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1.  Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study 
BMC Immunology  2006;7:29.
Cryopreservation of peripheral blood mononuclear cells has been used to preserve and standardize immunologic measurements for multicenter studies, however, effects of cryopreservation on cytokine responses are incompletely understood. In designing immunologic studies for a new multicenter birth cohort study of childhood asthma, we performed a series of experiments to determine the effects of two different methods of cryopreservation on the cytokine responses of cord and peripheral blood mononuclear cells.
Paired samples of PBMC were processed freshly, or after cryopreservation in a Nalgene container (NC) or a controlled-rate freezer (CRF). Although there were some differences between the methods, cryopreservation inhibited PHA-induced IL-10 secretion and Der f 1-induced IL-2 secretion, and augmented PHA-induced IL-2 secretion and spontaneous secretion of TNF-α. In separate experiments, NC cryopreservation inhibited secretion of several cytokines (IL-13, IL-10, IFN-γ, TNF-α) by PHA-stimulated cord blood mononuclear cells. With the exception of PHA-induced IL-13, results from fresh and cryopreserved cord blood samples were not significantly correlated. Finally, in reproducibility studies involving processing of identical cell samples in up to 4 separate laboratories, variances in cytokine responses of fresh cells stimulated at separate sites did not exceed those in cryopreserved cells stimulated at a central site.
Collectively, these studies indicate that cryopreservation can affect mononuclear cell cytokine response profiles, and that IL-10 secretion and antigen-induced responses may be especially vulnerable. These studies also demonstrate that mononuclear cell responses can be standardized for performance in a small number of laboratories for multicenter studies, and underscore the importance of measuring reproducibility and of testing whether cryopreservation techniques alter specific immunologic outcomes.
PMCID: PMC1762025  PMID: 17156490
2.  Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes 
BMC Immunology  2006;7:28.
Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection.
Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX) domain of p47phox localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47phox, is translocated to the membrane of mature phagosomes.
We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils.
PMCID: PMC1712351  PMID: 17150107
3.  Direct and indirect effects of retinoic acid on human Th2 cytokine and chemokine expression by human T lymphocytes 
BMC Immunology  2006;7:27.
Vitamin A (VA) deficiency induces a type 1 cytokine response and exogenously provided retinoids can induce a type 2 cytokine response both in vitro and in vivo. The precise mechanism(s) involved in this phenotypic switch are inconsistent and have been poorly characterized in humans. In an effort to determine if retinoids are capable of inducing Th2 cytokine responses in human T cell cultures, we stimulated human PBMCs with immobilized anti-CD3 mAb in the presence or absence of all-trans retinoic acid (ATRA) or 9-cis-RA.
Stimulation of human PBMCs and purified T cells with ATRA and 9-cis-RA increased mRNA and protein levels of IL-4, IL-5, and IL-13 and decreased levels of IFN-γ, IL-2, IL-12p70 and TNF-α upon activation with anti-CD3 and/or anti-CD28 mAbs. These effects were dose-dependent and evident as early as 12 hr post stimulation. Real time RT-PCR analysis revealed a dampened expression of the Th1-associated gene, T-bet, and a time-dependent increase in the mRNA for the Th2-associated genes, GATA-3, c-MAF and STAT6, upon treatment with ATRA. Besides Th1 and Th2 cytokines, a number of additional proinflammatory and regulatory cytokines including several chemokines were also differentially regulated by ATRA treatment.
These data provide strong evidence for multiple inductive roles for retinoids in the development of human type-2 cytokine responses.
PMCID: PMC1665462  PMID: 17118196
4.  Targeted in vitro and in vivo gene transfer into T Lymphocytes: potential of direct inhibition of allo-immune activation 
BMC Immunology  2006;7:26.
Successful inhibition of alloimmune activation in organ transplantation remains one of the key events in achieving a long-term graft survival. Since T lymphocytes are largely responsible for alloimmune activation, targeted gene transfer of gene of cyclin kinase inhibitor p21 into T cells might inhibit their aberrant proliferation. A number of strategies using either adenoviral or lentiviral vectors linked to mono or bispecific antibodies directed against T cell surface markers/cytokines did not yield the desired results. Therefore, this study was designed to test if a CD3promoter-p21 chimeric construct would in vitro and in vivo transfer p21 gene to T lymphocytes and result in inhibition of proliferation. CD3 promoter-p21 chimeric constructs were prepared with p21 in the sense and antisense orientation. For in vitro studies EL4-IL-2 thyoma cells were used and for in vivo studies CD3p21 sense and antisense plasmid DNA was injected intramuscularly in mice. Lymphocyte proliferation was quantified by 3H-thymidine uptake assay; IL-2 mRNA expression was studied by RT-PCR and using Real Time PCR assay, we monitored the CD3, p21, TNF-α and IFN-γ mRNA expression.
