Recombinant Fab expression and secretion in Escherichia coli continuous culture at medium cell densities: Influence of temperature

Production of recombinant antibody fragments (Fabs) in Escherichia coli has gained interest because of the recognised advantages of this expression system and because Fabs do not require glycosylation. However, more comprehensive studies on the factors that influence expression conditions and product yield are still required for full process development. In this work, the effect of growth temperature on the periplasmatic expression of the 3H6 Fab in E. coli was studied in carbon-limited continuous cultures operated at medium cell densities. Three different temperatures were assayed, namely 37, 33 and 30 °C. Results showed that biomass yield was not affected within this temperature range whilst product yield increased as temperature decreased. Periplasmic Fab secretion corresponded to 30% of the produced Fab protein and its efficiency was irrespective of the process temperature. Moreover, considerable product leakage to the culture supernatant was detected in all cases, ranging from about 40% at 37 °C to almost 70% at 30 °C. Besides, plasmid loss was observed along process time indicating a selective pressure against plasmid-bearing cells. This study supports the potential of continuous cultivations of E. coli at medium cell densities under well controlled conditions as a tool for characterising the impact of environmental parameters and cell physiology under protein production conditions.     

Combined effect of temperature and zinc on Caenorhabditis elegans wild type and daf-21 mutant strains

Heavy metal pollution in aquatic ecosystems is a far reaching environmental problem. The possible influences of heavy metal exposure and the potential harm to organisms when combined with other environmental stressors such as temperature have been largely unexplored. An aquatic toxicity test of Caenorhabditis elegans was performed to estimate the 24 h median lethal concentration (LC₅₀) of different zinc concentrations at different temperatures (15 °C, 20 °C, 25 °C, and 30 °C). We also examined the time course thermotolerance on wild type (N2) and daf-21 null (JT6130) adults exposed to 6.1 mM zinc at 37 °C. Hsp90 protein expression level in response to the combined effect of temperature and zinc toxicity was also investigated by both Western blots and ELISA. Our results show that C. elegans wild type nematodes exhibit severe lethal toxicity after a 24 h exposure to zinc at higher temperatures. In addition, the expression level of Hsp90 was highly inhibited in adult worms subjected to zinc stress. This toxicity assay at different temperatures provides insight into organism response to combined effects of temperature and zinc toxicity.     

Enterococcus faecalis immunoregulates osteoclastogenesis of macrophages

Persistent apical periodontitis (PAP) is characterized by refractory inflammation and progressive bone destruction. Enterococcus faecalis infection is considered an important etiological factor for the development of PAP, although the exact mechanisms remain unknown. This study aimed at investigating the role of E. faecalis in cell proliferation, inflammatory reactions and osteoclast differentiation of macrophages using an in vitro infection model of osteoclast precursor RAW264.7 cells. A cell viability assay of cultured RAW264.7 cells exposed to live E. faecalis at a multiplicity of infection of 100 for 2 h, indicated that the infection exhibited no cytotoxic effect. Transmission electron microscopy images revealed no apoptotic changes but a rise of metabolic activity and phagocytic features in the infected RAW264.7 cells. Confocal laser scanning microscopic and flow cytometric analysis indicated that the phagocytosis of RAW264.7 cells was activated by E. faecalis infection. Furthermore, quantitative real-time PCR assays demonstrated that the expression of inflammatory cytokines was remarkably elevated in infected RAW264.7 cells. Differentiation of infected RAW264.7 cells into osteoclasts was remarkably attenuated, and expression of osteoclast marker genes as well as fusogenic genes significantly dropped. In summary, E. faecalis appears to attenuate osteoclastic differentiation of RAW264.7 precursor cells, rather stimulates them to function as macrophages.     

The in vitro effect of temperature on motility and antioxidant response of common carp Cyprinus carpio spermatozoa

The effect of temperature on Cyprinus carpio spermatozoa in vitro was investigated with spermatozoa activated at 4, 14, and 24 °C. At 30 s post-activation, motility rate was significantly higher at 4 °C compared to 14 and 24 °C, whereas highest swimming velocity was observed at 14 °C. The thiobarbituric acid-reactive substance (TBARS) content was significantly higher at 14 °C and 24 °C than at 4 °C in motile spermatozoa. No significant differences in catalase and superoxide dismutase activity relative to temperature were observed. This study provides new information regarding effect of temperature on lipid peroxidation intensity and spermatozoon motility parameters in carp. The elevation of TBARS seen at higher temperatures could be due to inadequate capacity of antioxidant enzymes to protect the cell against the detrimental effects of oxidative stress induced by higher temperatures.     

