Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions

Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein (LMP) 1, which is consistently expressed in multiple EBV-associated malignancies, has been proposed as a potential target antigen for any future vaccine designed to control these malignancies. However, the high degree of genetic variation in the LMP1 sequence has been considered a major impediment for its use as a potential immunotherapeutic target for the treatment of EBV-associated malignancies. In the present study, we have employed a highly efficient strategy, based on ex vivo functional assays, to conduct an extensive sequence-wide analysis of LMP1-specific T-cell responses in a large panel of healthy virus carriers of diverse ethnic origin and nasopharyngeal carcinoma patients. By comparing the frequencies of T cells specific for overlapping peptides spanning LMP1, we mapped a number of novel HLA class I- and class II-restricted LMP1 T-cell epitopes, including an epitope with dual HLA class I restriction. More importantly, extensive sequence analysis of LMP1 revealed that the majority of the T-cell epitopes were highly conserved in EBV isolates from Caucasian, Papua New Guinean, African, and Southeast Asian populations, while unique geographically constrained genetic variation was observed within one HLA A2 supertype-restricted epitope. These findings indicate that conserved LMP1 epitopes should be considered in designing epitope-based immunotherapeutic strategies against EBV-associated malignancies in different ethnic populations.     

Triterpenoids from walsura piscidia

A series of tirucallane (piscidinol A and B) and apotirucallane (piscidinol C–E) derivatives has been isolated from the leaves of Walsura piscidia. The fruit yielded a new tetranortriterpenoid, piscidofuran. The structures were assigned on the basis of ¹H NMR and ¹³C NMR evidence.     

Thrombin activation of factor XI on activated platelets requires the interaction of factor XI and platelet glycoprotein Ib alpha with thrombin anion-binding exosites I and II,respectively

Activation of factor XI (FXI) by thrombin on stimulated platelets plays a physiological role in hemostasis, providing additional thrombin generation required in cases of severe hemostatic challenge. Using a collection of 53 thrombin mutants, we identified 16 mutants with <50% of the wild-type thrombin FXI-activating activity in the presence of dextran sulfate. These mutants mapped to anion-binding exosite (ABE) I, ABE-II, the Na+-binding site, and the 50-insertion loop. Only the ABE-II mutants showed reduced binding to dextran sulfate-linked agarose. Selected thrombin mutants in ABE-I (R68A, R70A, and R73A), ABE-II (R98A, R245A, and K248A), the 50-insertion loop (W50A), and the Na+-binding site (E229A and R233A) with <10% of the wild-type activity also showed a markedly reduced ability to activate FXI in the presence of stimulated platelets. The ABE-I, 50-insertion loop, and Na+-binding site mutants had impaired binding to FXI, but normal binding to glycocalicin, the soluble form of glycoprotein Ibalpha (GPIb alpha). In contrast, the ABE-II mutants were defective in binding to glycocalicin, but displayed normal binding to FXI. Our data support a quaternary complex model of thrombin activation of FXI on stimulated platelets. Thrombin bound to one GPIb alpha molecule, via ABE-II on its posterior surface, is properly oriented for its activation of FXI bound to a neighboring GPI alpha molecule, via ABE-I on its anterior surface. GPIb alpha plays a critical role in the co-localization of thrombin and FXI and the resultant efficient activation of FXI.     

The role of factor XIa (FXIa) catalytic domain exosite residues in substrate catalysis and inhibition by the Kunitz protease inhibitor domain of protease nexin 2

To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal K(m) values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of k(cat) for S-2366 hydrolysis. All six Ala mutants displayed deficient k(cat) values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of K(i) except for K192A, and Y5901A, which displayed increased values of K(i). The integrity of the S1 binding site residue, Asp(189), utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr(143), Ile(151), Arg(3704), and Tyr(5901)) are important for S-2366 hydrolysis; Glu(98) and Lys(192) are essential for FIX but not S-2366 hydrolysis; and Lys(192) and Tyr(5901) are required for both inhibitor and macromolecular substrate interactions.     

Prevalence and Determinants of Adult Under-Nutrition in Botswana

Background

To estimate the prevalence and determinants of adult under-nutrition in Botswana.

Methods

A cross-sectional survey was conducted where a nationally representative sample of people aged 20 to 49 years was used for the analysis. The outcome measure of under-nutrition was measured as BMI<18.5 kg/m².

