High molecular weight polysaccharides are key immunomodulators in North American ginseng extracts: characterization of the ginseng genetic signature in primary human immune cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng (GS) has played a pivotal role in traditional Chinese medicine for thousands of years. Its use has become increasingly popular in North America, in part due to the many claims of its immune-enhancing properties. The immunopharmacology of the North American variety of GS and its extracts is needed to substantiate these claims. MATERIALS AND METHODS: Human peripheral blood mononuclear cells were exposed to different North American GS extracts and microarray analysis was performed. The profile of cytokine response to GS extracts was established by ELISA, and Ingenuity Pathway Analysis was used to identify potential signaling pathways responsible for the transcriptional profile induced by GS. Fractionation of the aqueous and polysaccharide extracts was done to determine the molecular weight of the active immune modulatory ingredient(s). RESULTS: We found that GS induced a transcriptional profile of immunomodulation characterized by a net T(h)1 immune response, with up-regulation of multiple pro-inflammatory cytokines (e.g., IFN-γ, IL-23A and IL-6) and down-regulation of TGF-ß, IL-13 and the LPS co-receptor CD14. Ingenuity Pathway Analysis (IPA) revealed that the MAPK (ERK-1/2), PI3K, p38 and NF-κB cascades were key signaling pathways through which GS may trigger its immunomodulatory action. Furthermore, induction of such an immunomodulatory signature was recapitulated with the high molecular weight polysaccharides found in aqueous and polysaccharide GS extracts. CONCLUSIONS: Based on our results, we conclude that high molecular weight polysaccharides in North American GS aqueous and polysaccharide extracts likely trigger the MAPK (ERK-1/2), PI3K, p38 and NF-κB signaling pathways in PBMC resulting in the induction of a T(h)1 transcriptional profile. Our results may assist in optimizing GS-mediated immunomodulation and focus the search for compounds in GS extracts with specific immunomodulatory activities.

Stomatin-like protein 2 binds cardiolipin and regulates mitochondrial biogenesis and function.

Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipin-enriched microdomains in which electron transport complexes are optimally assembled. Likely through the prohibitin functional interactome, SLP-2 then regulates mitochondrial biogenesis and function.

Impaired GFR is the most important determinant for FGF-23 increase in chronic kidney disease.

OBJECTIVES: It is unclear whether fibroblast growth factor-23 (FGF-23) increases in response to phosphate accumulation or to decrease clearance in chronic kidney disease (CKD) as is the case with other low molecular weight proteins such as cystatin C (CysC). DESIGN AND METHODS: This cross-sectional study measured serum FGF-23, CysC, and other serum markers of bone metabolism in 69 patients, aged 18 months-24 years, with various stages of CKD (eGFR=11-214mL/min). RESULTS: FGF-23 levels were significantly correlated with CysC and parathyroid hormone levels (PTH) on univariate non-linear regression analysis. In multivariate linear regression analysis, log (CysC) (ß=0.660, p<0.0001), log (PTH) (ß=0.038, p=0.37), and phosphate (ß=0.222, p=0.028) explained 69.1% of the variance of FGF-23. CONCLUSIONS: CysC had the largest unique contribution to FGF-23 variance in this model, supporting the hypothesis that renal clearance may be the most responsible factor for elevated FGF-23 levels in early stages of CKD.

Tumor suppression by phospholipase C-beta3 via SHP-1-mediated dephosphorylation of Stat5.

Given its catalytic activity to generate diacylglycerol and inositol 1,4,5-trisphosphate, phospholipase C (PLC) is implicated in promoting cell growth. However, we found that PLC-beta3-deficient mice develop myeloproliferative disease, lymphoma, and other tumors. The mutant mice have increased numbers of hematopoietic stem cells with increased proliferative, survival, and myeloid-differentiative abilities. These properties are dependent on Stat5 and can be antagonized by the protein phosphatase SHP-1. Stat5-dependent cooperative transformation by active c-Myc and PLC-beta3 deficiency was suggested in mouse lymphomas in PLC-beta3(-/-) and in Emicro-myc;PLC-beta3(+/-) mice and human Burkitt's lymphoma cells. The same mechanism for malignant transformation seems to be operative in other human lymphoid and myeloid malignancies. Thus, PLC-beta3 is likely a tumor suppressor.

Modulation of T cell activation by stomatin-like protein 2.

T cell activation through the Ag receptor (TCR) requires sustained signaling from signalosomes within lipid raft microdomains in the plasma membrane. In a proteomic analysis of lipid rafts from human T cells, we identified stomatin-like protein (SLP)-2 as a candidate molecule involved in T cell activation through the Ag receptor. In this study, we show that SLP-2 expression in human primary lymphocytes is up-regulated following in vivo and ex vivo activation. In activated T cells, SLP-2 interacts with components of TCR signalosomes and with polymerized actin. More importantly, up-regulation of SLP-2 expression in human T cell lines and primary peripheral blood T cells increases effector responses, whereas down-regulation of SLP-2 expression correlates with loss of sustained TCR signaling and decreased T cell activation. Our data suggest that SLP-2 is an important player in T cell activation by ensuring sustained TCR signaling, which is required for full effector T cell differentiation, and point to SLP-2 as a potential target for immunomodulation.

