An evaluation of enforced rapid proteasomal degradation as a means of enhancing vaccine-induced CTL responses

The HIV-1 Gag protein is an attractive target for CTL-based vaccine strategies because it shows less sequence variability than other HIV-1 proteins. In an attempt to increase the immunogenicity of HIV-1 Gag, we created Gag variants that were targeted to the proteasomal pathway for rapid degradation. This enhanced rate of degradation was associated with increased presentation of MHC class I-associated antigenic peptides on the cell surface. Despite this, immunizing mice with either plasmid DNA or recombinant vaccinia vectors expressing unstable Gag failed to produce significant increases in bulk CTL responses or Ag-specific production of IFN-gamma by CD8(+) T cells compared with mice immunized with stable forms of Gag. Production of IFN-gamma by CD4(+) T cells was also impaired, and we speculate that the abrogation of CD4(+) T cell help was responsible for the impaired CTL response. These results suggest that vaccine strategies designed to increase the density of peptide-MHC class I complexes on the surfaces of APC may not necessarily enhance immunogenicity with respect to CTL responses.     

Disparate in vitro and in vivo requirements for IL-2 during antigen-independent CD8 T cell expansion

Transient TCR stimulation induces multiple rounds of CD8 T cell division without further requirement for Ag. The mechanism driving Ag-independent proliferation, however, remains unclear. In this study, we show that the initial duration of TCR stimulation positively correlates with the number of divisions that CD8 T cells subsequently undergo. We find that increased periods of Ag stimulation result in enhanced CD25 up-regulation and greater IL-2 production by CD8 T cells. Depletion of IL-2 from T cell cultures with specific Abs dramatically impairs programmed proliferation. Consistent with this result, IL-2-deficient T cells undergo markedly attenuated Ag-independent proliferation in vitro. Although IL-2 production by stimulated CD8 T cells appears to be essential for in vitro proliferation, upon transfer into recipient mice, IL-2-deficient CD8 T cells undergo extensive proliferation in vivo after transient stimulation. Furthermore, the extent of in vivo proliferation correlates with the duration of in vitro Ag stimulation. These results indicate that the requirements for autocrine IL-2 production by CD8 T cells differs between in vitro and in vivo conditions and suggests that factors in addition to IL-2 can support Ag-independent CD8 T cell proliferation.     

Changes in retinal expression of neurotrophins and neurotrophin receptors induced by ocular hypertension

Open angle glaucoma is defined as a progressive and time-dependent death of retinal ganglion cells concomitant with high intraocular pressure, leading to loss of visual field. Because neurotrophins are a family of growth factors that support neuronal survival, we hypothesized that quantitative and qualitative changes in neurotrophins or their receptors may take place early in ocular hypertension, preceding extensive cell death and clinical features of glaucoma. We present molecular, biochemical, and phenotypic evidence that significant neurotrophic changes occur in retina, which correlate temporally with retinal ganglion cell death. After 7 days of ocular hypertension there is a transient up-regulation of retinal NGF, while its receptor TrkA is up-regulated in a sustained fashion in retinal neurons. After 28 days of ocular hypertension there is sustained up-regulation of retinal BDNF, but its receptor TrkB remains unchanged. Throughout, NT-3 levels remain unchanged but there is an early and sustained increase of its receptor TrkC in Müller cells but not in retinal ganglion cells. These newly synthesized glial TrkC receptors are truncated, kinase-dead isoforms. Expression of retinal p75 also increases late at day 28. Asymmetric up-regulation of neurotrophins and neurotrophin receptors may preclude efficient neurotrophic rescue of RGCs from apoptosis. A possible rationale for therapeutic intervention with Trk receptor agonists and p75 receptor antagonists is proposed.     

Epitope scanning reveals gain and loss of strain specific antibody binding epitopes associated with the conversion of normal cellular prion to scrapie prion

