Once daily 300 mg aspirin with compression versus compression alone in patients with chronic venous leg ulcers (ASPiVLU): A randomised, double-blinded, multicentre, placebo-controlled, clinical trial.

AIM: Venous leg ulcers are lower limb skin ulcers characterised by a cycle of healing and recurrence due to underlying chronic venous insufficiency. While compression improves healing outcomes, many ulcers do not heal. As a daily 300 mg oral dose of aspirin in conjunction with compression may improve healing outcomes, we investigated the effect of adjuvant aspirin on venous leg ulcer healing in participants already receiving compression. MATERIALS AND METHODS: We conducted a prospective, randomised, double-blinded, placebo-controlled, clinical trial (known as ASPiVLU). Participants were recruited from six wound clinics in Australia. We screened 844 participants. Community-dwelling adult participants identified at six hospital outpatient clinics and clinically diagnosed with a venous leg ulcer present for 6+ weeks were eligible between April 13, 2015 to June 30, 2018. We randomised 40 participants (n = 19 aspirin, n = 21 placebo) and evaluated against the primary outcome. There were no dropouts. Ten serious adverse events in six participants were recorded. None were study related. The primary outcome measure was healing at 12 weeks based on blinded assessment. RESULTS: We found no difference in the number of ulcers healed at 12 weeks between the intervention and control groups. CONCLUSION: This study could not detect whether or not aspirin affected VLU healing speed. This is likely because we recruited fewer participants than expected due to the high number of people with venous leg ulcers in Australia who were already taking Aspirin; future research should investigate other adjuvant therapies or different study designs.

Expression of the plasminogen system in the physiological mouse ovary and in the pathological polycystic ovary syndrome (PCOS) state.

BACKGROUND: The fibrinolytic system and its inhibitors play a number of roles, apart from their function in blood haemostasis and thrombosis, namely in ovarian folliculogenesis and in ovulation. Plasminogen is converted to active plasmin at the time of follicular rupture through a decrease in plasminogen activator inhibitor-1 (PAI-1) and an increase in plasminogen activators. Oligo-/anovulation and follicle arrest are key characteristics of PCOS, but studies evaluating fibrinolytic/proteolytic markers within human or animal PCOS ovaries are lacking. We aimed to investigate and compare the expression and distribution of the plasminogen system markers in PCOS and control ovaries. METHODS: A hyperandrogenised PCOS mouse model was used that mimics the ovarian, endocrine and metabolic features of the human condition. Immunohistochemistry and digital image analysis were used to investigate and compare fibrinolytic/proteolytic markers plasminogen, plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator and inhibitor PAI-1 in PCOS and control ovaries. Student's t-test was used to compare data sets for normally distributed data and Wilcoxon-Mann Whitney test for non-normally distributed data. RESULTS: We noted differences in the ovarian distribution of PAI-1 that was expressed throughout the PCOS ovary, unlike the peripheral distribution observed in control ovaries. Plasminogen was present in small follicles only in PCOS ovaries but not in small follicles of control ovaries. When we assessed and compared PAI-1 expression within follicles of different developmental stages we also noted significant differences for both the PCOS and control ovaries. While we noted differences in distribution and expression within specific ovarian structures, no differences were noted in the overall ovarian expression of markers assessed between acyclical PCOS mice and control mice at the diestrus stage of the estrous cycle. CONCLUSIONS: Our novel study, that comprehensively assessed the fibrinolytic/proteolytic system in the mouse ovary, showed the expression, differential localisation and a potential role for the plasminogen system in the physiological mouse ovary and in PCOS. Androgens may be involved in regulating expression of the ovarian plasminogen system. Further studies evaluating these markers at different time-points of ovulation may help to further clarify both physiological and potential pathological actions these markers play in ovulatory processes distorted in PCOS.

Combined Effects of Gold Nanoparticles and Ionizing Radiation on Human Prostate and Lung Cancer Cell Migration.

