搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Cytoplasmic APOBEC3G has been reported to block wild-type human immunodeficiency virus type 1 (HIV-1) infection in some primary cells. It is not known whether cytoplasmic APOBEC3G has residual activity in activated T cells,even though virion-packaged APOBEC3G does restrict HIV-1 in activated T cells. Because we found that APOBEC3G expression is greater in activated CD4(+) T-helper type 1 (Th1) lymphocytes than in T-helper type 2 (Th2) lymphocytes,we hypothesized that residual target cell restriction of incoming Vif-positive virions that lack APOBEC3G,if present,would be greater in Th1 than Th2 lymphocytes. Infection of activated Th1 cells with APOBEC3-negative virions did result in decreased amounts of early and late reverse transcription products and integrated virus relative to infection of activated Th2 cells. Two-long terminal repeat (2-LTR) circles,which are formed in the nucleus when reverse transcripts do not integrate,were increased after APOBEC3-negative virus infection of activated Th1 cells relative to infection of activated Th2 cells. In contrast,2-LTR circle forms were decreased after infection of APOBEC3G-negative cells with APOBEC3G-containing virions relative to APOBEC3G-negative virions and with Th1 cell-produced virions relative to Th2 cell-produced virions. Increasing APOBEC3G in Th2 cells and decreasing APOBEC3G in Th1 cells modulated the target cell phenotypes,indicating causation by APOBEC3G. The comparison between activated Th1 and Th2 cells indicates that cytoplasmic APOBEC3G in activated Th1 cells partially restricts reverse transcription and integration of incoming Vif-positive,APOBEC3G-negative HIV-1. The differing effects of cytoplasmic and virion-packaged APOBEC3G on 2-LTR circle formation indicate a difference in their antiviral mechanisms. View Publication

NK cells express different TLRs,such as TLR3,TLR7,and TLR9,but little is known about their role in NK cell stimulation. In this study,we used specific agonists (poly(I:C),loxoribine,and synthetic oligonucleotides containing unmethylated CpG sequences to stimulate human NK cells without or with suboptimal doses of IL-12,IL-15,or IFN-alpha,and investigated the secretion of IFN-gamma,cytotoxicity,and expression of the activating receptor NKG2D. Poly(I:C) and loxoribine,in conjunction with IL-12,but not IL-15,triggered secretion of IFN-gamma. Inhibition of IFN-gamma secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also,secretion of IFN-gamma was dependent on MEK1/ERK,p38 MAPK,p70(S6) kinase,and NF-kappaB,but not on calcineurin. IFN-alpha induced a similar effect,but promoted lesser IFN-gamma secretion. However,cytotoxicity (51Cr release assays) against MHC class I-chain related A (MICA)- and MICA+ tumor targets remained unchanged,as well as the expression of the NKG2D receptor. Excitingly,IFN-gamma secretion was significantly increased when NK cells were stimulated with poly(I:C) or loxoribine and IL-12,and NKG2D engagement was induced by coculture with MICA+ tumor cells in a PI3K-dependent manner. We conclude that resting NK cells secrete high levels of IFN-gamma in response to agonists of TLR3 or TLR7 and IL-12,and this effect can be further enhanced by costimulation through NKG2D. Hence,integration of the signaling cascades that involve TLR3,TLR7,IL-12,and NKG2D emerges as a critical step to promote IFN-gamma-dependent NK cell-mediated effector functions,which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer. View Publication

An urgent need exists to devise strategies to augment antiviral immune responses in patients with HIV who are virologically well controlled and immunologically stable on highly active antiretroviral therapy (HAART). The objective of this study was to compare the immunomodulatory effects of the cytokines interleukin (IL)-21 with IL-15 on CD8 T cells in patients with HIV RNA of less than 50 copies/mL and CD4 counts greater than 200 cells/mm.(3) Patient CD8 T cells displayed skewed maturation and decreased perforin expression compared with healthy controls. Culture of freshly isolated patient peripheral-blood mononuclear cells (PBMCs) for 5 hours to 5 days with IL-21 resulted in up-regulation of perforin in CD8 T cells,including memory and effector subsets and virus-specific T cells. IL-21 did not induce T-cell activation or proliferation,nor did it augment T-cell receptor (TCR)-induced degranulation. Treatment of patient PBMCs with IL-15 resulted in induction of perforin in association with lymphocyte proliferation and augmentation of TCR-induced degranulation. Patient CD8 T cells were more responsive to cytokine effects than the cells of healthy volunteers. We conclude that CD8 T cells of patients with HIV can be modulated by IL-21 to increase perforin expression without undergoing overt cellular activation. IL-21 could potentially be useful for its perforin-enhancing properties in anti-HIV immunotherapy. View Publication