Hypoglycemic effect of aspalathin,a rooibos tea component from Aspalathus linearis,in type 2 diabetic model db/db mice

Effects of aspalathin, a green rooibos tea component, on glucose metabolism were studied in vitro and in vivo. We first examined the effect of aspalathin on glucose uptake by cultured L6 myotubes and on insulin secretion from cultured RIN-5F pancreatic β-cells in vitro, and then investigated the effect of dietary aspalathin on fasting blood glucose level and conducted an intraperitoneal glucose tolerance test (IPGTT) using type 2 diabetes model mice in vivo. Aspalathin dose-dependently and significantly increased glucose uptake by L6 myotubes at concentrations 1–100 μM. It also significantly increased insulin secretion from cultured RIN-5F cells at 100 μM. Dietary aspalathin (0.1–0.2%) suppressed the increase in fasting blood glucose levels of db/db mice for 5 weeks. In IPGTT, aspalathin improved impaired glucose tolerance at 30, 60, 90, and 120 min in db/db mice. These results suggest that aspalathin has beneficial effects on glucose homeostasis in type 2 diabetes through stimulating glucose uptake in muscle tissues and insulin secretion from pancreatic β-cells.     

Proteomic analysis of the adaptation to warming in the Antarctic bacteria Shewanella frigidimarina

Antarctica is subjected to extremely variable conditions, but the importance of the temperature increase in cold adapted bacteria is still unknown. To study the molecular adaptation to warming of Antarctic bacteria, cultures of Shewanella frigidimarina were incubated at temperatures ranging from 0 °C to 30 °C, emulating the most extreme conditions that this strain could tolerate. A proteomic approach was developed to identify the soluble proteins obtained from cells growing at 4 °C, 20 °C and 28 °C. The most drastic effect when bacteria were grown at 28 °C was the accumulation of heat shock proteins as well as other proteins related to stress, redox homeostasis or protein synthesis and degradation, and the decrease of enzymes and components of the cell envelope. Furthermore, two main responses in the adaptation to warm temperature were detected: the presence of diverse isoforms in some differentially expressed proteins, and the composition of chaperone interaction networks at the limits of growth temperature. The abundance changes of proteins suggest that warming induces a stress situation in S. frigidimarina forcing cells to reorganize their molecular networks as an adaptive response to these environmental conditions.     

Berberine in combination with doxorubicin suppresses growth of murine melanoma B16F10 cells in culture and xenograft

Melanoma is very aggressive and major cause of mortality due to skin cancer. Herein, we studied the anticancer effects of berberine, a plant alkaloid, in combination with doxorubicin on murine melanoma B16F10 cells in vitro and in vivo. This drug combination strongly inhibited cell growth and induced cell death, and caused G2/M arrest in cell cycle together with a decrease in Kip1/p27. Berberine showed stronger inhibitory effect on ERK1/2 phosphorylation as compared to Akt phosphorylation, whereas the combination of the drugs showed greater inhibitory effect on Akt phosphorylation. In murine B16F10 xenograft, cells were implanted into mice and treated with vehicle (methyl cellulose) or berberine (100 mg/kg of body weight/day by oral gavage) or doxorubicin (4 mg/kg of body weight/week by intraperitoneal injection) or combination of berberine and doxorubicin. Berberine alone did not show any considerable effect on tumor growth as observed with doxorubicin, however, the combination of the two drugs resulted in a significant and strong decrease in tumor volume (85%, p < 0.005) and tumor weight (78%, p < 0.05) as compared to control. Immunohistochemical analysis of tumor samples showed that drug combination decreased PCNA-positive cells (82%, p < 0.001) and increased cleaved caspase-3 positive cells (3-fold, p < 0.05) indicating inhibition of proliferation and an increase in apoptosis, respectively. Overall, our findings suggest that berberine and doxorubicin could be a novel combination to inhibit melanoma tumor growth.     