The effect of 15 nm-sized gold nanoparticles (AuNPs) and/or ionizing radiation (IR) on the migration and adhesion of human prostate (DU145) and lung (A549) cancer cell lines was investigated. Cell migration was measured by observing the closing of a gap created by a pipette tip on cell monolayers grown in 6-well plates. The ratio of the gap areas at 0 h and 24 h were used to calculate the relative migration. The relative migration of cells irradiated with 5 Gy was found to be 89% and 86% for DU145 and A549 cells respectively. When the cells were treated with 1 mM AuNPs this fell to ~75% for both cell lines. However, when the cells were treated with both AuNPs and IR an additive effect was seen, as the relative migration rate fell to ~60%. Of interest was that when the cells were exposed to either 2 or 5 Gy IR, their ability to adhere to the surface of a polystyrene culture plate was significantly enhanced, unlike that seen for AuNPs. The delays in gap filling (cell migration) in cells treated with IR and/or AuNPs can be attributed to cellular changes which also may have altered cell motility. In addition, changes in the cytoskeleton of the cancer cells may have also affected adhesiveness and thus the cancer cell's motility response to IR.

Immunogenicity of a Tripartite Cell Penetrating Peptide Containing a MUC1 Variable Number of Tandem Repeat (VNTR) and A T Helper Epitope.

Peptide-based vaccines for cancer have many advantages however, for optimization these immunogens should incorporate peptide epitopes that induce CD8, as well as CD4 responses, antibody and long term immunity. Cell penetrating peptides (CPP) with a capacity of cytosolic delivery have been used to deliver antigenic peptides and proteins to antigen presenting cells to induce cytotoxic T cell, helper T cell and humoral responses in mice. For this study, a tripartite CPP including a mucin 1 (MUC1) variable number of tandem repeat (VNTR) containing multiple T cell epitopes and tetanus toxoid universal T helper epitope peptide (tetCD4) was synthesised (AntpMAPMUC1tet) and immune responses investigated in mice. Mice vaccinated with AntpMAPMUC1tet + CpG show enhanced antigen-specific interferon-gamma (IFN-γ) and IL-4 T cell responses compared with AntpMAPMUC1tet vaccination alone and induced a Th1 response, characterised by a higher ratio of IgG2a antibody/IgG1 antibodies. Furthermore, vaccination generated long term MUC1-specific antibody and T cell responses and delayed growth of MUC1+ve tumours in mice. This data demonstrates the efficient delivery of branched multiple antigen peptides incorporating CPP and that the addition of CpG augments immune responses.

Greater endurance capacity and improved dyspnoea with acute oxygen supplementation in idiopathic pulmonary fibrosis patients without resting hypoxaemia.

BACKGROUND AND OBJECTIVE: Supplemental oxygen is commonly prescribed in patients with idiopathic pulmonary fibrosis (IPF), although its benefits have not been proven. The aims of this study were to investigate the effect of oxygen on oxidative stress, cytokine production, skeletal muscle metabolism and physiological response to exercise in IPF. METHODS: Eleven participants with IPF received either oxygen, at an FiO2 of 0.50, or compressed air for 1 h at rest and during a cycle endurance test at 85% of peak work rate. Blood samples collected at rest and during exercise were analysed for markers of oxidative stress, skeletal muscle metabolism and cytokines. The protocol was repeated a week later with the alternate intervention. RESULTS: Compared with air, oxygen did not adversely affect biomarker concentrations at rest and significantly improved endurance time (mean difference = 99 ± 81s, P = 0.002), dyspnoea (-1 ± 1 U, P = 0.02), systolic blood pressure (BP; -11 ± 11 mm Hg, P = 0.006), nadir oxyhaemoglobin saturation (SpO2 ; 8 ± 6%, P = 0.001), SpO2 at 2-min (7 ± 6%, P = 0.003) and 5-min isotimes (5 ± 3, P < 0.001) and peak exercise xanthine concentrations (-42 ± 73 µmol/L, P = 0.03). Air significantly increased IL-10 (5 ± 5 pg/mL, P = 0.04) at 2-min isotime. Thiobarbituric acid-reactive substances (TBARs), IL-6, TNF-α, creatine kinase, lactate, heart rate and fatigue did not differ between the two interventions at any time point. CONCLUSION: In patients with IPF, breathing oxygen at FiO2 of 0.50 at rest seems safe. During exercise, oxygen improves exercise tolerance, alleviates exercise-induced hypoxaemia and reduces dyspnoea. A potential relationship between oxygen administration and improved skeletal muscle metabolism should be explored in future studies.