Transfection of CD3p21 sense and antisense in mouse thyoma cell line (EL4-IL-2) resulted in modulation of mitogen-induced proliferation. The intramuscular injection of CD3p21 sense and antisense plasmid DNA into mice also modulated lymphocyte proliferation and mRNA expression of pro-inflammatory cytokines.
These results demonstrate a novel strategy of in vitro and in vivo transfer of p21 gene to T cells using CD3-promoter to achieve targeted inhibition of lymphocyte proliferation and immune activation.
PMCID: PMC1657031  PMID: 17096842
5.  Immunogenicity of a polyvalent HIV-1 candidate vaccine based on fourteen wild type gp120 proteins in golden hamsters 
BMC Immunology  2006;7:25.
One of the major obstacles in the design of an effective vaccine against HIV-1 is the hypervariability of the HIV-1 envelope glycoprotein. Most HIV-1 vaccine candidates have utilized envelope glycoprotein from a single virus isolate, but to date, none of them elicited broadly reactive humoral immunity. Herein, we hypothesised that a cocktail of HIV-1 gp120 proteins containing multiple epitopes may increase the breadth of immune responses against HIV-1. We compared and evaluated the immunogenicity of HIV-1 vaccines containing either gp120 protein alone or in combinations of four or fourteen gp120s from different primary HIV-1 isolates in immunized hamsters.
We amplified and characterized 14 different gp120s from primary subtype B isolates with both syncytium and non-syncytium inducing properties, and expressed the proteins in Chinese Hamster Ovary (CHO) cell lines. Purified proteins were used either alone or in combinations of four or fourteen different gp120s to vaccinate golden hamsters. The polyvalent vaccine showed higher antibody titers to HIV-1 subtype B isolates MN and SF162 compared to the groups that received one or four gp120 proteins. However, the polyvalent vaccine was not able to show higher neutralizing antibody responses against HIV-1 primary isolates. Interestingly, the polyvalent vaccine group had the highest proliferative immune responses and showed a substantial proportion of cross-subtype CD4 reactivity to HIV-1 subtypes B, C, and A/E
Although the polyvalent approach achieved only a modest increase in the breadth of humoral and cellular immunity, the qualitative change in the vaccine (14 vs. 1 gp120) resulted in a quantitative improvement in vaccine-induced immunity.
PMCID: PMC1636068  PMID: 17076905
6.  A novel assay for monitoring internalization of nanocarrier coupled antibodies 
BMC Immunology  2006;7:24.
Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA.
The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay.
The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers.
PMCID: PMC1633733  PMID: 17014727
7.  Novel insights in the regulation of CCL18 secretion by monocytes and dendritic cells via cytokines, Toll-like receptors and rheumatoid synovial fluid 
BMC Immunology  2006;7:23.
The T cell attracting chemokine CCL18 is produced by antigen presenting cells and a role for CCL18 has been suggested in the pathogenesis of a variety of diseases. Rheumatoid arthritis (RA) is one of these conditions, in which abundant CCL18 production is present. Although Th2 cytokines and IL-10 are known to have an effect on CCL18 production, there are several gaps in our knowledge regarding the exact regulation of CCL18 secretion, both in general and in RA. In this study we provide new insights in the regulation of CCL18 secretion by monocytes and dendritic cells.
In contrast to a large panel of pro-inflammatory stimuli (IL-1β, TNF-α, IL-10, IL-13, IL-15, IL-17, IL-18, IFN-γ), T cell mimicking molecules (RANKL, CD40L) or TLR driven maturation, the anti-inflammatory IL-10 strongly stimulated DC to secrete CCL18. On freshly isolated monocytes, CCL18 secretion was induced by IL-4 and IL-13, in strong synergy with IL-10. This synergistic effect could already be observed after only 24 hours, indicating that not only macrophages and dendritic cells, but also monocytes secrete CCL18 under these stimulatory conditions. A high CCL18 expression was detected in RA synovial tissue and incubation of monocytes with synovial fluid from RA patients clearly enhanced the effects of IL-4, IL-13 and IL-10. Surprisingly, the effect of synovial fluid was not driven by IL-10 of IL-13, suggesting the presence of another CCL18 inducing factor in synovial fluid.
In summary, IL-10 synergistically induces CCL18 secretion in combination with IL-4 of IL-13 on monocytes and monocyte derived cells. The effects of IL-14, IL-13 and IL-10 are strongly enhanced by synovial fluid. This synergy may contribute to the high CCL18 expression in RA.