Effect of environmental and nutritional factors on growth and azaspiracid production of the dinoflagellate Azadinium spinosum

Azadinium spinosum, a small dinoflagellate isolated from the North Sea, is a producer of azaspiracids (AZAs), a group of biotoxins associated with human illness following ingestion of contaminated shellfish. Using batch and continuous cultures of A. spinosum, the present study investigated the effects of different environmental and nutritional factors (salinity, temperature, photon flux density, aeration, culture media, nitrogen sources, phosphate source, and N/P ratios) on growth, maximum cell concentration, and AZA cell quota.Azadinium spinosum grew in a wide range of conditions; from 10 ̊C to 26 ̊C and salinities from 30 to 40, under irradiances ranging from 50 μmol m⁻² s⁻¹ to 250 μmol m⁻² s⁻¹, with or without aeration. Growth and maximum cell concentration were highest at a salinity of 35, at temperatures between 18 ̊C and 22 ̊C, and with aeration. Concerning AZA cell quota, the most significant effect was observed at low temperature; the AZA cell quota was more than 20 times higher at 10 ̊C (220 fg cell⁻¹) than at temperatures between 18 ̊C and 26 ̊C. A. spinosum grew on all media tested with only slight differences in growth rate and AZA cell quota. In continuous culture, lowering the concentration of nutrients (0.5 strength of a modified K-medium) in the inflow improved AZA cell quota whereas higher concentration (doubling the normal strength of K-medium) improved maximal cell concentration. A. spinosum grew on different sources of nitrogen tested (nitrate, urea, ammonium) with almost no effect on toxin cell quota and growth, except that adding ammonium caused a decrease in growth.These first experiments on Azadinium spinosum increased our knowledge on factors affecting its growth and toxin production; furthermore, these results allowed and improved particularly A. spinosum production in pilot scale photobioreactors for AZA isolation.     

A novel anti-inflammatory mechanism of high density lipoprotein through up-regulating annexin A1 in vascular endothelial cells

High density lipoprotein (HDL) as well as annexin A1 have been reported to be associated with cardiovascular protection. However, the correlation between HDL and annexin A1 was still unknown. In this study, HDL increased endothelial annexin A1 and prevented the decrease of annexin A1 in TNF-α-activated endothelial cells in vitro and in vivo, and above effects were attenuated after knockdown of annexin A1. Annexin A1 modulation affected HDL-mediated inhibition of monocyte adhesion to TNF-α-activated endothelium (45.2 ± 13.7% decrease for annexin A1 RNA interference; 78.7 ± 16.3% decrease for anti-Annexin A1 antibody blocking; 11.2 ± 6.9% increase for Ad-ANXA1 transfection). Additionally, HDL up-regulated annexin A1 through scavenger receptor class B type I, involving ERK, p38MAPK, Akt and PKC signaling pathways, and respective inhibitors of these pathways attenuated HDL-induced annexin A1 expression as well as impaired HDL-mediated inhibition of monocyte–endothelial cell adhesion. Apolipoprotein AI also increased annexin A1 and activated similar signaling pathways. Endothelial annexin A1 from apolipoprotein AI knockout mice was decreased in comparison to that from wild type mice. Finally, HDL-induced annexin A1 inhibited cell surface VCAM-1, ICAM-1 and E-selectin, and secretion of MCP-1, IL-8, VCAM-1 and E-selectin, thereby inhibiting monocyte adhesion.     

Rising temperatures may increase growth rates and microcystin production in tropical Microcystis species

Rising temperatures (1.4–6 °C) due to climate change have been predicted to increase cyanobacterial bloom occurrences in temperate water bodies; however, the impacts of warming on tropical cyanobacterial blooms are unknown. We examined the effects of four different temperatures on the growth rates and microcystin (MC) production of five tropical Microcystis isolates (M. ichthyoblabe (two strains), M. viridis, M. flos-aquae, and M. aeruginosa). The temperature treatments are based on current temperature range in Singapore's reservoirs (27 °C and 30 °C), as well as projected mean (33 °C) and maximum temperatures (36 °C) based on tropical climate change estimates of +6 °C in air temperature. Increasing temperatures did not significantly affect the maximum growth rates of most Microcystis strains. Higher growth rates were only observed in one M. ichthyoblabe strain at 33 °C and M. flos-aquae at 30 °C where both were isolated from the same reservoir. MC-RR and MC-LR were produced in varying amounts by all four species of Microcystis. Raised temperatures of 33 °C were found to boost total MC cell quota for three Microcystis strains although further increase to 36 °C led to a sharp decrease in total MC cell quota for all five Microcystis strains. Increasing temperature also led to higher MC-LR:MC-RR cell quota ratios in M. ichthyoblabe. Our study suggests that higher mean water temperatures resulting from climate change will generally not influence growth rates of Microcystis spp. in Singapore except for increases in M. ichthyoblabe strains. However, toxin cell quota may increase under moderate warming scenarios depending on the species.     

