Carbohydrate induced modulation of cell membrane VII. Binding of exogenous lectin increases osmofragility of erythrocytes

AbstractDue to their multivalent binding character, lectins when added exogenously will cross-link membrane surface receptors leading to lateral molecular reorganizations in the plane of the bilayer. This study reports for the first time that agglutination of rabbit erythrocytes by lentil lectin and concanavalin A increases their osmofragility. Increase in osmofragility was detected by measuring the hemolysis of erythrocytes in hypotonic as well as in isotonic solutions. It was also found that agglutination per se does not increase osmofragility but the binding of legume lectin is essential since human Rh+ cells agglutinated by a monoclonal antibody do not exhibit hemolysis

Linear motion correction in three dimensions applied to dynamic gadolinium enhanced breast imaging

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134776/1/mp8576.pd

JunB Mediates Basal- and TGFβ1-Induced Smooth Muscle Cell Contractility

Smooth muscle contraction is a dynamic process driven by acto-myosin interactions that are controlled by multiple regulatory proteins. Our studies have shown that members of the AP-1 transcription factor family control discrete behaviors of smooth muscle cells (SMC) such as growth, migration and fibrosis. However, the role of AP-1 in regulation of smooth muscle contractility is incompletely understood. In this study we show that the AP-1 family member JunB regulates contractility in visceral SMC by altering actin polymerization and myosin light chain phosphorylation. JunB levels are robustly upregulated downstream of transforming growth factor beta-1 (TGFβ1), a known inducer of SMC contractility. RNAi-mediated silencing of JunB in primary human bladder SMC (pBSMC) inhibited cell contractility under both basal and TGFβ1-stimulated conditions, as determined using gel contraction and traction force microscopy assays. JunB knockdown did not alter expression of the contractile proteins α-SMA, calponin or SM22α. However, JunB silencing decreased levels of Rho kinase (ROCK) and myosin light chain (MLC20). Moreover, JunB silencing attenuated phosphorylation of the MLC20 regulatory phosphatase subunit MYPT1 and the actin severing protein cofilin. Consistent with these changes, cells in which JunB was knocked down showed a reduction in the F:G actin ratio in response to TGFβ1. Together these findings demonstrate a novel function for JunB in regulating visceral smooth muscle cell contractility through effects on both myosin and the actin cytoskeleton

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

K -space acquisition method for dynamic contrast -enhanced MRI: Application to breast tumors.

Dynamic contrast-enhanced (DCE)-MRI is increasingly being used for detection and diagnosis of tumors. The objective of DCE-MRI is to elicit diagnostically significant architectural and pharmacokinetic features of lesions. Hence, DCE-MRI of tumors is ideally performed at high spatial resolution while sampling a time-varying process at high temporal resolution. A variety of variable rate sampling strategies and associated reconstructive schemes have been developed to resolve the conflicting demands of simultaneous high resolution sampling of temporal and spatial detail. In this work a novel method, termed spatio-temporal bandwidth-based (STBB) acquisition, is developed to address the trade-off inherent in DCE-MRI. This technique is constrained only by the overall scan duration, within which the temporal changes are expected to reach steady-state, the imaging sequence repetition time (TR), and a targeted spatio-temporal object. Neither the spatial nor temporal resolution is preselected. The STBB formalism, which is applicable to any dynamic contrast-enhanced imaging condition, is demonstrated using a priori modeling of breast tumors. The k-space traversal scheme is obtained by maximizing energy coverage of the Fourier space that encompasses the instantaneous spectral energy of the temporally enhancing object during the DCE-MRI experiment. A method to use the energy maximization concept in designing an acquisition scheme that is adequate for a class of space-time objects is also demonstrated in this work. This concept was tested in computer simulations under a range of object spatial features and enhancement conditions. In addition, two issues that closely impact the accuracy in quantification of pharmacokinetic parameters measured using DCE-MRI are addressed: motion artifact and B1-field inhomogeneity. A linear three-dimensional motion-correction algorithm to compensate for patient motion over the course of the dynamic acquisition is developed. The errors in parameter estimation due to B1-field inhomogeneity are investigated and a correction method is proposed.Ph.D.Applied SciencesBiomedical engineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124463/2/3138205.pd

ESR studies on the effect of ionic radii on displacement of Mn2+ bound to a soluble β-galactoside binding hepatic lectin

AbstractBinding of divalent metal ions to hepatic soluble β-galactoside binding lectin was studied using electron spin resonance (ESR) spectroscopy. The Mn2+ bound to hepatic lectin could be displaced by Mg2+, Cu2+, Ni2+ and Ca2+ but not by Sr2+. As the ionic radii of Mg2+ (0.65 Å), Cu2+ (0.73 Å) and Ni2+ (0.72 Å) are appreciably smaller than Ca Mn2+ binding site is more accessible to Mg2+, Cu2+, and Ni2+ as compared to Ca2+, the ionic radius of Mn2+ being 0.80 Å. Sr2+ with an ionic radius of 1.13 is thus unable to displace bound Mn2+. Surprisingly, the presence of specific sugars like α-lactose, or α-d-galactose facilitated the displacement of bound Mn2+ by metal ions whereas non-specific sugars, i.e. α-d-glucose, β-d-fructose and α-d-ribose had no effect. It appears that minor perturbations in the saccharide binding site significantly affect the ability of the metal binding site to ligate bivalent metals

Inhibiting Airway Smooth Muscle Contraction Using Pitavastatin: A Role for the Mevalonate Pathway in Regulating Cytoskeletal Proteins.

Despite maximal use of currently available therapies, a significant number of asthma patients continue to experience severe, and sometimes life-threatening bronchoconstriction. To fill this therapeutic gap, we examined a potential role for the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitor, pitavastatin. Using human airway smooth muscle (ASM) cells and murine precision-cut lung slices, we discovered that pitavastatin significantly inhibited basal-, histamine-, and methacholine (MCh)-induced ASM contraction. This occurred via reduction of myosin light chain 2 (MLC2) phosphorylation, and F-actin stress fiber density and distribution, in a mevalonate (MA)- and geranylgeranyl pyrophosphate (GGPP)-dependent manner. Pitavastatin also potentiated the ASM relaxing effect of a simulated deep breath, a beneficial effect that is notably absent with the β2-agonist, isoproterenol. Finally, pitavastatin attenuated ASM pro-inflammatory cytokine production in a GGPP-dependent manner. By targeting all three hallmark features of ASM dysfunction in asthma-contraction, failure to adequately relax in response to a deep breath, and inflammation-pitavastatin may represent a unique asthma therapeutic