PMCID: PMC1590050  PMID: 16984635
8.  Expression of P2 receptors in human B cells and Epstein-Barr virus-transformed lymphoblastoid cell lines 
BMC Immunology  2006;7:22.
Epstein-Barr virus (EBV) infection immortalizes primary B cells in vitro and generates lymphoblastoid cell lines (LCLs), which are used for several purposes in immunological and genetic studies. Purinergic receptors, consisting of P2X and P2Y, are activated by extracellular nucleotides in most tissues and exert various physiological effects. In B cells, especially EBV-induced LCLs, their expression and function have not been well studied. We investigated the expression of P2 receptors on primary human B cells and LCLs using the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for revealing the gene expression profile of the P2 receptor subtypes and their changes during transformation.
The mRNA transcripts of most P2 receptors were detected in primary B cells; the expression of P2X3 and P2X7 receptors was the lowest of all the P2 receptors. By contrast, LCLs expressed several dominant P2 receptors – P2X4, P2X5, and P2Y11 – in amounts similar to those seen in B cells infected with EBV for 2 weeks. The amount of most P2 subtypes in LCLs or EBV-infected B cells was lower than in normal B cells. However, the amount of P2X7 receptor expressed in LCLs was higher. Protein expression was studied using Western blotting to confirm the mRNA findings for P2X1, P2X4, P2X7, P2Y1, and P2Y11 receptors. ATP increased the intracellular free Ca2+ concentration ([Ca2+]i) by enhancing the Ca2+ influx in both B cells and LCLs in a dose-dependent manner.
These findings describe P2 receptor expression profiles and the effects of purinergic stimuli on B cells and suggest some plasticity in the expression of the P2 receptor phenotype. This may help explain the nature and effect of P2 receptors on B cells and their role in altering the characteristics of LCLs.
PMCID: PMC1609185  PMID: 16970829
9.  Long-term changes of serum chemokine levels in vaccinated military personnel 
BMC Immunology  2006;7:21.
Members of the United States Armed Forces receive a series of vaccinations during their course of service. To investigate the influence of multiple vaccinations on innate immunity, we measured concentrations of a panel of immunomodulatory and pro-inflammatory cytokines in serum samples from a group of such individuals.
Significantly increased levels of macrophage inflammatory protein 1α (MIP-1α), MIP-1β and interleukin 8 (IL-8) were detected. Since these cytokines are known to have anti-human immunodeficiency virus (HIV) activity, we tested the effect of serum from these individuals on HIV-1 infectivity and susceptibility of their peripheral blood mononuclear cells (PBMCs) to HIV-1 infection in vitro. Sera from vaccinated military personnel inhibited, and their PBMCs were partially resistant to, infection by HIV-1 strains tropic to CCR5 (R5), but not to CXCR4 (X4), chemokine receptor.
These findings demonstrate that increased anti-HIV chemokines can be detected in vaccine recipients up to 68 weeks following immunization.
PMCID: PMC1578581  PMID: 16965634
10.  Optimized detection of circulating anti-nuclear envelope autoantibodies by immunofluorescence 
BMC Immunology  2006;7:20.
Antinuclear antibodies are useful diagnostic tools in several autoimmune diseases. However, the routine detection of nuclear envelope autoantibodies using immunofluorescence (IF) is not always easy to perform in patients' sera because of the presence of autoantibodies to other nuclear and cytoplasmic components which could mask the characteristic rim-like pattern of nuclear envelope autoantibodies. This is particularly common in sera from patients with primary biliary cirrhosis (PBC), which generaly have high titres of anti-mitochondrial antibodies. Therefore, we have assayed a number of commercial slides and alternative fixation conditions to optimize the detection of anti-nuclear envelope antibodies (ANEA) in PBC sera.
We have explored the presence of ANEA in 33 sera from patients with established PBC using three different Hep2 commercial slides and home-made slides with HeLa and Hep2 cells fixed with methanol, ethanol, 1% or 4% formaldehyde.
We observed that the IF pattern was related to the cell type used (Hep2 or HeLa), the manufacturer and the cell fixation scheme. When both cell lines were fixed with 1% formaldehyde, the intensity of the cytoplasmic staining was considerably decreased regardless to the serum sample, whereas the prevalence of cytoplasmic autoantibodies was significantly lowered, as compared to any of the Hep2 commercial slide and fixation used. In addition, the prevalence of ANEA was importantly increased in formaldehyde-fixed cells.
Immunofluorescence using appropriately fixed cells represent an easy, no time-consuming and low cost technique for the routine screening of sera for ANEA. Detection of ANEA is shown to be more efficient using formaldehyde-fixed cells instead of commercially available Hep2 cells.