Synthesis and in vitro cytotoxic evaluation of new 1H-benzo[d]imidazole derivatives of dehydroabietic acid

A series of new 1H-benzo[d]imidazole derivatives of dehydroabietic acid were designed and synthesized as potent antitumor agents. Structures of the target molecules were characterized using MS, IR, ¹H NMR, ¹³C NMR and elemental analyses. In the in vitro cytotoxic assay, most compounds showed significant cytotoxic activities against two hepatocarcinoma cells (SMMC-7721 and HepG2) and reduced cytotoxicity against noncancerous human hepatocyte (LO2). Among them, compound 7b exhibited the best cytotoxicity against SMMC-7721 cells (IC₅₀: 0.36 ± 0.13 μM), while 7e was most potent to HepG2 cells (IC₅₀: 0.12 ± 0.03 μM). The cell cycle analysis indicated that compound 7b caused cell cycle arrest of SMMC-7721 cells at G2/M phase. Further, compound 7b also induced the apoptosis of SMMC-7721 cells in Annexin V-APC/7-AAD binding assay.     

Efficacy of the antagonist Aureobasidium pullulans PL5 against postharvest pathogens of peach,apple and plum and its modes of action

The efficacy of Aureobasidium pullulans PL5 against different postharvest pathogens of fruits (Monilinia laxa on plums and peaches, Botrytis cinerea and Penicillium expansum on apples) were evaluated under storage conditions when applied at 10⁸ cells ml⁻¹ and their interactions were studied in vitro and in vivo to discover the possible modes of action. Under 1.2 °C and 95% relative humidity (RH) for 28 days, the efficacy of PL5 against M. laxa on plums was 45%, reducing disease incidence from 78% to 43%. Under 1 °C and 95% RH for 21 days, the efficacy against M. laxa on peaches was 63%, reducing disease incidence from 79% to 29%. Under 4 °C and 95% RH for 45 days, the efficacy against B. cinerea and P. expansum on apples was 56% and 46%, respectively. In Lilly–Barnett minimal salt medium with the fungal cell walls of pathogens as sole carbon source, PL5 produced β-1,3-glucanase, exo-chitinase and endo-chitinase. Nutrient concentrations had significant effect on pathogen growth reduction by PL5. No attachment was observed in antagonist–pathogen interactions in vitro or in vivo. PL5 completely inhibited pathogen spore germination in PDB at 10⁸ cells ml⁻¹, whereas at 10⁶ cells ml⁻¹ the efficacy was significantly decreased. However, inactivated cells and culture filtrate of PL5 had no effect on pathogen spore germination and germ tube elongation. Our results showed that A. pullulans PL5 could be introduced in commercial formulations to control postharvest pathogens on fruits and its activity was based on secretion of lytic enzymes and competition for nutrients.     

Immunotherapy with Dendritic Cells Modified with Tumor-Associated Antigen Gene Demonstrates Enhanced Antitumor Effect Against Lung Cancer

BACKGROUND: Immunotherapy using dendritic cell (DC) vaccine has the potential to overcome the bottleneck of cancer therapy. METHODS: We engineered Lewis lung cancer cells (LLCs) and bone marrow–derived DCs to express tumor-associated antigen (TAA) ovalbumin (OVA) via lentiviral vector plasmid encoding OVA gene. We then tested the antitumor effect of modified DCs both in vitro and in vivo. RESULTS: The results demonstrated that in vitro modified DCs could dramatically enhance T-cell proliferation (P < .01) and killing of LLCs than control groups (P < .05). Moreover, modified DCs could reduce tumor size and prolong the survival of LLC tumor-bearing mice than control groups (P < .01 and P < .01, respectively). Mechanistically, modified DCs demonstrated enhanced homing to T-cell–rich compartments and triggered more naive T cells to become cytotoxic T lymphocytes, which exhibited significant infiltration into the tumors. Interestingly, modified DCs also markedly reduced tumor cells harboring stem cell markers in mice (P < .05), suggesting the potential role on cancer stem-like cells. CONCLUSION: These findings suggested that DCs bioengineered with TAA could enhance antitumor effect and therefore represent a novel anticancer strategy that is worth further exploration.     