Results

Of the total sample, 19.5% of males and 10.1% of females were underweight (BMI<18.5 kg/m²). The wealth index showed that 30.9% of the adult population with low a BMI belongs to the poorest 20% of the households while only 9.6% comprised of the richest 20% of the households. Results from logistic regression analysis indicated that both adult men and women who had no education and belonged to the low socioeconomic group had a statistically significant association with low BMI. Among the female adult population, being young and not having watched TV at least once a week were significantly associated with low BMI. For the male adult population, being unmarried was significantly associated with low BMI.

Conclusions

Programme interventions aimed at improving the nutritional status of adults can use these findings to make appropriate policy, to establish baselines and study nutritional changes over time and its covariates.     

Immunotherapeutic strategies for EBV-associated malignancies

Advances in our understanding of the role of cytotoxic T lymphocytes (CTLs) in the control of Epstein-Barr virus (EBV)-associated malignancies and the overall biology of these diseases have led to the development of novel therapeutic strategies designed to specifically target viral antigens expressed in these malignancies. Long-term success of many of these strategies is constrained by the latency phenotypes adopted by different diseases. Adoptive transfer of polyclonal virus-specific CTLs has been used successfully to reverse the outgrowth of malignancies such as post-transplant lymphoproliferative disease (PTLD). On the other hand, limited viral gene expression in other EBV-associated malignancies such as Burkitt's lymphoma, Hodgkin's disease and nasopharyngeal carcinoma limits the efficacy of immunotherapeutic strategies used for PTLD. Preclinical studies based on specific targeting of viral antigens expressed in these malignancies have provided very encouraging results and thus are likely to serve as an important platform for the treatment of human patients.     

Blood flow in stented arteries: a parametric comparison of strut design patterns in three dimensions

BACKGROUND: Restenosis after stent implantation varies with stent design. Alterations in secondary flow patterns and wall shear stress (WSS) can modulate intimal hyperplasia via their effects on platelet and inflammatory cell transport toward the wall, as well as direct effects on the endothelium. METHOD OF APPROACH: Detailed flow characteristics were compared by estimating the WSS in the near-strut region of realistic stent designs using three-dimensional computational fluid dynamics (CFD), under pulsatile high and low flow conditions. The stent geometry employed was characterized by three geometric parameters (axial strut pitch, strut amplitude, and radius of curvature), and by the presence or lack of the longitudinal connector. RESULTS: Stagnation regions were localized around stent struts. The regions of low WSS are larger distal to the strut. Under low flow conditions, the percentage restoration of mean axial WSS between struts was lower than that for the high flow by 10-12%. The largest mean transverse shear stresses were 30-50% of the largest mean axial shear stresses. The percentage restoration in WSS in the models without the longitudinal connector was as much as 11% larger than with the connector The mean axial WSS restoration between the struts was larger for the stent model with larger interstrut spacing. CONCLUSION: The results indicate that stent design is crucial in determining the fluid mechanical environment in an artery. The sensitivity of flow characteristics to strut configuration could be partially responsible for the dependence of restenosis on stent design. From a fluid dynamics point of view, interstrut spacing should be larger in order to restore the disturbed flow; struts should be oriented to the flow direction in order to reduce the area of flow recirculation. Longitudinal connectors should be used only as necessary, and should be parallel to the axis. These results could guide future stent designs toward reducing restenosis.     

Polymer‐coated viral vectors: hybrid nanosystems for gene therapy

The advantages and critical aspects of nanodimensional polymer‐coated viral vector systems potentially applicable for gene delivery are reviewed. Various viral and nonviral vectors have been explored for gene therapy. Viral gene transfer methods, although highly efficient, are limited by their immunogenicity. Nonviral vectors have a lower transfection efficiency as a result of their inability to escape from the endosome. To overcome these drawbacks, novel nanotechnology‐mediated interventions that involve the coating or modification of virus using polymers have emerged as a new paradigm in gene therapy. These alterations not only modify the tropism of the virus, but also reduce their undesirable interactions with the biological system. Also, co‐encapsulation of other therapeutic agents in the polymeric coating may serve to augment the treatment efficacy. The viral particles can aid endosomal escape, as well as nuclear targeting, thereby enhancing the transfection efficiency. The integration of the desirable properties of both viral and nonviral vectors has been found beneficial for gene therapy by enhancing the transduction efficiency and minimizing the immune response. However, it is essential to ensure that these attempts should not compromise on the inherent ability of viruses to target and internalize into the cells and escape the endosomes.