PMCID: PMC1574344  PMID: 16956395
11.  The innate interferon gamma response of BALB/c and C57BL/6 mice to in vitro Burkholderia pseudomallei infection 
BMC Immunology  2006;7:19.
Burkholderia pseudomallei is the causative agent for melioidosis. For many bacterial infections, cytokine dysregulation is one of the contributing factors to the severe clinical outcomes in the susceptible hosts. The C57BL/6 and BALB/c mice have been established as a differential model of susceptibility in murine melioidosis. In this study, we compared the innate IFN-γ response to B. pseudomallei between the C57BL/6 and BALB/c splenocytes and characterized the hyperproduction of IFN-γ in the relatively susceptible BALB/c mice in vitro.
Naïve BALB/c splenocytes were found to produce more IFN-γ in response to live bacterial infection compared to C57BL/6 splenocytes. Natural killer cells were found to be the major producers of IFN-γ, while T cells and Gr-1intermediate cells also contributed to the IFN-γ response. Although anti-Gr-1 depletion substantially reduced the IFN-γ response, this was not due to the contribution of Gr-1high, Ly-6G expressing neutrophils. We found no differences in the cell types making IFN-γ between BALB/c and C57BL/6 splenocytes. Although IL-12 is essential for the IFN-γ response, BALB/c and C57BL/6 splenocytes made similar amounts of IL-12 after infection. However, BALB/c splenocytes produced higher proinflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-18 than C57BL/6 splenocytes after infection with B. pseudomallei.
Higher percentages of Gr-1 expressing NK and T cells, poorer ability in controlling bacteria growth, and higher IL-18 could be the factors contributing to IFN-γ hyperproduction in BALB/c mice.
PMCID: PMC1559720  PMID: 16919160
12.  Profiling helper T cell subset gene expression in deer mice 
BMC Immunology  2006;7:18.
Deer mice (Peromyscus maniculatus) are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV), the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS) in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses.
We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNγ, TNF, LT), Th2 cells (GATA-3, STAT6, IL-4, IL-5) and regulatory T cells (Fox-p3, IL-10, TGFβ1). These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice.
We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice.
PMCID: PMC1559719  PMID: 16916450
13.  Regulatory role of pro-Th1 and pro-Th2 cytokines in modulating the activity of Th1 and Th2 cells when B cell and macrophages are used as antigen presenting cells 
BMC Immunology  2006;7:17.
Presence of antigen presenting cells, expression of costimulatory molecules, the strength of first signal and cytokine milieu are quite important in influencing the reactivation of differentiated Th1 and Th2 cells.
In the present study, we have analyzed the concerted action of pro-Th1 and pro-Th2 cytokines in the presence of B cells, peritoneal and splenic macrophages as antigen presenting cells and varied concentration of first (anti-CD3 Ab) and second (B7-1 transfectant) signals on the proliferation and cytokine secretion by Th1 and Th2 cells. Interesting observations were made that IFN-γ significantly augmented the secretion of IL-4 by Th2 cells when either B cells or splenic or peritoneal macrophages were used as APC. Further, IFN-γ significantly inhibited the proliferation of Th1 cells only in the presence of peritoneal macrophages. We have also observed that B cells could significantly respond to cytokines to further enhance the proliferation and cytokine release by Th1 and Th2 cells. But not much effect on addition of exogenous cytokines IL-1, IL-4, IL-5, IL-12 was observed on the proliferation of Th1 and Th2 cells in the presence of macrophages. In contrast, both IFN-γ and IL-2 significantly enhanced the production of IL-4 and IL-5 respectively, by Th2 cells in presence of B cells, splenic and peritoneal macrophages. Another important observation was that the addition of B7-1 transfectants in the cultures, which were stimulated with low dose of anti-CD3 Ab significantly, enhanced the proliferation and cytokine secretion.
This study indicates involvement of different type of APCs, cytokine milieu, dose of first and second signals in a concerted manner in the outcome of the immune response. The significance of this study is that the immunization with antigen along with costimulatory molecules may significantly reduce the dose of antigen and can generate better immune response than antigen alone.
PMCID: PMC1550426  PMID: 16889674
14.  Enumeration of leukocyte infiltration in solid tumors by confocal laser scanning microscopy 
BMC Immunology  2006;7:16.
Leukocytes commonly infiltrate solid tumors, and have been implicated in the mechanism of spontaneous regression in some cancers. Conventional techniques for the quantitative estimation of leukocyte infiltrates in tumors rely on light microscopy of immunostained thin tissue sections, in which an arbitrary assessment (based on low, medium or high levels of infiltration) of antigen density is made by the pathologist. These estimates are relatively subjective and often require the opinion of a second pathologist. In addition, since thin tissue sections are cut, no data regarding the three-dimensional distribution of antigen can be obtained.
