood from twin and general population subject registries revealed significant relationships between AChE activity and certain CV endpoints. Also, the analysis enabled us to ascertain the impact of genetic variation and environmental influences on AChE activity. To date, we identified 19 SNPs of which 4 are non‐synonymous coding SNPs (cSNPs). Mutagenesis was conducted to introduce the non‐synonymous cSNPs into a human AChE cDNA vector with subsequent transfection into HEK cells for protein expression. Ongoing characterization of the purified mutant enzymes revealed significant thermal stability differences when compared with the predominant AChE species. With the structure of cholinesterase enzyme proteins and genes delineated, this research is relevant particularly in CV diseases and individual risks associated with chemical agent exposure affecting cholinergic function. Supported by NIH R37‐GM18360
Single nucleotide polymorphisms in cytochrome P450 oxidoreductase influence drug metabolism
Authors: Hart Steven , Wang Shuang , Nakamoto Kaori , Wesselman Chris , Li Ye , Zhong Xiao‐bo
Published: June 2021

Cytochrome P450 oxidoreductase (POR) is the only protein that donates electrons to all microsomal P450 enzymes, which metabolize more than 80% of prescription drugs. Our objective was to determine if polymorphisms in POR could affect gene expression and activity of POR, and/or P450‐catalyzed drug metabolism. POR polymorphisms were identified from non‐diseased human livers (n=99) by sequencing the POR gene and mRNA levels were quantified by the branched DNA method. POR activity was quantified by measuring cytochrome c reduction in liver microsomes and activities of ten drug‐metabolizing P450 enzymes were quantified by HPLC. Of the 34 identified polymorphisms, 9 were novel, 9 were in exons, and 4 changed an amino acid: K49N, L420M, A503V, and L577P. We report a novel exonic polymorphism, L577P (patent pending), which is associated with decreased POR (p=0.003) and several P450 activities. Our data suggest that [1] POR is functionally polymorphic in the general population and [2] has the potential to serve as a biomarker to optimize pharmacologic dosing strategies. This work was supported by NIH COBRE 5P20 RR021940 and Pharmaceutical Research and Manufacturers of America Foundation (PhRMA).
Inhibitory effect of berberine on the TNF‐α‐induced Expression of IL‐8 and MCP‐1 is mediated through suppression of NF‐κB activation in HT29 Human Colon Epithelial Cells
Authors: Lee Gwang Ik , Thapa Dinesh , Lee Jong Suk , Babu Dinesh , Kim Kyoung‐Jin , Kim Jung‐Ae
Published: June 2021

Berberine, an isoquinoline alkaloid, has a wide range of pharmacological effects, including anti‐inflammation. In this study, we examined the effects of berberine on the TNF‐α‐induced chemokine expression in HT29 human colon adenocarcinoma cells that were used as an in vitro model of inflammatory bowel disease (IBD). Since the activation and recruitment of leukocytes to the inflamed tissue is an important step initiating IBD, we evaluated the effect of berberine on the adhesion of U937 monocytic cells to HT29 colon epithelial cells. Berberine significantly inhibited the TNF‐α‐induced monocyte adhesion to HT29 cells in a concentration‐dependent manner. Co‐treatment of the TNF‐α‐stimulated HT29 cells with berberine concentration‐dependently inhibited the mRNA expression of IL‐8 and MCP‐1, important mediators involved in the pathologic process of IBD. In addition, the ratio of the firefly and renilla luciferase activities (F/R) was used as an indicator of the NF‐κB activity in the cells. TNF‐α significantly increased the F/R ratio in the cells, and this was significantly inhibited by berberine in a concentration‐dependent manner. The results suggest that berberine may be a potential candidate to suppress the colon inflammation by inhibiting TNF‐α‐induced pro‐inflammatory cytokines.
Anti‐inflammatory properties of KY087a and KY087 in three animal models
Authors: Zheng Taoye , Yang Jieying , Zhang Yan , Gao Ling , Zhao Chunsheng , Zhou Yanjiao , Tan Ningzhi , Zhu Jia‐Shi
Published: June 2021

