Rapid Collection of Human Rectal Secretions Using OriCol Devices Is Suitable for Measurement of Mucosal Ig without Blood Contamination.

Measurements of IgG and IgA in human rectal secretions are used to evaluate the Abs elicited by HIV vaccines or the bioaccumulation following immunoprophylaxis at the sites of HIV exposure. To improve sampling methods and tolerability of the procedure, we optimized a balloon device (OriCol) for rectal microbiome sampling requiring 10 second inflation and compared this method to a 5 minute collection using sponges. Lubrication of the device did not interfere with IgG, IgA, or hemoglobin ELISA. Lubricated OriCols inflated to 30 cc minimized hemoglobin contamination (<4.68 ng/ml) compared with collections with two sponge types (Weck-Cel: 267.2 ng/ml, p < 0.0001; and Merocel: 59.38 ng/ml, p = 0.003). Median human serum albumin for OriCols was 14.9 µg/ml, whereas Merocels and Weck-Cels were 28.57 µg/ml (p = 0.0005) and 106.2 µg/ml (p = 0.0002), respectively. Consistent with reduced systemic contamination, the median IgG measured in OriCol-collected rectal secretions (986 ng) was lower than secretions from sponges (Weck-Cel: 8588 ng, p < 0.0001; Merocel: 2509 ng, p = 0.0389). The median IgA yield of samples using the OriCol method (75,253 ng) was comparable to that using Merocel (71,672 ng; p = 0.6942) but significantly higher than Weck-Cel sponges (16,173 ng, p = 0.0336). Median recovery volumes for OriCols were 800 µl, whereas Merocels and Weck-Cels were 615 µl (p = 0.0010) and 655 µl (p = 0.0113), respectively. The balloon device was acceptable among 23 participants, as 85.1% experiencing their first collection ranked it as "seven: acceptable - a lot" or "six: acceptable - somewhat" in a seven-point Likert scale. Therefore, lubricated OriCols inflated to 30 cc allowed for a rapid, well-tolerated, blood-free collection of human rectal secretions.

Neutralization Takes Precedence Over IgG or IgA Isotype-related Functions in Mucosal HIV-1 Antibody-mediated Protection.

HIV-1 infection occurs primarily through mucosal transmission. Application of biologically relevant mucosal models can advance understanding of the functional properties of antibodies that mediate HIV protection, thereby guiding antibody-based vaccine development. Here, we employed a human ex vivo vaginal HIV-1 infection model and a rhesus macaque in vivo intrarectal SHIV challenge model to probe the protective capacity of monoclonal broadly-neutralizing (bnAb) and non-neutralizing Abs (nnAbs) that were functionally modified by isotype switching. For human vaginal explants, we developed a replication-competent, secreted NanoLuc reporter virus system and showed that CD4 binding site bnAbs b12 IgG1 and CH31 IgG1 and IgA2 isoforms potently blocked HIV-1JR-CSF and HIV-1Bal26 infection. However, IgG1 and IgA nnAbs, either alone or together, did not inhibit infection despite the presence of FcR-expressing effector cells in the tissue. In macaques, the CH31 IgG1 and IgA2 isoforms infused before high-dose SHIV challenge were completely to partially protective, respectively, while nnAbs (CH54 IgG1 and CH38 mIgA2) were non-protective. Importantly, in both mucosal models IgG1 isotype bnAbs were more protective than the IgA2 isotypes, attributable in part to greater neutralization activity of the IgG1 variants. These findings underscore the importance of potent bnAb induction as a primary goal of HIV-1 vaccine development.

Development of a Minor Histocompatibility Antigen Vaccine Regimen in the Canine Model of Hematopoietic Cell Transplantation.