To overcome these problems, we have designed a method to enumerate leukocyte infiltration into tumors, using confocal laser scanning microscopy of fluorescently immunostained leukocytes in thick tissue sections. Using image analysis software, a threshold was applied to eliminate unstained tissue and residual noise. The total antigen volume in the scanned tissue was calculated and divided by the mean cell volume (calculated by "seeding" ten individual cells) to obtain the cell count. Using this method, we compared the calculated leukocyte counts with those obtained manually by ten laboratory personnel. There was no significant difference (P > 0.05) between the cell counts obtained by either method.
We then compared leukocyte infiltration into seven tumors and matched non-malignant tissue obtained from the periphery of the resected tissue. There was a significant increase in the infiltration of all leukocyte subsets into the tumors compared to minimal numbers in the non-malignant tissue.
From these results we conclude that this method may be of considerable use for the enumeration of cells in tissues. Furthermore, since it can be performed by laboratory technical staff, less time input is required by the pathologist in assessing the degree of leukocyte infiltration into tumors.
PMCID: PMC1544349  PMID: 16859557
15.  Structure function analysis of SH2D2A isoforms expressed in T cells reveals a crucial role for the proline rich region encoded by SH2D2A exon 7 
BMC Immunology  2006;7:15.
The activation induced T cell specific adapter protein (TSAd), encoded by SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd.
TSAd was found to interact with Lck in human CD4+ T cells ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239–256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239–334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239–334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells.
These data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239–334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined.
PMCID: PMC1553471  PMID: 16839418
16.  Human cerebrospinal fluid contains CD4+ memory T cells expressing gut- or skin-specific trafficking determinants: relevance for immunotherapy 
BMC Immunology  2006;7:14.
Circulating memory T cells can be divided into tissue-specific subsets, which traffic through distinct tissue compartments during physiologic immune surveillance, based on their expression of adhesion molecules and chemokine receptors. We reasoned that a bias (either enrichment or depletion) of CSF T cell expression of known organ-specific trafficking determinants might suggest that homing of T cells to the subarachnoid space could be governed by a CNS-specific adhesion molecule or chemokine receptor.
The expression of cutaneous leukocyte antigen (CLA) and CC-chemokine receptor 4 (CCR4; associated with skin-homing) as well as the expression of integrin α4β7 and CCR9 (associated with gut-homing) was analyzed on CD4+ memory T cells in CSF from individuals with non-inflammatory neurological diseases using flow cytometry. CSF contained similar proportions of CD4+ memory T cells expressing CLA, CCR4, integrin α4β7 and CCR9 as paired blood samples.
The results extend our previous findings that antigen-experienced CD4+ memory T cells traffic through the CSF in proportion to their abundance in the peripheral circulation. Furthermore, the ready access of skin- and gut-homing CD4+ memory T cells to the CNS compartment via CSF has implications for the mechanisms of action of immunotherapeutic strategies, such as oral tolerance or therapeutic immunization, where immunogens are administered using an oral or subcutaneous route.
PMCID: PMC1539023  PMID: 16824229
17.  Wnt expression and canonical Wnt signaling in human bone marrow B lymphopoiesis 
BMC Immunology  2006;7:13.
The early B lymphopoiesis in mammals is regulated through close interactions with stromal cells and components of the intracellular matrix in the bone marrow (BM) microenvironment. Although B lymphopoiesis has been studied for decades, the factors that are implicated in this process, both autocrine and paracrine, are inadequately explored. Wnt signaling is known to be involved in embryonic development and growth regulation of tissues and cancer. Wnt molecules are produced in the BM, and we here ask whether canonical Wnt signaling has a role in regulating human BM B lymphopoiesis.
Examination of the mRNA expression pattern of Wnt ligands, Fzd receptors and Wnt antagonists revealed that BM B progenitor cells and stromal cells express a set of ligands and receptors available for induction of Wnt signaling as well as antagonists for fine tuning of this signaling. Furthermore, different B progenitor maturation stages showed differential expression of Wnt receptors and co-receptors, β-catenin, plakoglobin, LEF-1 and TCF-4 mRNAs, suggesting canonical Wnt signaling as a regulator of early B lymphopoiesis. Exogenous Wnt3A induced stabilization and nuclear accumulation of β-catenin in primary lineage restricted B progenitor cells. Also, Wnt3A inhibited B lymphopoiesis of CD133+CD10- hematopoietic progenitor cells and CD10+ B progenitor cells in coculture assays using a supportive layer of stromal cells. This effect was blocked by the Wnt antagonists sFRP1 or Dkk1. Examination of early events in the coculture showed that Wnt3A inhibits cell division of B progenitor cells.