Comparative Immunogenicity of a Cytotoxic T Cell Epitope Delivered by Penetratin and TAT Cell Penetrating Peptides.

Cell penetrating peptides (CPP), including the TAT peptide from the human immunodeficiency virus transactivator of transcription (HIV-TAT) protein and penetratin from Drosophila Antennapedia homeodomain protein, translocate various cargos including peptides and proteins across cellular barriers. This mode of delivery has been harnessed by our group and others to deliver antigenic proteins or peptides into the cytoplasm of antigen processing cells (APC) such as monocyte-derived dendritic cells (MoDC). Antigens or T cell epitopes delivered by CPP into APC in vivo generate antigen-specific cytotoxic T cell and helper T cell responses in mice. Furthermore, mice immunised with these peptides or proteins are protected from a tumour challenge. The functional properties of CPP are dependent on the various cargos being delivered and the target cell type. Despite several studies demonstrating superior immunogenicity of TAT and Antp-based immunogens, none has compared the immunogenicity of antigens delivered by TAT and Antp CPP. In the current study we demonstrate that a cytotoxic T cell epitope from the mucin 1 (MUC1) tumour associated antigen, when delivered by TAT or Antp, generates identical immune responses in mice resulting in specific MUC1 T cell responses as measured by in vivo CTL assays, IFNγ ELISpot assays and prophylactic tumour protection.

Cell density impacts on Candida glabrata survival in hypo-osmotic stress.

Candida glabrata cells suspended in water are under hypo-osmotic stress and undergo cell death in 1-2 days, unless they are at a density of more than 10(5 ) CFU mL(-1) . The dying cells exhibit FITC-annexin V staining, indicative of programmed cell death (apoptosis). In a higher cell density, cells are protected and survive at least for 4 days. Filtrates from cells at high density can protect those at lower density, indicating that cells release substances, amounting to c. 5 mg L(-1) of cell suspension, that protect each other against hypo-osmotic stress. In a concentrated form, the released materials can support growth, indicating that the protective material includes carbon and nitrogen sources, as well as vitamins that are required by C. glabrata for growth. We conclude that cell death from hypo-osmotic stress can be alleviated by small amounts of nutrients.

Siah2-deficient mice show impaired skin wound repair.

Hypoxia is associated with the dermal wound healing process and hypoxia signaling is presumed to be crucial for normal wound repair. The Siah2 ubiquitin ligase controls the abundance of hypoxia-inducible factor-1 alpha, and loss of Siah2 results in destabilization of hypoxia-inducible factor-1 alpha under hypoxia. Utilizing Siah2(-/-) mice we demonstrate that cutaneous wound healing is impaired in these mice. Wounds in Siah2(-/-) mice heal slower and are associated with delayed induction of myofibroblast infiltration and reduced collagen deposition. This coincides with delayed angiogenesis and reduced macrophage infiltration into the wounds of Siah2(-/-) mice. We furthermore demonstrate that primary Siah2(-/-) dermal fibroblasts have reduced migratory capacities and produce less collagen than wild-type fibroblasts. Additionally, Siah2(-/-) fibroblasts showed conserved responses to transforming growth factor-ß at the receptor level (pSmad 2C activation) but reduced responses downstream. Together, our data show, for the first time, that Siah2 is involved as a positive regulator in the wound healing response. Understanding the role of hypoxia signaling in tissue repair and fibrosis and interference with the hypoxia signaling pathway via regulation of Siah2 may provide new targets for clinical regulation of fibrosis and scarring.