Structurally and functionally characterized in vitro model of rabbit vocal fold epithelium

In this paper, we describe a method for primary culture of a well differentiated electrically tight rabbit vocal fold epithelial cell multilayer and the measurement of transepithelial electrical resistance (TEER) for the evaluation of epithelial barrier function in vitro. Rabbit larynges were harvested and enzymatically treated to isolate vocal fold epithelial cells and to establish primary culture. Vocal fold epithelial cells were co-cultured with mitomycin C-treated feeder cells on collagen-coated plates. After 10–14 days in primary culture, cells were passaged and cultured until they achieved 70–90% confluence on collagen-coated plates. Epithelial cells were then passaged onto collagen-coated cell culture inserts using 4.5 cm² membrane filters (1.0 μm pore size) with 10% fetal bovine serum or 30 μg/mL bovine pituitary extract to investigate the effects of growth-promoting additives on TEER. Additional experiments were performed to investigate optimal seeding density (1.1, 2.2, 4.4, or 8.9 × 10⁵ cells/cm²), the effect of co-culture with feeder cells, and the effect of passage number on epithelial barrier function. Characterization of in vitro cultures was performed using hematoxylin and eosin staining and immunostaining for vocal fold epithelial cell markers and tight junctions. Results revealed higher TEER in cells supplemented with fetal bovine serum compared to bovine pituitary extract. TEER was highest in cells passaged at a seeding density of 2.2 × 10⁴ cells/cm², and TEER was higher in cells at passage two than passage three. Ultrastructural experiments revealed a well-differentiated epithelial cell multilayer, expressing the epithelial cell markers CK13, CK14 and the tight junction proteins occludin and ZO-1.     

Survivin and its spliced isoform gene expression is associated with proliferation of renal cancer cells and clinical stage of renal cancer

Background: Survivin has been implicated in inhibition of apoptosis. To date, alternatively spliced isoforms, Survivin-2α, -2B, -ΔEx3, -3B, have been described. We assessed the effect of survivin gene expression on the proliferation of renal cancer (RCC) cells, and studied the association of survivin and its spliced isoform gene expression levels with the clinical stage of RCC. Methods: Gene expression of survivin and its spliced isoform in RCC cells, Caki-1, were performed by RT-PCR. We knocked down the gene expression of Survivin using small interfering RNA (siRNA), and assessed the cell proliferation by MTS assay. Next, we quantified the gene expression levels of survivin and its isoform in nephrectomy samples using quantitative real-time PCR. Results: In Caki-1 cells, survivin and survivin-2α, -2B were expressed higher than survivin-ΔEx3. Decrease of Survivin gene expression by transfection of siRNA was accompanied by inhibition of the proliferation of Caki-1 cell with 36% decrease in comparison with negative control transfected cells (p < 0.01). In clinical RCC tissues, survivin expression levels in metastatic stage were significantly higher compared with those in distant metastasis stage (M0:M1 = 1:4.81, p = 0.014); survivin 2B gene expression levels in pT3 tumors were associated significantly higher than those in pT1 (pT1:pT3 = 1:4.50, p = 0.043). No significant differences were found in survivin-2α expression levels and the ratio of survivin-2B/survivin gene expression levels among any clinical stages. Conclusion: We first demonstrated the gene expression of survivin-2α in renal cancer cells, and also showed that survivin and its spliced isoforms had associations with renal cancer cell proliferation and distant metastases.     

Effect of oregano essential oil and carvacrol on Cryptosporidium parvum infectivity in HCT-8 cells

Cryptosporidium parvum is the second leading cause of persistent diarrhea among children in low-resource settings. This study examined the effect of oregano essential oil (OEO) and carvacrol (CV) on inhibition of C. parvum infectivity in vitro. HCT-8 cells were seeded (1 × 10⁶) in 96-well microtiter plates until confluency. Cell viability and infectivity were assessed by seeding HCT-8 cell monolayers with C. parvum oocysts (1 × 10⁴) in two modalities: 1) 4 h co-culture with bioactive (0–250 μg/mL) followed by washing and incubation (48 h, 37 °C, 5% CO₂) in bioactive-free media; and 2) 4 h co-culture of C. parvum oocysts followed by washing and treatment with bioactive (0–250 μg/mL) during 48-h incubation. Cell viability was tested using Live/Dead? assay whereas infectivity was measured using C. parvum-specific antibody staining via immunofluorescence detection. Loss of cell viability was observed starting at 125 μg/mL and 60 μg/mL for OEO and CV, respectively. Neither OEO nor CV modulated the invasion of C. parvum sporozoites in HCT-8 cells. Treatment with bioactive after invasion reduced relative C. parvum infectivity in a dose-dependent manner to 55.6 ± 10.4% and 45.8 ± 4.1% at 60 and 30 μg/mL of OEO and CV, respectively. OEO and CV are potential bioactives to counteract C. parvum infection in children.     