These results indicate that canonical Wnt signaling is involved in human BM B lymphopoiesis where it acts as a negative regulator of cell proliferation in a direct or stroma dependent manner.
PMCID: PMC1543656  PMID: 16808837
18.  Induction of PNAd and N-acetylglucosamine 6-O-sulfotransferases 1 and 2 in mouse collagen-induced arthritis 
BMC Immunology  2006;7:12.
Leukocyte recruitment across blood vessels is fundamental to immune surveillance and inflammation. Lymphocyte homing to peripheral lymph nodes is mediated by the adhesion molecule, L-selectin, which binds to sulfated carbohydrate ligands on high endothelial venules (HEV). These glycoprotein ligands are collectively known as peripheral node addressin (PNAd), as defined by the function-blocking monoclonal antibody known as MECA-79. The sulfation of these ligands depends on the action of two HEV-expressed N-acetylglucosamine 6-O-sulfotransferases: GlcNAc6ST-2 and to a lesser degree GlcNAc6ST-1. Induction of PNAd has also been shown to occur in a number of human inflammatory diseases including rheumatoid arthritis (RA).
In order to identify an animal model suitable for investigating the role of PNAd in chronic inflammation, we examined the expression of PNAd as well as GlcNAc6ST-1 and -2 in collagen-induced arthritis in mice. Here we show that PNAd is expressed in the vasculature of arthritic synovium in mice immunized with collagen but not in the normal synovium of control animals. This de novo expression of PNAd correlates strongly with induction of transcripts for both GlcNAc6ST-1 and GlcNAc6ST-2, as well as the expression of GlcNAc6ST-2 protein.
Our results demonstrate that PNAd and the sulfotransferases GlcNAc6ST-1 and 2 are induced in mouse collagen-induced arthritis and suggest that PNAd antagonists or inhibitors of the enzymes may have therapeutic benefit in this widely-used mouse model of RA.
PMCID: PMC1533857  PMID: 16772045
19.  VACUTAINER® CPT™ and Ficoll density gradient separation perform equivalently in maintaining the quality and function of PBMC from HIV seropositive blood samples 
BMC Immunology  2006;7:11.
For immune monitoring studies during HIV vaccine clinical trials, whole blood specimens from HIV seropositive (HIV+) patients may be collected at multiple sites and sent to a central location for peripheral blood mononuclear cell (PBMC) isolation, cryopreservation and functional evaluation. In this study we show a comparison of two PBMC preparation options, Ficoll density gradient separation (Ficoll) and Cell Preparation Tubes (CPT) using shipped whole blood specimens from 19 HIV+ patients (CD4 > 350, viral load < 50). The pre- and post- cryopreservation performance of samples collected by these two methods were compared by assessment of antigen-specific IFNγ expression in CD8+ and CD8- T cells, cellular viability, and cellular recovery.
The results indicate that cryopreserved PBMC samples tested for CMV- and HIV- specific interferon-gamma (IFNγ) expression performed equivalent to the respective fresh PBMC processed under both collection conditions. Compared to fresh PBMC, the viability was significantly lower for cryopreserved PBMC derived using Ficoll, although it was never less than 90%. There were no significant differences in the IFNγ response, viability, or recovery between cryopreserved PBMC derived by Ficoll and by CPT.
These data suggest that CPT is an efficient system for the collection and cryopreservation of functionally active HIV+ PBMC, as well as a viable alternative to Ficoll gradient separation.
PMCID: PMC1481571  PMID: 16725038
20.  Bovine CD2-/NKp46+ cells are fully functional natural killer cells with a high activation status 
BMC Immunology  2006;7:10.
Natural killer (NK) cells in the cow have been elusive due to the lack of specific NK cell markers, and various criteria including a CD3-/CD2+ phenotype have been used to identify such cells. The recent characterization of the NK-specific NKp46 receptor has allowed a more precise definition of bovine NK cells. NK cells are known as a heterogeneous cell group, and we here report the first functional study of bovine NK cell subsets, based on the expression of CD2.
Bovine CD2- NK cells, a minor subset in blood, proliferated more rapidly in the presence of IL-2, dominating the cultures after a few days. Grown separately with IL-2, CD2- and CD2+ NK cell subsets did not change CD2 expression for at least two weeks. In blood, CD2- NK cells showed a higher expression of CD44 and CD25, consistent with a high activation status. A higher proportion of CD2- NK cells had intracellular interferon-gamma in the cytoplasm in response to IL-2 and IL-12 stimulation, and the CD2- subset secreted more interferon-gamma when cultured separately. Cytotoxic capacity was similar in both subsets, and both carried transcripts for the NK cell receptors KIR, CD16, CD94 and KLRJ. Ligation by one out of two tested anti-CD2 monoclonal antibodies could trigger interferon-gamma production from NK cells, but neither of them could alter cytotoxicity.