Molecular basis of TCR selectivity, cross-reactivity, and allelic discrimination by a bacterial superantigen: integrative functional and energetic mapping of the SpeC-Vbeta2.1 molecular interface.

Superantigens activate large fractions of T cells through unconventional interactions with both TCR beta-chain V domains (Vbetas) and MHC class II molecules. The bacterial superantigen streptococcal pyrogenic exotoxin C (SpeC) primarily stimulates human Vbeta2(+) T cells. Herein, we have analyzed the SpeC-Vbeta2.1 interaction by mutating all SpeC residues that make contact with Vbeta2.1 and have determined the energetic and functional consequences of these mutations. Our comprehensive approach, including mutagenesis, functional readouts from both bulk T cell populations, and an engineered Vbeta2.1(+) Jurkat T cell, as well as surface plasmon resonance binding analysis, has defined the SpeC "functional epitope" for TCR engagement. Although only two SpeC residues (Tyr(15) and Arg(181)) are critical for activation of virtually all human CD3(+) T cells, a larger cluster of four hot spot residues are required for interaction with Vbeta2.1. Three of these residues (Tyr(15), Phe(75), and Arg(181)) concentrate their binding energy on the CDR2 loop residue Ser(52a), a noncanonical residue insertion found only in Vbeta2 and Vbeta4 chains. Plasticity of this loop is important for recognition by SpeC. Although SpeC interacts with the Vbeta2.1 hypervariable CDR3 loop, our data indicate these contacts have little to no influence on the functional interaction with Vbeta2.1. These studies also provide a molecular basis for selectivity and cross-reactivity of SpeC-TCR recognition and reveal a degree of fine specificity in these interactions, whereby certain SpeC mutants are capable of distinguishing between different alleles of the same Vbeta domain subfamily.

Mechanism of modulation of T cell responses by N-palmitoylated peptides.

Small structural changes in the antigenic peptides recognized by TCR can alter the biological properties of those peptides and convert them into weak agonists, partial agonists, or antagonists of these receptors. These altered peptide ligands (APL) are usually generated by conservative amino acid substitutions at TCR contact residues. Here, we show that APL with therapeutic properties can also be generated by attachment of palmitic acid at the N terminus of the peptide without the need to modify the peptide's primary sequence. Using N-palmitoylated pigeon cytochrome-c peptide 81-104 (PALPCC(81-104)), we were able to induce T cell hyporesponsiveness to the wild-type peptide in vitro. More importantly, administration of the PALPCC(81-104 )to mice reduced the responsiveness to the native peptide when tested ex vivo. Biochemical and functional experiments indicated that the action of N-palmitoylated peptides was due to the conversion of the native peptide into a weak agonist that could then induce T cell anergy. Our results demonstrate that N-palmitoylation of antigenic peptides is a feasible strategy to generate APL, as it avoids the need to screen multiple amino acid variants of each specific antigen to identify those with therapeutic properties.

Conversion of CTLA-4 from inhibitor to activator of T cells with a bispecific tandem single-chain Fv ligand.

Abs or their recombinant fragments against surface receptors of the Ig superfamily can induce or block the receptors' native function depending on whether they induce or prevent the assembly of signalosomes on their cytoplasmic tails. In this study, we introduce a novel paradigm based on the observation that a bispecific tandem single-chain variable region fragment ligand of CTLA-4 by itself converts this inhibitory receptor into an activating receptor for primary human T lymphocytes. This reversal of function results from increased recruitment of the serine/threonine phosphatase 2A to the cytoplasmic tail of CTLA-4, consistent with a role of this phosphatase in the regulation of CTLA-4 function, and assembly of a distinct signalosome that activates an lck-dependent signaling cascade and induces IL-2 production. Our data demonstrate that the cytoplasmic domain of CTLA-4 has an inherent plasticity for signaling that can be exploited therapeutically with recombinant ligands for this receptor.

Genomic organization and evolution of the CX3CR1/CCR8 chemokine receptor locus.

The chemokine receptors CCR8 and CX3CR1 are key players in adaptive immunity and are co-receptors for human immunodeficiency virus. We describe here the genomic organization and evolutionary history of both of these genes. CX3CR1 has three promoters that transcribe three separate exons that are spliced with a fourth exon containing the coding region. CCR8 has two promoters. One promoter produces a transcript of two spliced exons, and the other promoter transcribes an exon containing the coding region and lacks introns. We analyzed these promoters in the context of a luciferase reporter and identified several positive and negative regulatory elements. Identification of the genomic organization of these genes in mouse demonstrates a similar organization for CCR8, but mouse CX3CR1 lacks two of the human promoters and has an additional mouse-specific promoter that transcribes only the exon containing the coding region and therefore resembles the organization of the human and mouse CCR8 genes. We also identify two nontranscribed regions that are highly conserved between human and mouse CX3CR1 containing possible regulatory elements. Examination of the CX3CR1 and CCR8 genes and surrounding genomic regions indicates that these genes are the result of the duplication of an ancestral gene prior to the divergence of teleost fish. We characterize single nucleotide polymorphisms in the promoters of human CCR8 and CX3CR1 and establish linkage relationships between CX3CR1 promoter polymorphisms and two previously described CX3CR1 coding polymorphisms associated with human immunodeficiency virus disease progression and arteriosclerosis susceptibility.