Human peripheral blood V alpha 24(+) V beta 11(+) NKT cells expand following administration of alpha-galactosylceramide-pulsed dendritic cells

Background and Objectives We have undertaken the first clinical trial involving the administration of alpha-GalactosylCeramine (alpha-GalCer)-pulsed dendritic cells (DCs) to human subjects, to determine safety, optimal dose, optimal administration route and immunological effects. Materials and Methods Subjects (n = 4) with metastatic malignancy received two infusions of alpha-GalCer-pulsed DCs intravenously, and two infusions intradermally. The percentages of Valpha24 Vbeta11 NKT cells in peripheral blood (PB) were determined by three-colour flow cytometry and the PB NKT cell numbers were calculated using the total number of PB lymphocytes/ml determined by automated full-blood counts. Results No serious treatment related adverse events were observed during the study period. Administration of alpha-GalCer-pulsed DCs in vivo can significantly (P < 0.03) increase PB Valpha24(+) Vbeta11(+) NKT cell numbers above pretreatment baseline levels after the transient fall in the NKT numbers within 48 h. Conclusions Administration of alpha-GalCer-pulsed DCs is well tolerated, modulates PB Valpha24(+) Vbeta11(+) NKT cells and may have a role in the therapy of malignancies sensitive to activities of Valpha24(+) Vbeta11(+) NKT cells, or for autoimmune diseases.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts

Cadherin cell adhesion molecules are major determinants of tissue patterning which function in cooperation with the actin cytoskeleton [1-4]. In the context of stable adhesion [1], cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis [2]. Here actin polymerization has been proposed to drive cell surfaces together [5], although F-actin reorganization also occurs as cell contacts mature [6]. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. We sought to analyze the relationship between cadherin homophilic binding and cytoskeletal activity during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere [7]. We therefore activated homophilic ligation using a specific functional recombinant protein. We report the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator and demonstrate that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface.

Cellular responses to Schistosoma japonicum cathepsin D aspartic protease

Lymphocyte proliferation and cytokine production were measured in groups of mice vaccinated (but not subsequently challenge infected) with recombinant forms of Schistosoma japonicum cathepsin D aspartic protease, rSjASP1 (expressed in bacteria; enzymatically inactive) and rSjASP2 (expressed in insect cells; enzymatically active). Both forms of the schistosome enzyme induced significant proliferation of splenocytes recovered from vaccinated mice, and expression of interferon (IFN)-gamma, interleukin (IL)-4 and IL-10 mRNA in these cells was detected using reverse transcriptase-polymerase chain reaction. Secretion of IFN-gamma, IL-4 and IL-10 by splenocytes from vaccinated mice was confirmed and quantified using enzyme-linked immunosorbent assay. IFN-gamma was the most abundant cytokine produced, followed by IL-4 and IL-10 in rank order. These findings indicated that vaccination of mice with the schistosome protease induces a mixed Th1/Th2 cytokine response, which may explain the modest level of protection after challenge infection in cathepsin d-vaccinated mice, reported previously.

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: Formation of the 4-hydroxy, 4′-hydroxy andN-desmethyl metabolites and isomerization oftrans-4-hydroxytamoxifen

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 muM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 muM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 muM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 muM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.

Role of PGF(2 alpha) and oxytocin in parturition in the brushtail possum (Trichosurus vulpecula)

Maturation of the fetal pituitary and adrenal glands allows the secretion of cortisol, which in turn leads to an increase in prostaglandin and mesotocin production. The production of prostaglandin and mesotocin results in an increase in uterine contractions and initiates birth in marsupials. The major metabolite of PGF(2alpha), 13,14-dihydro-15-keto-prostaglandin F-2alpha (PGFM), has been found in the plasma of the possum at the time of birth and administration of PGF(2alpha) to female possums induced the adoption of the birth position. Evidence that mesotocin is an integral hormone of birth in the tammar wallaby indicates that both PGF(2alpha) and mesotocin or oxytocin are required for marsupial birth. The presence of PGF(2alpha) receptors in the uterus and corpus luteum of the possum, and the in vitro uterine responsiveness to PGF(2alpha) or oxytocin, were examined. PGF(2alpha) receptors were not observed in possum uteri and the inability of PGF(2alpha) to cause contractions indicates that PGF(2alpha) is not involved directly in contraction of the uterus at parturition. The presence of oxytocin and mesotocin receptors in the uterus of possoms and the ability of oxytocin to induce uterine contraction in vitro supports the view that mesotocin is required for expulsion of the young from the uterus. Low numbers of PGF(2alpha) receptors were found in the possum corpus luteum at birth, indicating an involvement of PGF(2alpha) in regression of the corpus luteum.

Microstructural evidence for the mechanism of the alpha-beta phase transformation in ytterbium SiAlON ceramics

Detailed microstructural evidence for the mechanism of the alpha-beta phase transformation in ytterbium SiAlON ceramics is presented. Grains, which show partial transformation, have been examined using transmission electron microscopy. We suggest that the transformation proceeds as a discernable reaction front and the accompanying lattice mismatch is accommodated be a series of complex dislocations. The stabilizing cation is ejected from the transformed alpha- phase and diffuse along the dislocation to accumulate as isolated pockets in a way similar to that observed in metal systems and termed pipe diffusion. High-resolution electron microscopy reveals the details of each of these features.

