Electrospray Synthesis of Poly(lactide-co-glycolide) Nanoparticles Encapsulating Peptides to Enhance Proliferation of Antigen-Specific CD8+ T Cells.

Polymer nanoparticles (NP) are of escalating interest for their application as immune stimulatory pharmaceutics. The production of nanosized carrier systems is currently being widely investigated, but commonly used techniques, such as the double emulsion technique, are limited by shortcomings of low encapsulation efficiency and poor control over size distribution. In this study, the electrospray technique was successfully implemented and optimized to produce monodisperse 200-nm poly(lactide-co-glycolide) (PLGA) NP. For cytomegalovirus (CMV) pp65 and IE-1 peptides, a consistent encapsulation efficiency of approximately 85% was achieved. In vitro stimulation of peripheral blood mononuclear cells (PBMCs) from CMV+ donors using electrosprayed pp65489-503 peptide-loaded NP revealed a significantly increased proliferation rate and frequency of antigen-specific CD8+ T cells as compared to the soluble peptide. The results of this study demonstrate the suitability of the electrospray technique for production of monodisperse PLGA NP with high drug encapsulation efficiency as promising peptide-based vaccine carriers.

Decrease of murine cytomegalovirus-induced retinitis by intravenous delivery of immediate early protein-3-specific siRNA.

PURPOSE: Retinitis induced by both human and murine cytomegaloviruses following immunosuppression is characterized by progressive loss of retinal architecture, due to necrosis of virus-infected cells as well as widespread apoptosis of uninfected bystander cells. Because small inhibitory RNA molecules (siRNA) can reduce murine cytomegalovirus (MCMV) gene expression and thereby inhibit virus replication in vitro, we tested siRNAs directed against MCMV immediate early protein-3 (IE-3) to determine if MCMV-induced retinitis could be alleviated in vivo. METHODS: Immunosuppressed Balb/c mice (2.0 mg methylprednisolone acetate every 3 days beginning on day -2) were infected with 5 × 10(3) pfu of the K181 strain of MCMV via the supraciliary route. At day 2 post infection, mice were treated with various doses of IE-3-specific siRNA ranging from 0.1 nmol to 10 nmol, in a volume of 20 µL PBS via tail vein injection. Injected eyes were collected at various times post inoculation and subjected to plaque assay for virus titer, MCMV antigen staining, H&E staining, TUNEL assay, and Western blot for MCMV IE-3 protein. RESULTS: Small but significant amounts of fluorescently labeled IE-3-specific siRNA localized to the RPE layer 48 hours after intravenous injection. IE-3-specific siRNA significantly reduced virus titers at all concentrations tested (ranging from 0.1 nmol to 10 nmol), but the most potent effect of siRNA was observed at a dose of 1 nmol. We also observed that IE-3-specific siRNA produced a substantial decrease in MCMV titers and a substantial reduction in bystander cell apoptosis over the time course of virus infection. CONCLUSIONS: Systemic administration of IE-3-specific siRNA could alleviate MCMV retinitis by inhibiting virus replication and subsequent death of uninfected retinal cells.

Varicella zoster-specific CD4+Foxp3+ T cells accumulate after cutaneous antigen challenge in humans.

We investigated the relationship between varicella zoster virus (VZV)-specific memory CD4(+) T cells and CD4(+)Foxp3(+) regulatory T cells (Tregs) that accumulate after intradermal challenge with a VZV skin test Ag. VZV-specific CD4(+) T cells were identified with a MHC class II tetramer or by intracellular staining for either IFN-γ or IL-2 after Ag rechallenge in vitro. VZV-specific T cells, mainly of a central memory (CD45RA(-)CD27(+)) phenotype, accumulate at the site of skin challenge compared with the blood of the same individuals. This resulted in part from local proliferation because >50% of tetramer defined Ag-specific CD4(+) T cells in the skin expressed the cell cycle marker Ki67. CD4(+)Foxp3(+) T cells had the characteristic phenotype of Tregs, namely CD25(hi)CD127(lo)CD39(hi) in both unchallenged and VZV challenged skin and did not secrete IFN-γ or IL-2 after antigenic restimulation. The CD4(+)Foxp3(+) T cells from unchallenged skin had suppressive activity, because their removal led to an increase in cytokine secretion after activation. After VZV Ag injection, Foxp3(+)CD25(hi)CD127(lo)CD39(hi) T cells were also found within the VZV tetramer population. Their suppressive activity could not be directly assessed by CD25 depletion because activated T cells in the skin were also CD25(+). Nevertheless, there was an inverse correlation between decreased VZV skin responses and proportion of CD4(+)Foxp3(+) T cells present, indicating indirectly their inhibitory activity in vivo. These results suggest a linkage between the expansion of Ag-specific CD4(+) T cells and CD4(+) Tregs that may provide controlled responsiveness during Ag-specific stimulation in tissues.