BACKGROUND: Minor histocompatibility antigen (miHA) vaccines have the potential to augment graft-versus-tumor effects without graft-versus-host disease (GVHD). We used mixed hematopoietic chimerism in the canine model of major histocompatibility complex-matched allogeneic hematopoietic cell transplantation as a platform to develop a miHA vaccination regimen. METHODS: We engineered DNA plasmids and replication-deficient human adenovirus type 5 constructs encoding large sections of canine SMCY and the entire canine SRY gene. RESULTS: Priming with replication-deficient human adenovirus type 5 constructs and boosting with ex vivo plasmid-transfected dendritic cells and cutaneous delivery of plasmids with a particle-mediated epidermal delivery device (PMED) in 2 female dogs induced antigen-specific T-cell responses. Similar responses were observed after a prime-boost vaccine regimen in three female hematopoietic cell transplantation donors. Subsequent donor lymphocyte infusion resulted in a significant change of chimerism in 1 of 3 male recipients without any signs of graft-versus-host disease. The change in chimerism in the recipient occurred in association with the development of CD4+ and CD8+ T-cell responses to the same peptide pools detected in the donor. CONCLUSIONS: These studies describe the first in vivo response to miHA vaccination in a large, outbred animal model without using recipient cells to sensitize the donor. This model provides a platform for ongoing experiments designed to define optimal miHA targets and develop protocols to directly vaccinate the recipient.

Protease-activated receptor 2 signaling upregulates angiogenic growth factors in renal cell carcinoma.

Renal cell carcinoma (RCC) is a highly vascular tumor associated with expression of various angiogenic growth factors. The precise process of how these growth factors are regulated in RCC is not fully understood. Recent evidence suggests that protease activated receptors (PARs), a new family of G-protein coupled receptors, play a crucial role in vascular development and tumor progression through a variety of mechanisms. However, the nature of PAR expression in human RCC tissues and its function in regulating angiogenesis in RCC are largely unknown. In this study, we investigated the expression and function of PAR-2 in RCC. RT-PCR and immunohistochemistry assays show that PAR-2 expression is significantly increased in human RCC tissue compared with the adjacent non-neoplastic kidney tissue. In RCC derived cells, PAR-2 is functional as evidenced by robust signaling through MAP kinases including ERK1/2 and JNK. Furthermore, activation of PAR-2 significantly upregulates several angiogenic cytokines, including interleukin-6 (IL-6), IL-8, monocytes chemotactic protein-1 (MCP-1) and growth-related oncogene (GRO). To our knowledge, this is the first report that characterized PAR-2 expression in RCC tissue and further demonstrated that PAR-2 has a critical role in regulating angiogenesis in RCC.

A novel HIV vaccine adjuvanted by IC31 induces robust and persistent humoral and cellular immunity.

The HIV vaccine strategy that, to date, generated immune protection consisted of a prime-boost regimen using a canarypox vector and an HIV envelope protein with alum, as shown in the RV144 trial. Since the efficacy was weak, and previous HIV vaccine trials designed to generate antibody responses failed, we hypothesized that generation of T cell responses would result in improved protection. Thus, we tested the immunogenicity of a similar envelope-based vaccine using a mouse model, with two modifications: a clade C CN54gp140 HIV envelope protein was adjuvanted by the TLR9 agonist IC31®, and the viral vector was the vaccinia strain NYVAC-CN54 expressing HIV envelope gp120. The use of IC31® facilitated immunoglobulin isotype switching, leading to the production of Env-specific IgG2a, as compared to protein with alum alone. Boosting with NYVAC-CN54 resulted in the generation of more robust Th1 T cell responses. Moreover, gp140 prime with IC31® and alum followed by NYVAC-CN54 boost resulted in the formation and persistence of central and effector memory populations in the spleen and an effector memory population in the gut. Our data suggest that this regimen is promising and could improve the protection rate by eliciting strong and long-lasting humoral and cellular immune responses.

Kallikrein-related peptidase-4 initiates tumor-stroma interactions in prostate cancer through protease-activated receptor-1.