Kunjin virus replicon vectors for human immunodeficiency virus vaccine development

We have previously demonstrated the ability of the vaccine vectors based on replicon RNA of the Australian flavivirus Kunjin (KUN) to induce protective antiviral and anticancer CD8(+) T-cell responses using murine polyepitope as a model immunogen (I. Anraku, T. J. Harvey, R. Linedale, J. Gardner, D. Harrich, A. Suhrbier, and A. A. Khromykh, J. Virol. 76:3791-3799, 2002). Here we showed that immunization of BALB/c mice with KUN replicons encoding HIV-1 Gag antigen resulted in induction of both Gag-specific antibody and protective Gag-specific CD8(+) T-cell responses. Two immunizations with KUNgag replicons in the form of virus-like particles (VLPs) induced anti-Gag antibodies with titers of greater than or equal to1:10,000. Immunization with KUNgag replicons delivered as plasmid DNA, naked RNA, or VLPs induced potent Gag-specific CD8(+) T-cell responses, with one immunization of KUNgag VLPs inducing 4.5-fold-more CD8(+) T cells than the number induced after immunization with recombinant vaccinia virus carrying the gag gene (rVVgag). Two immunizations with KUNgag VLPs also provided significant protection against challenge with rVVgag. Importantly, KUN replicon VLP vaccinations induced long-lasting immune responses with CD8(+) T cells able to secrete gamma interferon and to mediate protection 6 to 10 months after immunization. These results illustrate the potential value of the KUN replicon vectors for human immunodeficiency virus vaccine design.

A structural basis for the selection of dominant alpha beta T cell receptors in antiviral immunity

We have examined the basis for immunodominant or public TCR usage in an antiviral CTL response. Residues encoded by each of the highly selected genetic elements of an immunodominant clonotype recognizing Epstein-Barr virus were critical to the antigen specificity of the receptor. Upon recognizing antigen the immunodominant TCR undergoes extensive conformational changes in the complementarity determining regions (CDRs), including the disruption of the canonical structures of the germline-encoded CDR1alpha and CDR2alpha loops to produce an enhanced fit with the HLA-peptide complex. TCR ligation induces conformational changes in the TCRalpha constant domain thought to form part of the docking site for CD3epsilon. These findings indicate that TCR immunodominance is associated with structural properties conferring receptor specificity and suggest a novel structural link between TCR ligation and intracellular signaling.

Explicit/implicit partitioning and a new explicit form of the generalized alpha method

Most finite element packages use the Newmark algorithm for time integration of structural dynamics. Various algorithms have been proposed to better optimize the high frequency dissipation of this algorithm. Hulbert and Chung proposed both implicit and explicit forms of the generalized alpha method. The algorithms optimize high frequency dissipation effectively, and despite recent work on algorithms that possess momentum conserving/energy dissipative properties in a non-linear context, the generalized alpha method remains an efficient way to solve many problems, especially with adaptive timestep control. However, the implicit and explicit algorithms use incompatible parameter sets and cannot be used together in a spatial partition, whereas this can be done for the Newmark algorithm, as Hughes and Liu demonstrated, and for the HHT-alpha algorithm developed from it. The present paper shows that the explicit generalized alpha method can be rewritten so that it becomes compatible with the implicit form. All four algorithmic parameters can be matched between the explicit and implicit forms. An element interface between implicit and explicit partitions can then be used, analogous to that devised by Hughes and Liu to extend the Newmark method. The stability of the explicit/implicit algorithm is examined in a linear context and found to exceed that of the explicit partition. The element partition is significantly less dissipative of intermediate frequencies than one using the HHT-alpha method. The explicit algorithm can also be rewritten so that the discrete equation of motion evaluates forces from displacements and velocities found at the predicted mid-point of a cycle. Copyright (C) 2003 John Wiley Sons, Ltd.

Immunogenicity of recombinant HBsAg/HCV particles in mice pre-immunised with hepatitis B virus-specific vaccine

Due to their spatial structure virus-like particles (VLPs) generally induce effective immune responses. VLPs derived from the small envelope protein (HBsAg-S) of hepatitis B virus (HBV) comprise the HBV vaccine. Modified HBsAs-S VLPs, carrying the immunodominant hypervariable region (HVR1) of the hepatitis C virus (HCV) envelope protein E2 within the exposed 'a'-determinant region (HBsAg/HVR1-VLPs), elicited HVR1-specific antibodies in mice. A high percentage of the human population is positive for anti-HBsAg antibodies (anti-HBs), either through vaccination or natural infection. We, therefore, determined if pre-existing anti-HBs could influence immunisation with modified VLPs. Mice were immunised with a commercial HBV vaccine, monitored to ensure an anti-HBs response, then immunised with HBsAg/HVR1-VLPs. The resulting anti-HVR1 antibody titre was similar in mice with or without pre-existing anti-HBs. This suggests that HBsAg/HVR1-VLPs induce a primary immune response to HVR1 in anti-HBs positive mice and, hence, they may be used successfully in individuals already immunised with the HBV vaccine. (C) 2003 Elsevier Science Ltd. All rights reserved.

Biochemical characterization of two mutants of human pyruvate dehydrogenase, F205L and T231A of the E1 alpha subunit

Mutations in the E1alpha subunit of the pyruvate dehydrogenase multienzyme complex may result in congenital lactic acidosis, but little is known about the consequences of these mutations at the enzymatic level. Here we characterize two mutants (F205L and T231A) of human pyruvate dehydrogenase in vitro, using the enzyme expressed in Escherichia coli. Wild-type and mutant proteins were purified successfully and their kinetic parameters were measured. F205L shows impaired binding of the thiamin diphosphate cofactor, which may explain why patients carrying this mutation respond to high-dose vitamin B-1 therapy. T231A has very low activity and a greatly elevated K-m for pyruvate, and this combination of effects would be expected to result in severe lactic acidosis. The results lead to a better understanding of the consequences of these mutations on the functional and structural properties of the enzyme, which may lead to improved therapies for patients carrying these mutations.