These results provide evidence that bovine CD2- as well as CD2+ cells of the NKp46+ phenotype are fully functional NK cells, the CD2- subset showing signs of being more activated in the circulation.
PMCID: PMC1482717  PMID: 16643649
21.  Co-receptor and co-stimulation blockade for mixed chimerism and tolerance without myelosuppressive conditioning 
BMC Immunology  2006;7:9.
A major challenge in the application of marrow transplantation as a route to immunological tolerance of a transplanted organ is to achieve hematopoietic stem cell (HSC) engraftment with minimal myelosuppressive treatments.
We here describe a combined antibody protocol which can achieve long-term engraftment with clinically relevant doses of MHC-mismatched bone marrow, without the need for myelosuppressive drugs. Although not universally applicable in all strains, we achieved reliable engraftment in permissive strains with a two-stage strategy: involving first, treatment with anti-CD8 and anti-CD4 in advance of transplantation; and second, treatment with antibodies targeting CD4, CD8 and CD40L (CD154) at the time of marrow transplantation. Long-term mixed chimerism through co-receptor and co-stimulation blockade facilitated tolerance to donor-type skin grafts, without any evidence of donor-antigen driven regulatory T cells.
We conclude that antibodies targeting co-receptor and co-stimulatory molecules synergise to enable mixed hematopoietic chimerism and central tolerance, showing that neither cytoreductive conditioning nor 'megadoses' of donor bone marrow are required for donor HSC to engraft in permissive strains.
PMCID: PMC1463008  PMID: 16638128
22.  CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: Use of multiple peptide sets increases the detectable breadth of the CTL response 
BMC Immunology  2006;7:8.
Subtype A is a major strain in the HIV-1 pandemic in eastern Europe, central Asia and in certain regions of east Africa, notably in rural Kenya. While considerable effort has been focused upon mapping and defining immunodominant CTL epitopes in HIV-1 subtype B and subtype C infections, few epitope mapping studies have focused upon subtype A.
We have used the IFN-γ ELIspot assay and overlapping peptide pools to show that the pattern of CTL recognition of the Gag and Nef proteins in subtype A infection is similar to that seen in subtypes B and C. The p17 and p24 proteins of Gag and the central conserved region of Nef were targeted by CTL from HIV-1-infected Kenyans. Several epitope/HLA associations commonly seen in subtype B and C infection were also observed in subtype A infections. Notably, an immunodominant HLA-C restricted epitope (Gag 296–304; YL9) was observed, with 8/9 HLA-CW0304 subjects responding to this epitope. Screening the cohort with peptide sets representing subtypes A, C and D (the three most prevalent HIV-1 subtypes in east Africa), revealed that peptide sets based upon an homologous subtype (either isolate or consensus) only marginally improved the capacity to detect CTL responses. While the different peptide sets detected a similar number of responses (particularly in the Gag protein), each set was capable of detecting unique responses not identified with the other peptide sets.
Hence, screening with multiple peptide sets representing different sequences, and by extension different epitope variants, can increase the detectable breadth of the HIV-1-specific CTL response. Interpreting the true extent of cross-reactivity may be hampered by the use of 15-mer peptides at a single concentration and a lack of knowledge of the sequence that primed any given CTL response. Therefore, reagent choice and knowledge of the exact sequences that prime CTL responses will be important factors in experimentally defining cross-reactive CTL responses and their role in HIV-1 disease pathogenesis and validating vaccines aimed at generating broadly cross-reactive CTL responses.
PMCID: PMC1464141  PMID: 16620386
23.  CED: a conformational epitope database 
BMC Immunology  2006;7:7.
Antigen epitopes provide valuable information useful for disease prevention, diagnosis, and treatment. Recently, more and more databases focusing on different types of epitopes have become available. Conformational epitopes are an important form of epitope formed by residues that are sequentially discontinuous but close together in three-dimensional space. These epitopes have implicit structural information, making them attractive for both theoretical and applied biomedical research. However, most existing databases focus on linear rather than conformational epitopes.