We used anti-prion (PrP) monoclonal antibodies (Mabs) in different combinations to scan changes in the availability of antibody binding epitopes--using an epitope scanning assay--in brain homogenates from normal mice, and from mice infected with either ME7 or 139 A strains of infectious scrapie prion (PrPSc). In ME7-infected brains, the epitope detected by the Mab pair 8B4/8H4 is reduced, while the epitope detected by the Mab pair 8F9/11G5 is increased. Mab 8F9/11G5 detect a conformational epitope on PrPSc because the rise in Mab 8F9/11G5 binding is sensitive to a denaturing agent but resistant to proteinase K (PK). While the increase in Mab 8F9/11G5 binding correlates with the presence of PK-resistant PrP and clinical signs of infection, the reduction in Mab 8B4/8H4 binding is detected earlier. Fractionation of the ME7-infected brain homogenate in sucrose gradient revealed that the PrPSc species detected by the epitope scanning assay are heterogeneous in size, with a molecular mass of approximately > or = 2000-kDa. We also investigated whether these findings were applicable to two other strains of PrPSc, namely 87 V and 22 L. We found that the decrease in Mab 8B4/8H4 binding detected in ME7-infected brains was also detected in 87 V-infected brains but not in 22 L-infected brains. In contrast, the increase in Mab 8F9/11G5 binding detected in ME7- and 139 A-infected brains was also detected in 22 L-infected brains but not in 87 V-infected brains. Therefore, each prion strain has its unique conformation, and we can monitor the conversion of normal cellular prion (PrPC) to PrPSc based on the changes in the antibody binding patterns. The epitope can be decreased or increased, linear or conformational, detected late or early during infection, in a strain specific manner.     

The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite ‘scavenger’?

Hydrogen sulfide (H2S) is a well-known cytotoxic gas. Recently it has been shown to stimulate N-methyl-D-aspartate (NMDA) receptors to enhance long-term potentiation suggesting a novel neuromodulatory role in vivo. Endogenous levels of H2S in the brain are reported to range between 10 and 160 microm. Considerably lower H2S levels are reported in the brains of Alzheimer's disease (AD) patients, where levels of brain protein nitration (probably mediated by peroxynitrite) are markedly increased. Activation of NMDA receptors leads to intracellular tyrosine nitration by peroxynitrite. Because H2S and peroxynitrite are important mediators in brain function and disease, we investigated the effects of the H2S 'donor', sodium hydrogen sulfide (NaSH) on peroxynitrite-mediated damage to biomolecules and to cultured human SH-SY5Y cells. H2S significantly inhibited peroxynitrite-mediated tyrosine nitration and inactivation of alpha1-antiproteinase to a similar extent to reduced glutathione at each concentration tested (30-250 microm). H2S also inhibited peroxynitrite-induced cytotoxicity, intracellular protein nitration and protein oxidation in human neuroblastoma SH-SY5Y cells. These data suggest that H2S has the potential to act as an inhibitor of peroxynitrite-mediated processes in vivo and that the potential antioxidant action of H2S deserves further study, given that extracellular GSH levels in the brain are very low.     

Optimal design of experiments with anticipated pattern of missing observations

We propose a general method of designing an experiment when there are potentially failing trials. We use polynomial models and the Michaelis-Menten model as examples and construct different types of optimal designs under a broad class of response probability functions. We show that the usual optimal designs, that assume all observations are available at the end of the experiment, can be quite inefficient if the anticipated missingness pattern is not accounted for at the design stage. We also investigate robustness properties of the proposed designs to specification of their nominal values and the response probability functions.     

T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus spike protein elicit a specific T-cell immune response in patients who recover from SARS

The immunogenicity of HLA-A2-restricted T-cell epitopes in the S protein of the Severe acute respiratory syndrome coronavirus (SARS-CoV) and of human coronavirus strain 229e (HCoV-229e) was analyzed for the elicitation of a T-cell immune response in donors who had fully recovered from SARS-CoV infection. We employed online database analysis to compare the differences in the amino acid sequences of the homologous T epitopes of HCoV-229e and SARS-CoV. The identified T-cell epitope peptides were synthesized, and their binding affinities for HLA-A2 were validated and compared in the T2 cell system. The immunogenicity of all these peptides was assessed by using T cells obtained from donors who had fully recovered from SARS-CoV infection and from healthy donors with no history of SARS-CoV infection. HLA-A2 typing by indirect immunofluorescent antibody staining showed that 51.6% of SARS-CoV-infected patients were HLA-A2 positive. Online database analysis and the T2 cell binding test disclosed that the number of HLA-A2-restricted immunogenic epitopes of the S protein of SARS-CoV was decreased or even lost in comparison with the homologous sequences of the S protein of HCoV-229e. Among the peptides used in the study, the affinity of peptides from HCoV-229e (H77 and H881) and peptides from SARS-CoV (S978 and S1203) for binding to HLA-A2 was higher than that of other sequences. The gamma interferon (IFN-gamma) release Elispot assay revealed that only SARS-CoV-specific peptides S1203 and S978 induced a high frequency of IFN-gamma-secreting T-cell response in HLA-A2(+) donors who had fully recovered from SARS-CoV infection; such a T-cell epitope-specific response was not observed in HLA-A2(+) healthy donors or in HLA-A2(-) donors who had been infected with SARS-CoV after full recovery. Thus, T-cell epitopes S1203 and S978 are immunogenic and elicit an overt specific T-cell response in HLA-A2(+) SARS-CoV-infected patients.     

Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2

Infection of receptor-bearing cells by coronaviruses is mediated by their spike (S) proteins. The coronavirus (SARS-CoV) that causes severe acute respiratory syndrome (SARS) infects cells expressing the receptor angiotensin-converting enzyme 2 (ACE2). Here we show that codon optimization of the SARS-CoV S-protein gene substantially enhanced S-protein expression. We also found that two retroviruses, simian immunodeficiency virus (SIV) and murine leukemia virus, both expressing green fluorescent protein and pseudotyped with SARS-CoV S protein or S-protein variants, efficiently infected HEK293T cells stably expressing ACE2. Infection mediated by an S-protein variant whose cytoplasmic domain had been truncated and altered to include a fragment of the cytoplasmic tail of the human immunodeficiency virus type 1 envelope glycoprotein was, in both cases, substantially more efficient than that mediated by wild-type S protein. Using S-protein-pseudotyped SIV, we found that the enzymatic activity of ACE2 made no contribution to S-protein-mediated infection. Finally, we show that a soluble and catalytically inactive form of ACE2 potently blocked infection by S-protein-pseudotyped retrovirus and by SARS-CoV. These results permit studies of SARS-CoV entry inhibitors without the use of live virus and suggest a candidate therapy for SARS.     

Attenuated endothelium-mediated relaxation by acteoside in rat aorta: Role of endothelial [Ca2+]i and nitric oxide/cyclic GMP pathway

Acteoside and other phenylethanoid glycoside are contained in many plants that are widely used in traditional Chinese herbal medicine. Acteoside possesses multiple biological actions. Its effect on the vascular system is, however, incompletely understood. This study was aimed to investigate the role of endothelial [Ca²⁺]_i, nitric oxide (NO), and cyclic GMP in acteoside-induced inhibition of endothelial NO-mediated relaxation in rat aorta. Acteoside reduced endothelial NO-dependent relaxation induced by acetylcholine (Ach) or A23187. Acteoside inhibited Ach-stimulated increase in tissue content of cyclic GMP in endothelium-intact rings. L-NNA abolished the stimulatory effect of Ach. Treatment with acteoside significantly suppressed bradykinin-induced increase in [Ca²⁺]_i of cultured rat aortic endothelial cells. Acute exposure to acteoside (30 μM) did not affect the expression of eNOS mRNA in endothelium-intact rings. In summary, acteoside impairs endothelial NO-mediated aortic relaxation partially through inhibition of agonist-induced endothelial Ca²⁺ mobilization and Ca²⁺-dependent NO production and subsequent suppression of cyclic GMP formation. This novel pharmacological action if occurring in small vessels in vivo, may contribute to the reported anti-inflammatory effect of acteoside against NO-mediated vascular permeability-related acute edema.     

Interaction between the polyol pathway and non-enzymatic glycation on aortic smooth muscle cell migration and monocyte adhesion

We investigated for the interaction between the polyol pathway and enhanced non-enzymatic glycation, both implicated in the pathogenesis of diabetic atherosclerosis, in the activation of aortic smooth muscle cell (SMC) function. Mouse aortas and primary cultures of SMCs from wildtype (WT) mice and transgenic (TG) mice expressing human aldose reductase (AR) were studied regarding changes in AR activity, and SMC gene activation, migration and monocyte adhesion, in response to advanced glycation end-product modified BSA (AGE-BSA). Results showed that AGE-BSA increased AR activity in both WT and TG aortas, with greater increments (p < 0.05) in TG aortas which, basally, had elevated AR activity (2.8 fold of WT). These increments were attenuated by zopolrestat, an AR inhibitor. Similar AGE-induced increments in AR activity were observed in primary cultures of aortic SMCs from WT and TG mice (60% and 100%, respectively, P < 0.01). Such increments were accompanied by increases in intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) mRNA levels (both P < 0.05), activation of membrane-associated PKC-beta1 (P < 0.05) as well as increased SMC migration and Tamm-Horsfall protein (THP)-1 monocyte adhesion to SMCs (both p < 0.01), with all changes being significantly greater in TG SMCs (P < 0.05) and suppressible by either zopolrestat or transfection with an AR antisense oligonucleotide. Our findings suggest that the effects of AGEs on SMC activation, migration and monocyte adhesion are mediated partly through the polyol pathway and, possibly, PKC activation. The greater AGE-induced changes in the TG SMCs have provided further support for the dependency of such changes on polyol pathway hyperactivity.