Promotion of attachment of human bone marrow stromal cells by CCN2.

Cell attachment is a crucial step in tissue regeneration. In this study, human bone marrow stromal cells (hBMSCs) were isolated, and the effects of CCN2 on their attachment were examined. CCN2 significantly enhanced the hBMSC attachment, and this enhanced cell attachment was mainly regulated by the C-terminal module of CCN2. This enhancement was negated by the anti-integrin alpha(v)beta(3) antibody and p38 MAPK inhibitor, and phosphorylation of p38 MAPK was detected upon the enhanced cell attachment mediated by CCN2. We thus conclude that CCN2 enhances hBMSC attachment via integrin-p38 MAPK signal pathway. Enhanced hBMSC attachment on hydroxyapatite plates by CCN2 further indicated the utility of CCN2 in bone regeneration.

The repairing effect of a recombinant human connective-tissue growth factor in a burn-wounded rhesus-monkey (Macaca mulatta) model.

CTGF (connective-tissue growth factor) has been characterized as an extracellular-matrix-associated protein that modulates basic-fibroblast-growth-factor signalling and angiogenesis. In the present paper, the cloning of the ctgf gene from human umbilical-vein endothelial cells and expression of the protein in Escherichia coli as an N-terminal hexahistidine fusion protein is described. Recombinant human CTGF (rhCTGF) was expressed and purified so that we could investigate its effect on the proliferation of human embryo fibroblast KMB-17 and NIH3T3 cells. The results indicated not only that the protein was properly folded, but also that it had the same specific activity and stability as the native protein. Furthermore, we administered this recombinant protein in a non-human primate [rhesus monkey (Macaca mulatta)] burn-wound model and report the clinical findings and structural effects. Epitheliotrophic effects were conspicuous in wounded tissues at 10-100 ng of CTGF/cm(2), suggesting that administered rhCTGF can play a normal physiological role in wound repairing in a non-human primate model.

Four distinct patterns of memory CD8 T cell responses to chronic murine cytomegalovirus infection.

CMVs are beta herpesviruses that establish lifelong latent infection of their hosts. Acute infection of C57BL/6 mice with murine CMV elicits a very broad CD8 T cell response, comprising at least 24 epitopes from 18 viral proteins. In contrast, we show here that the CD8 T cell response in chronically infected mice was dominated by only five epitopes. Altogether, four distinct CD8 T cell kinetic patterns were evident. Responses to some epitopes, including M45, which dominates the acute response, contracted sharply after day 7 and developed into stable long-term memory. The response to m139 underwent rapid expansion and contraction, followed by a phase of memory inflation, whereas the response to an M38 epitope did not display any contraction phase. Finally, responses against two epitopes encoded by the immediate early gene IE3 were readily detectable in chronically infected mice but near the limit of detection during acute infection. CD8 T cells specific for the noninflationary M45 epitope displayed a classic central memory phenotype, re-expressing the lymph node homing receptor CD62L and homeostatic cytokine receptors for IL-7 and IL-15, and produced low levels of IL-2. Responses to two inflationary epitopes, m139 and IE3, retained an effector memory surface phenotype (CD62L(low), IL-7Ralpha(-), IL-15Rbeta(-)) and were unable to produce IL-2. We suggest that immunological choices are superimposed on altered viral gene expression profiles to determine immunodominance during chronic murine CMV infection.

An egr-1 (zif268) antisense oligodeoxynucleotide infused into the amygdala disrupts fear conditioning.

Studies of gene expression following fear conditioning have demonstrated that the inducible transcription factor, egr-1, is increased in the lateral nucleus of the amygdala shortly following fear conditioning. These studies suggest that egr-1 and its protein product Egr-1 in the amygdala are important for learning and memory of fear. To directly test this hypothesis, an egr-1 antisense oligodeoxynucleotide (antisense-ODN) was injected bilaterally into the amygdala prior to contextual fear conditioning. The antisense-ODN reduced Egr-1 protein in the amygdala and interfered with fear conditioning. A 250-pmole dose produced an 11% decrease in Egr-1 protein and reduced long-term memory of fear as measured by freezing in a retention test 24 h after conditioning, but left shock-induced freezing intact. A larger 500-pmole dose produced a 25% reduction in Egr-1 protein and significantly decreased both freezing immediately following conditioning and freezing in the retention test. A nonsense-ODN had no effect on postshock or retention test freezing. In addition, 500 pmole of antisense-ODN infused prior to the retention test in previously trained rats did not reduce freezing, indicating that antisense-ODN did not suppress conditioned fear behavior. Finally, rats infused with 500 pmole of antisense-ODN displayed unconditioned fear to a predator odor, demonstrating that unconditioned freezing was unaffected by the antisense-ODN. The data indicate that the egr-1 antisense-ODN interferes with learning and memory processes of fear without affecting freezing behavior and suggests that the inducible transcription factor Egr-1 within the amygdala plays important functions in long-term learning and memory of fear.