We describe CED, a special database of well defined conformational epitopes. CED provides a collection of conformational epitopes and related information including the residue make up and location of the epitope, the immunological property of the epitope, the source antigen and corresponding antibody of the epitope. All entries in this database are manually curated from articles published in peer review journals. The database can be browsed or searched through a user-friendly web interface. Most epitopes in CED can also be viewed interactively in the context of their 3D structures. In addition, the entries are also hyperlinked to various databases such as Swiss-Prot, PDB, KEGG and PubMed, providing wide background information.
A conformational epitope database called CED has been developed as an information resource for investigators involved in both theoretical and applied immunology research. It complements other existing specialised epitope databases. The database is freely available at
PMCID: PMC1513601  PMID: 16603068
24.  Delayed functional maturation of natural regulatory T cells in the medulla of postnatal thymus: role of TSLP 
BMC Immunology  2006;7:6.
Generation of functional CD4+CD8-CD25+ regulatory T cells (Treg) in the murine thymus depends on FoxP3. Removal of the thymus from neonatal mice has been shown to result in a multiple organ autoimmune disease phenotype that can be prevented by introducing the FoxP3+ Treg population to the animal. It has therefore, been proposed that functional FoxP3+ Treg cells are not made in the neonatal thymus; however, it remains unclear when and where functional FoxP3+CD4+CD8-CD25+ thymocytes are generated in postnatal thymus.
We report that neither FoxP3 mRNA nor protein is expressed in CD4+CD8-CD25+, or CD4+CD8-CD25- thymocytes until 3–4 days post birth, despite the presence of mature CD4+CD8-CD25+/- thymocytes in the thymus by 1–2 days after birth. FoxP3-CD4+CD8-CD25+ thymocytes from day 2 newborn mice show no Treg activity. Interestingly, we are able to detect low numbers of FoxP3+ thymocytes dispersed throughout the medullary region of the thymus as early as 3–4 days post birth. Expression of FoxP3 is induced in embryonic day 17 fetal thymus organ culture (FTOC) after 4–6 days of in vitro culture. Treatment of FTOCs with thymic stromal derived lymphopoietin (TSLP) enhanced expression of FoxP3, and blocking the TSLP receptor reduces FoxP3 expression in FTOC. Furthermore, TSLP stimulates FoxP3 expression in purified CD4+CD8- thymocytes, but not in CD4+CD8+, CD4-CD8+ and CD4-CD8- thymocytes.
Expression of FoxP3 or Treg maturation is ontogenically distinct and kinetically delayed from the generation of CD4+CD8-CD25+ or CD4+CD8-CD25- thymocytes in the postnatal thymus. TSLP produced from medullary thymic epithelia cells (mTEC) contributes to the expression of FoxP3 and the maturation of natural regulatory T cells. Overall, these results suggest that the development of Treg cells requires paracrine signaling during late stages of thymocyte maturation that is distinct from signaling during positive or negative selection.
PMCID: PMC1450317  PMID: 16579866
25.  β-(1→3)-D-glucan modulates DNA binding of nuclear factors κB, AT and IL-6 leading to an anti-inflammatory shift of the IL-1β/IL-1 receptor antagonist ratio 
BMC Immunology  2006;7:5.
β-1→3-D-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses. Employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal β-1→3-D-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide (LPS) or toxic shock syndrome toxin 1 (TSST-1).
Despite an activation of nuclear factor (NF)κB, NFinterleukin(IL)-6 and NFAT similar to LPS or TSST-1, we observed no significant production of IL-1β, IL-6, tumor necrosis factor α or interferon γ induced by glucan phosphate. Glucan phosphate-treated leukocytes induced a substantial amount of IL-8 (peak at 18 h: 5000 pg/ml), likely due to binding of NFκB to a consensus site in the IL-8 promoter. An increase in IL-1receptor antagonist(RA) production (peak at 24 h: 12000 pg/ml) by glucan phosphate-treated cells positively correlated with IL-8 levels. Glucan phosphate induced significant binding to a known NFIL-6 site and a new NFAT site within the IL-1RA promoter, which was confirmed by inhibition experiments. When applied in combination with either LPS or TSST-1 at the same time points, we detected that glucan phosphate elevated the LPS- and the TSST-1-induced DNA binding of NFκB, NFIL-6 and NFAT, leading to a synergistic increase of IL-1RA. Further, glucan phosphate modulated the TSST-1-induced inflammatory response via reduction of IL-1β and IL-6. As a consequence, glucan phosphate shifted the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype. Subsequently, glucan phosphate decreased the TSST-1-induced, IL-1-dependent production of IL-2.
Thus, β-1→3-D-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory IL-1RA-mediated reaction. Our results also offer new insights into the complex regulation of the IL-1RA gene, which can be modulated by a β-1→3-D-glucan.
PMCID: PMC1472690  PMID: 16553947

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