In prostate cancer, the mechanism by which the stromal cells surrounding the cancer epithelium become reactive and overproduce growth factors is unclear. Furthermore, the precise process of how these stromal cells stimulate the cancer epithelium is not fully understood. We recently found that protease-activated receptor-1 (PAR-1) in these reactive stromal cells is upregulated. To investigate the role of PAR-1 in the stromal-epithelial interaction, WPMY-1 stromal myofibroblasts were stimulated with PAR-1 agonists including thrombin and PAR-1 activating peptide. We show that WPMY-1 cells have functional PAR-1 by signaling through ERK1/2. Conditioned media (CM) from PAR-1 agonists-treated WPMY-1 cells stimulate the epithelial LNCaP cells leading to ERK1/2 activation and cell proliferation. Cytokine array analysis of the CM demonstrates that PAR-1 induces stromal cells to release numerous cytokines, of which interleukin 6 (IL-6) is the major factor responsible for mitogenic signaling in LNCaP cells. CM further induces expression of prostate-specific kallikrein-related peptidase-3 (KLK3/PSA) and KLK4 in LNCaP cells via the IL-6 pathway. Moreover, KLK4 functions as a potent agonist of PAR-1 by cleaving the receptor at the proper site on cell surface. KLK4 triggers transmembrane signaling and upregulates IL-6 in WPMY-1 cells through PAR-1. Immunohistochemical analysis indicates that PAR-1 is predominantly expressed in peritumoral stroma while KLK4 is produced exclusively by the epithelial cancer cells. These data provide evidence for a novel double-paracrine mechanism whereby cancer epithelium produces KLK4 to activate PAR-1 in the surrounding stroma, which in-turn releases cytokines (IL-6) that stimulate cancer cells to proliferate and increase production of KLKs.

Prostate-specific kallikreins-2 and -4 enhance the proliferation of DU-145 prostate cancer cells through protease-activated receptors-1 and -2.

A major characteristic of prostate cancer is the elevation of serum levels of prostate-specific antigen (hK3) and hK2, which are tumor markers that correlate with advancing stages of disease. Including hK4, these three kallikrein serine proteases are almost exclusively produced by the prostate. Prostate cancer cells have been recently shown to overexpress protease-activated receptors (PAR), which can be potentially activated by kallikreins and can regulate tumor growth. Here, we show that recombinant hK2 and hK4 activate ERK1/2 signaling of DU-145, PC-3, and LNCaP prostate cancer cells, which express both PAR1 and PAR2. These kallikreins also stimulate the proliferation of DU-145 cells. Pretreatment of hK2 and hK4 with the serine protease inhibitor, aprotinin, blocks the responses in DU-145 cells, and small interfering RNA against PAR1 and PAR2 also inhibits ERK1/2 signaling. To determine which PAR is activated by hK2 and hK4, a cell line that expresses a single PAR, a PAR1 knockout mouse lung fibroblast cell line transfected with PAR1 (KOLF-PAR1) or PAR2 (KOLF-PAR2) was used. hK4 activates both PAR1 and PAR2, whereas hK2 activates PAR2. hK4 generates more phosphorylated ERK1/2 than hK2. These data indicate that prostatic kallikreins (hK2 and hK4) directly stimulate prostate cancer cell proliferation through PAR1 and/or PAR2 and may be potentially important targets for future drug therapy for prostate cancer.

Protease-activated receptor-1 upregulates fibroblast growth factor 7 in stroma of benign prostatic hyperplasia.