Most polyreactive and antinuclear antibodies are removed from the human antibody repertoire during B cell development. To elucidate how B cell receptor (BCR) signaling may regulate human B cell tolerance,we tested the specificity of recombinant antibodies from single peripheral B cells isolated from patients suffering from X-linked agammaglobulinemia (XLA). These patients carry mutations in the Bruton's tyrosine kinase (BTK) gene that encode an essential BCR signaling component. We find that in the absence of Btk,peripheral B cells show a distinct antibody repertoire consistent with extensive secondary V(D)J recombination. Nevertheless,XLA B cells are enriched in autoreactive clones. Our results demonstrate that Btk is essential in regulating thresholds for human B cell tolerance. View Publication

Bacterial lipopeptides (bLPs) are increasingly used as adjuvants to activate cell-mediated immune responses to foreign Ags. To explore mechanisms whereby bLPs adjuvant T cell responses,we stimulated human PBMCs with bLPs. We found that bLPs stimulate T cells to proliferate and produce IFN-gamma in an accessory cell-dependent manner and in the absence of exogenous protein Ags. The ability of bLPs to stimulate T cell proliferation was Toll-like receptor 2 dependent and required IL-12,interaction with costimulatory molecules,and MHC proteins. Our data suggest that bLPs adjuvant adaptive Th1 responses by enhancing Ag presentation of endogenous peptides. View Publication

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37 °C incubator to the -196 °C liquid nitrogen storage tank are free from the influences of time. Thus,cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner,and carefully handled from blood draw through cell isolation. Furthermore,proper equipment must be in place to ensure consistency,reproducibility,and sterility. In addition,the correct choice and amount of cryoprotectant agent must be added at the correct temperature,and a controlled rate of freezing (most commonly 1 °C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel. View Publication

In this study,we examined the effects of cryoprotectant,freezing and thawing,and adenovirus (Adv) transduction on the viability,transgene expression,phenotype,and function of human dendritic cells (DCs). DCs were differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and then transduced using an Adv vector with an IL-12 transgene. Fresh,cryopreserved,and thawed transduced immature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an allogeneic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and 4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could be held in cryoprotectant for as long as 75 min at 2-8°C prior to freezing with little effect on their viability and cellularity. Further,cryopreservation,storage,and thawing reduced the viability of the transduced DCs by an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary,cryopreservation,storage,and thawing had no significant effect on DC viability,function,and transgene expression by Adv-transduced DCs. View Publication

To investigate the influence of antibody structure and specificity on antibody efficacy against Streptococcus pneumoniae,human monospecific antibodies (MAbs) to serotype 3 pneumococcal capsular polysaccharide (PPS-3) were generated from transgenic mice reconstituted with human immunoglobulin loci (XenoMouse mice) vaccinated with a PPS-3-tetanus toxoid conjugate and their molecular genetic structures,epitope specificities,and protective efficacies in normal and complement-deficient mice were determined. Nucleic acid sequence analysis of three MAbs (A7,1A2,and 7C5) revealed that they use two different V(H)3 genes (A7 and 1A2 both use V3-15) and three different V(kappa) gene segments. The MAbs were found to have similar affinities for PPS-3 but different epitope specificities and CDR3 regions. Both A7 and 7C5 had a lysine at the V(H)-D junction,whereas 1A2 had a threonine. Challenge experiments with serotype 3 S. pneumoniae in BALB/c mice revealed that both 10- and 1- micro g doses of A7 and 7C5 were protective,while only a 10- micro g dose of 1A2 was protective. Both A7 and 7C5 were also protective in mice lacking either an intact alternative (FB(-/-)) or classical (C4(-/-)) complement pathway,but 1A2 was not protective in either strain. Our data suggest that PPS-3 consists of epitopes that can elicit both highly protective and less protective antibodies and that the superior efficacies of certain antibodies may be a function of their structures and/or specificities. Further investigation of relationships between structure,specificity,and efficacy for defined MAbs to PPS may identify antibody features that might be useful surrogates for antibody (and vaccine) efficacy. View Publication

We analyzed 112 patients with high-risk acute myeloid leukemia (61 in complete remission [CR]; 51 in relapse),who received human leukocyte-antigen (HLA)-haploidentical transplants from natural killer (NK) alloreactive (n = 51) or non-NK alloreactive donors (n = 61). NK alloreactive donors possessed HLA class I,killer-cell immunoglobulin-like receptor (KIR) ligand(s) which were missing in the recipients,KIR gene(s) for missing self recognition on recipient targets,and alloreactive NK clones against recipient targets. Transplantation from NK-alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in CR (3% versus 47%) (P textgreater .003),better event-free survival in patients transplanted in relapse (34% versus 6%,P = .04) and in remission (67% versus 18%,P = .02),and reduced risk of relapse or death (relative risk versus non-NK-alloreactive donor,0.48; 95% CI,0.29-0.78; P textgreater .001). In all patients we tested the missing ligand" model which pools KIR ligand mismatched transplants and KIR ligand-matched transplants from donors possessing KIR(s) for which neither donor nor recipient have HLA ligand(s). Only transplantation from NK-alloreactive donors is associated with a survival advantage." View Publication