IE1-pp65 recombinant protein from human CMV combined with a nanoparticulate carrier, SMBV, as a potential source for the development of anti-human CMV adoptive immunotherapy.

BACKGROUND: Human cytomegalovirus (HCMV) infection and reactivation following allogeneic bone marrow transplantation is a major source of complications in grafted patients including pneumonitis, graft rejection and even death. Adoptive immunotherapy consisting in transfer of CD4(+) and CD8(+) T cells directed against HCMV has proved its worth. Nevertheless, established procedures have to be improved in terms of safety and waiting period required to obtain specific T cells. METHODS: As an alternative to infectious virus used in current strategies, we purified a recombinant protein IE1-pp65 resulting from the fusion of the regulatory IE1 and matrix pp65 proteins, both known as the major targets of the overall anti-HCMV T cell response. Based on our previous data demonstrating its use for in vitro stimulation and expansion of anti-HCMV CD4(+) and CD8(+) T cells (Vaz-Santiago et al, 2001, J.Virol, 75:7840-47) from peripheral blood mononuclear cells (PBMC) of seropositive donors, we planned to improve its in vitro immunogenicity through association with a nanoparticulate carrier, SMBV. RESULTS: We demonstrated that using of SMBV/IE1-pp65 formulation allowed to potentiate in vitro activation of T cells and to expand more CD8(+) T cells than with soluble IE1-pp65, following stimulation of PBMC. DISCUSSION: These data suggest the use of SMBV/IE1-pp65 formulation as a potential source of antigen for efficient T cells expansion in the development of safe anti-HCMV immunotherapy.

Mitogen activated protein kinase plays a significant role in metaphase II arrest, spindle morphology, and maintenance of maturation promoting factor activity in bovine oocytes.

Mammalian oocytes are arrested at the G2/M transition of the first meiotic division from which, after reaching full size and subsequent to an LH surge, they undergo final maturation. Oocyte maturation, which involves germinal vesicle breakdown, progression through metaphase I (MI), and arrest at MII, is triggered and regulated by the coordinated action of two kinases, maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK). The importance of the role of MPF in mammalian oocyte maturation is well established, while the role of MAPK, although well understood in mouse oocytes, has not been fully elucidated in oocytes of large domestic species, especially bovine oocytes. Here we show that injection of MKP-1 mRNA, which encodes a dual specificity MAPK phosphatase, into germinal vesicle stage bovine oocytes prevents the activation of MAPK during maturation. Despite the lack of MAPK activity, MKP-1-injected oocytes resume and progress through meiosis, although they are unable to arrest at MII stage and, by 22-26-hour post-maturation, exhibit decondensed pronucleus-like chromatin, a clear sign of parthenogenetic activation. MKP-1-injected bovine oocytes exhibit normal activation of MPF activity; however, by 18-hour post-maturation, MPF activity starts to decline and by 22-26 hr MPF activity is absent. MKP-1-injected oocytes also show disorganized MII spindles with poorly aligned chromosomes. In summary, our results demonstrate that in bovine oocytes MAPK activity is required for MII arrest, maintenance of MPF activity, and spindle organization.

Comparison of primary sensitization of naive human T cells to varicella-zoster virus peptides by dendritic cells in vitro with responses elicited in vivo by varicella vaccination.

Dendritic cells (DC) are potent APC during primary and secondary immune responses. The first objective of this study was to determine whether human DC mediate in vitro sensitization of naive CD4+ T cells to epitopes of the immediate early 62 (IE62) protein of varicella zoster virus (VZV). The induction of CD4+ T cell proliferative responses to eight synthetic peptides representing amino acid sequences of the VZV IE62 protein was assessed using T cells and DC from VZV-susceptible donors. The second objective was to compare in vitro responses of naive T cells with responses to VZV peptides induced in vivo after immunization with varicella vaccine. T cell proliferation was induced by three peptides, P1, P4, and P7, in 71-100% of the donors tested before and after vaccination using DC as APC. Monocytes were effective APC for VZV peptides only after immunization. Two peptides, P2 and P8, induced naive T cell proliferation less effectively and were also less immunogenic for T cells from vaccinated or naturally immune donors. T cell recognition of specific peptides was concordant between naive, DC-mediated responses, and postvaccine responses using monocytes as APC in 69% of comparisons (p = 0.05; chi2); the predictive value of a positive response to an IE62 peptide before immunization for T cell sensitization in vivo was 82%. These observations indicate that primary T cell responses detected in vitro using DC as APC may be useful to characterize the potential immunogenicity of viral protein epitopes in vivo.