BACKGROUND: Benign prostatic hyperplasia (BPH) is characterized by abnormal epithelial and stromal proliferation causing urinary obstruction. Prostate growth is regulated by a variety of growth factors secreted from the stroma, including fibroblast growth factor 7 (FGF-7), a potent epithelial-specific growth factor which is increased in hyperplastic prostate. However, the mediator(s) of FGF-7 over-expression is unclear. Protease-activated receptor-1 (PAR-1) is a G-protein coupled receptor known to induce multiple biological processes, but its effect on BPH pathogenesis is mostly unknown. The aim of this study was to investigate the role of PAR-1 as a mediator of BPH development. METHODS: PAR-1 expression was investigated in BPH and normal prostate tissues by immunohistochemistry. Prostate stromal cells were isolated from BPH specimens, cultured and immunohistochemically characterized. Cultured stromal cells were stimulated with PAR-1 agonists, and extracellular-signal regulated kinase (ERK1/2) activation and cell proliferation were examined. PAR-1 mediated FGF-7 production by cultured stromal cells was assessed by RT-PCR and immunoassays, and verified by small interfering RNA (siRNA). RESULTS: PAR-1 expression was increased in BPH stroma. In stromal cells isolated from BPH tissues, PAR-1 agonists activated ERK1/2 in a time- and concentration-dependent manner and with resultant enhanced cell proliferation. Pertussis toxin-sensitive G protein/(betagamma-subunits)-phosphatidylinositol 3-kinase and protein kinase C pathways were involved in ERK1/2 phosphorylation. PAR-1 activation strikingly induced FGF-7 production from cultured stromal cells mediated predominantly via ERK1/2 signaling pathway, and PAR-1 siRNA decreased the elicited FGF-7 upregulation. CONCLUSIONS: The expression and function of PAR-1 in BPH stroma indicate PAR-1 may play important roles in BPH pathogenesis.

Regulated expression of active biotinylated G-protein coupled receptors in mammalian cells.

We have developed a mammalian expression system suitable for the production of enzymatically biotinylated integral membrane proteins. The key feature of this system is the doxycycline (dox)-regulated co-expression of a secreted variant of Escherichia coli biotin ligase (BirA) and a target protein with a 13-residue biotin acceptor peptide (BioTag) appended to its extracellular domain. Here we describe the expression and functional analysis of three G-protein coupled receptors (GPCRs): protease-activated receptors (PARs) 1 and 2, and the platelet ADP receptor, P2Y(12). Clonal Chinese hamster ovary (CHO) Tet-On cell lines that express biotinylated GPCRs were rapidly isolated by fluorescence-activated cell sorting following streptavidin-FITC staining, thereby circumventing the need for manual colony picking. Analysis by Western blotting with streptavidin-HRP following endoglycosidase treatment revealed that all three GPCRs undergo N-linked glycosylation. The expression of biotinylated GPCRs on the cell surface was regulated by the concentration of dox in the medium, reaching a maximum at approximately 1 microg/mL dox. Similarly, the extent of GPCR biotinylation was dependent on biotin concentration, with maximum and complete biotinylation achieved upon supplementation with 50 microM biotin. Biotinylated PAR1 and PAR2 were readily and specifically cleaved on the surface of intact cells by their cognate proteases, and were capable of transducing extracellular stimuli, resulting in the downstream phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, P2Y(12) mediated agonist-induced ERK phosphorylation only when it was expressed at low levels on the cell surface, highlighting the utility of regulated expression for the production of functionally active GPCRs in mammalian cells.

Overexpression of protease-activated receptors-1,-2, and-4 (PAR-1, -2, and -4) in prostate cancer.

BACKGROUND: Although protease-activated receptors (PARs) have been described to play a role in different malignancies, their expression and biological activity in prostate cancer are mostly unknown. METHODS: PAR expression in radical prostatectomy specimens was investigated by immunohistochemistry (IHC, 40 patients) and RT-PCR. Their role in LNCaP prostate cancer cell migration and Rac1/Cdc42 signaling was assessed with Boyden chamber analysis and Western blot, respectively. RESULTS: PAR mRNA expression was higher in cancer, and protein expression was increased in PAR-1 (45%), PAR-2 (42%), and PAR-4 (68%), compared to normal glands. Increased PAR-1 (periglandular stroma) was associated with higher rates of biochemical recurrence (median follow-up, 5 years; P = 0.006). LNCaP migration was enhanced twofold and Rac1/Cdc42 signaling was activated by stimulation of PAR-1 and PAR-2. CONCLUSIONS: PARs are overexpressed in prostate cancer and may serve as potential predictors of recurrence. The data suggest potential role of PARs in autocrine and paracrine mechanisms of prostate cancer.

The two upstream open reading frames of oncogene mdm2 have different translational regulatory properties.