We investigated the effects of KY087a and KY087 on anti‐inflammation in 3 animal models: adjuvant arthritis in rats (AA), ear swollen inflammation in mice (ES) and pain relief in mice (writhe test, PR). (1) In AA, KY087a (0.03g/kg) had no effect on primary inflammatory reaction, but inhibited secondary inflammatory reaction of rat paws (P=0.009 at Day 11; P=0.022 at Day 15). KY087 (0.2, 1.8g/kg) inhibited primary inflammatory reaction of rat paws (P=0.04 in 24 and 48 h.), but showed no significant effects on the secondary inflammatory reaction. KY087a (0.01, 0.03, 0.09g/kg) and KY087 (0.2, 0.6, 1.8g/kg) lowered serum TGF‐beta and CRP (P<0.001). (2) In ES, KY087a (0.06, 0.18g/kg) and KY087 (0.4, 1.2g/kg) reduced ear swollen by >50% (P<0.01). (3) In PR, KY087 (1.2g/kg) reduced the number of mouse writhe in the 15‐min observation period by 75% (P<0.05), compared to controls. Our results indicate the anti‐inflammatory effects of KY087a and KY087 in 3 inflammation models in rats and mice.
Percutaneous application of carbon dioxide affects skin microcirculatory dynamics in healthy subjects
Authors: Nagashima Yoshinao , Mori Hideki , Yokomichi Hideki , Yada Yukihiro , Tsuchiya Shuuichi , Suzuki Toshiyuki , Oh‐ishi Sachiko , Majima Masataka
Published: June 2021

Objective: Bathing in carbonated springs is considered to dilate skin blood vessels and increase blood flow. We developed a plaster‐type carbon dioxide CO2‐sheet to apply on a topical skin area, and examined the microcirculatory dynamics of its application.

Methods: CO2‐sheets containing different concentration of CO2 (250, 500, and 1000 ppm in solvent) were applied on the flexor aspect of the forearm of healthy subjects, and changes in the microcirculatory dynamics were studied by direct measurement of diameter of subepidermal microvessels using a Capillary Scope System, which irradiates the skin surface with indirect light. Local skin blood flow was measured by the venous occlusion method using near‐infrared spectroscopy, and also by two‐dimensional mapping using a full‐field laser profusion imager.

Results: Compared with sham sheets, application of the CO2‐sheets significantly increased the diameter of subepidermal microvessels and increased the blood flow concentration‐dependently.

Conclusions: This CO2‐sheets may be useful as a therapeutic device to improve skin microcirculation in the fields of health care as well as clinical medicine.
Mapping Microvascular Network Geometry in 3D
Authors: Fraser Graham Mathew , Goldman Daniel , Milkovich Stephanie , Ellis Christopher
Published: June 2021

The objective is to develop a novel mapping software package using MATLAB to reconstruct microvessels in 3‐dimensions for use in oxygen transport modeling schemes. A single optical imaging system is used to collect experimental in vivo data (hemodynamics and oxygen saturation). Vessels are selected from video sequences and functional still images using automated edge tracking and depth information, collected during the experiment, to construct the 3D network. Simple user driven commands allow the connection of vessel segments and the creation of bifurcations. Built in registration and calibration improves accuracy of relative vessel position and allows vessels to span across multiple focal planes and fields of view. Resulting output provides a simple and versatile array that accurately describes 3D network geometry. The final network visualization (see figure) represents vessels as variable diameter tubes that are scaled accurate to the bounding volume. Networks can be rotated and manipulated in 3D to verify network connections and vessel continuity. The network and corresponding hemodynamic and SaO2 data can then be easily integrated into computational oxygen transport models.
Aging and ovariectomy alter endothelium‐dependent and –independent vasodilation in the femoral principal nutrient artery
Authors: Prisby Rhonda D , Delp Michael , Muller‐Delp Judy
Published: June 2021

Enhanced bone resorption and bone blood flow (BBF) occurs subsequent to ovariectomy (OVX) whereas BBF declines with age. The potential of the osseous resistance vasculature to alter BBF in postmenopausal and age‐related osteoporosis is unclear. PURPOSE: We studied the effects of age, gender, and estrogen status on endothelium‐dependent and ‐independent vasodilation of the femoral principal nutrient artery (PNA). METHODS: INTACT female (6‐, 14‐, and 24‐ mon), OVX female (6‐, 14‐, and 24‐ mon), and male (6‐ and 24‐ mon) Fischer‐344 rats were studied. PNAs were isolated and cannulated. Endothelium‐dependent (ACh: 10−9 – 10−4 M) and ‐independent (DEA NONOate: 10−10 – 10−4 M) vasodilation were assessed. RESULTS: Declines in endothelial function occurred in aged rats but no differences in gender were observed. Endothelium‐dependent vasodilation in young or middle‐aged rats did not change following OVX. In aged OVX rats, vasodilation was enhanced to similar values observed in young INTACT and young OVX rats. Endothelium‐independent vasodilation was not altered with age or gender except in middle aged OVX rats vs. their INTACT controls. DISCUSSION: These data demonstrate that OVX influences both endothelium‐dependent and –independent vasodilator responses of the osseous resistance vasculature. Supported by the NIH Grant #1 R01 HL077224‐01 (JMD) and the NHLBI Postdoctoral Supplement (RDP).