Discovery of novel 4(1H)-quinolone derivatives as potential antiproliferative and apoptosis inducing agents

A series of novel 4(1H)-quinolone derivatives was synthesized and evaluated for antiproliferative activity in vitro. The results showed that these compounds exhibited more potent antiproliferative effect against a panel of human tumor cell lines than the lead compound 7-chloro-4(1H)-quinolone 1. Compound 7e was found to be the most potent antiproliferative agent and to exhibit selective cytotoxic activity against HepG2 cell lines with IC₅₀ value lower than 1.0 μM. Annexin V/FITC-PI assay showed that compound 7e induced apoptosis in HepG2 cells with a dose-dependent manner. Western blotting analysis indicated that compound 7e induced cell cycle arrest in G2/M phase by p53-depedent pathway.     

Tissue factor pathway inhibitor-2 induced hepatocellular carcinoma cell differentiation

To investigate the effect of over-expression of tissue factor pathway inhibitor-2 (TFPI-2) on the differentiation of hepatocellular carcinoma (HCC) cells (Hep3B and HepG2). The TFPI-2 recombinant adenovirus (pAd-TFPI-2) was constructed using the pAdeasy-1 vector system. Transfected by pAd-TFPI-2, the cell proliferation of HCC cells was evaluated by CCK-8 assay, flow cytometry was used to detect cell apoptosis and CD133 expression. Real-time PCR and Western blot were used to detect the expression levels of markers of hepatocellular cancer stem cells (CSC) and hepatocytes. The over-expression of TFPI-2 significantly suppressed cell proliferation, induced apoptosis, and dramatically decreased the percentage of CD133 cells, which was considered as CSC in HCC. Real-time PCR and Western blot showed that the expression of markers of CSC in Hep3B cells and HepG2 cells infected with pAd-TFPI-2 was markedly lower than those of the control group (P < 0.05), while the expression of markers of hepatocytes was significantly increased (P < 0.05). Hence, TFPI-2 could induce the differentiation of hepatocellular carcinoma cells into hepatocytes, and is expected to serve as a novel way for the treatment of HCC.     

Lipoic acid effects on established atherosclerosis

AimsAlpha-lipoic acid (LA) is a commonly used dietary supplement that exerts anti-oxidant and anti-inflammatory effects in vivo and in vitro. We investigated the mechanisms by which LA may confer protection in models of established atherosclerosis.Main methodsWatanabe heritable hyperlipidemic (WHHL) rabbits were fed with high cholesterol chow for 6 weeks and then randomized to receive either high cholesterol diet alone or combined with LA (20 mg/kg/day) for 12 weeks. Vascular function was analyzed by myography. The effects of LA on T cell migration to chemokine gradients was assessed by Boyden chamber. NF-κB activation was determined by measuring translocation and electrophoresis migration shift assay (EMSA).Key findingsLA decreased body weight by 15 ± 5% without alterations in lipid parameters. Magnetic Resonance Imaging (MRI) analysis demonstrated that LA reduced atherosclerotic plaques in the abdominal aorta, with morphological analysis revealing reduced lipid and inflammatory cell content. Consistent with its effect on atherosclerosis, LA improved vascular reactivity (decreased constriction to angiotensin II and increased relaxation to acetylcholine and insulin), inhibited NF-κB activation, and decreased oxidative stress and expression of key adhesion molecules in the vasculature. LA reduced T cell content in atherosclerotic plaque in conjunction with decreasing ICAM and CD62L (l-selectin) expression. These effects were confirmed by demonstration of a direct effect of LA in reducing T cell migration in response to CCL5 and SDF-1 and decreasing T cell adhesion to the endothelium by intra-vital microscopy.SignificanceThe present findings offer a mechanistic insight into the therapeutic effects of LA on atherosclerosis.