Few details are known of the mechanisms through which multiple upstream open reading frames (uORFs) interact to regulate translation in higher eukaryotes. The predominant transcript of oncogene mdm2 in normal human cells (L-mdm2) contains two upstream open reading frames in its 5' leader. Elimination of these two uORFs raises the translational efficiency of the transcript by over 10-fold in HeLa cells. The 5'-most uORF (uORF1) alone suppresses downstream translational activity by over 5-fold, whereas uORF2 contributes <2-fold to the inhibition by the intact leader. The different activities of the two uORFs do not depend on the nucleotide sequence surrounding the uORFs in the 5' leader, the order of the two uORFs in the 5' leader, or the occurrence of secondary structure or rare codons within the uORFs. Specific features of the amino acid sequence encoded by uORF1 contribute to its stronger suppressive activity, suggesting that it belongs to the class of "sequence-specific" uORFs. The weaker inhibitory activity inherent in uORF2 is potentiated by a sub-optimal nucleotide context surrounding its initiator AUG. The occurrence of two uORFs with differing activities in both the human gene and the mouse orthologue suggests that this pair of elements may play a fundamental role in regulating expression of the mdm2 gene.

Protease-activated receptor mediated RhoA signaling and cytoskeletal reorganization in LNCaP cells.

Thrombin and trypsin induce cell signaling through a subclass of G-protein-coupled receptors called the protease-activated receptors (PARs). In many cells, PAR signaling results in the activation of RhoA and other members of the Rho family of small GTPases which are involved in cytoskeletal reorganization. The expression of PARs and their role in the activation of Rho GTPases in prostate cancer cells are not clearly known. FACS analysis demonstrated that the androgen-dependent LNCaP cells express PAR1, PAR2, and PAR4 but not PAR3. Stimulation with thrombin and trypsin resulted in the rapid activation of RhoA in a dose-dependent manner with an EC(50) of 1.0 and 5 nM, respectively. Activation of RhoA was enhanced by, but not dependent on, the presence of 1 nM dihydrotestosterone. Inhibition of the proteolytic properties of thrombin by hirudin and trypsin by diisopropyl fluorophosphate abolished the observed RhoA activation. Stimulation with 150 microM PAR-activating peptides TFFLRN (PAR1), SLIGKV (PAR2), and AYPGKF (PAR4) demonstrated that PAR1 and PAR2 mediated protease-activated RhoA signaling. Fluorescent microscopy studies showed that LNCaP cells treated with either thrombin (10 nM) or trypsin (10 nM) developed an increased number of filopodia, stress fibers, and focal adhesions relative to untreated cells. These observations represent the first report of PAR signaling in prostate cancer cells as well as the ability of PAR2 to mediate RhoA activation. Since the activation of RhoA is important for cytoskeletal reorganization, we postulate that PAR-mediated RhoA activation may be a major signaling pathway in the biology of prostate cancer.

Regulated translation termination at the upstream open reading frame in s-adenosylmethionine decarboxylase mRNA.

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a cis-acting element that confers feedback control by cellular polyamines on translation of this message. Recent studies demonstrated that elevated polyamines inhibit synthesis of the peptide encoded by the uORF by stabilizing a ribosome paused in the vicinity of the termination codon. These studies suggested that polyamines act at the termination step of uORF translation. In this paper, we demonstrate that elevated polyamines stabilize an intermediate in the termination process, the complete nascent peptide linked to the tRNA that decodes the final codon. The peptidyl-tRNA molecule is found associated with the ribosome fraction, and decay of this molecule correlated with release of the paused ribosome from the message. Furthermore, the stability of this complex is influenced by the same parameters that influence regulation by the uORF in vivo, namely the concentration of polyamines and the sequence of the uORF-encoded peptide. These results suggest that the regulated step in uORF translation is after formation of the peptidyl-tRNA molecule but before hydrolysis of the peptidyl-tRNA bond. This regulation may involve an interaction between the peptide, polyamines, and a target in the translational apparatus.