Characterization of blood monocyte phenotype in patients with endometrial cancer.

BACKGROUND: Endometrial tumors induce various tumor escape mechanisms that result in immunosuppression in patients and, ultimately, tumor progression. Blood monocytes are able to exhibit potent cytotoxic action against tumor cells where novel immunotherapeutics targeting antigen-presenting cells including dendritic cells, and blood monocytes are being used as a means of delivering immunogens to stimulate an antitumor and, ultimately, therapeutic response. This study shows that peripheral blood monocytes from patients with endometrial cancer show functional deficiencies, and these deficiencies can be characterized by phenotypic changes as well as altered cytokine secretion. METHODS: This study assessed the phenotypic changes of peripheral blood monocytes by flow cytometry as well as the functional status via cytokine production measured by enzyme-linked immunosorbent assay in patients with endometrial cancer versus controls. RESULTS: Altered blood monocyte phenotype incorporating a decrease in costimulatory and adhesion factor expression and increased expression of vascular endothelial growth factor receptor 1 in patients with endometrial cancer versus controls. Increased interleukin 12 and decreased interleukin 10 secretion by blood monocytes in patients with endometrial cancer were also observed. CONCLUSIONS: These findings showed that peripheral blood monocytes from patients with endometrial cancer show an altered phenotype and cytokine secretion when compared with controls. Limitations to this study include the small sample size, the need to investigate the effect of phenotype and cytokine changes in functional assays, as well as future studies investigating the effect on tumor-associated macrophages from endometrial tissue from cancer versus control patients. Nevertheless, these findings suggest that peripheral blood monocyte induced immunosuppression in endometrial cancer and implications in the design of future immunotargeting therapies remain to be elucidated.

Self-Assembled Peptide Habitats to Model Tumor Metastasis.

Metastatic tumours are complex ecosystems; a community of multiple cell types, including cancerous cells, fibroblasts, and immune cells that exist within a supportive and specific microenvironment. The interplay of these cells, together with tissue specific chemical, structural and temporal signals within a three-dimensional (3D) habitat, direct tumour cell behavior, a subtlety that can be easily lost in 2D tissue culture. Here, we investigate a significantly improved tool, consisting of a novel matrix of functionally programmed peptide sequences, self-assembled into a scaffold to enable the growth and the migration of multicellular lung tumour spheroids, as proof-of-concept. This 3D functional model aims to mimic the biological, chemical, and contextual cues of an in vivo tumor more closely than a typically used, unstructured hydrogel, allowing spatial and temporal activity modelling. This approach shows promise as a cancer model, enhancing current understandings of how tumours progress and spread over time within their microenvironment.

A membrane penetrating multiple antigen peptide (MAP) incorporating ovalbumin CD8 epitope induces potent immune responses in mice.

Cell penetrating peptides (CPP) represent a novel approach to facilitate cytoplasmic delivery of macromolecules. The DNA binding domain of Drosophila Antennapedia contains 60 amino acids and consists of 3 α-helices, with internalizing activity mapped to a 16-amino acid peptide penetratin (Antp) within the third α-helix. Here, we report on the use of penetratin to deliver a multiple antigen peptide (MAP) incorporating the immunodominant CD8 epitope of ovalbumin, SIINFEKL (MAPOVACD8). We demonstrate that penetratin linked to the MAPOVACD8 construct either by a disulfide (SS) or thioether (SC) linkage promotes the uptake, cross presentation and subsequent in vivo proliferation and generation of OVACD8 (SIINFEKL)-specific T cells. The MAPOVACD8 construct without penetratin is not presented by MHC class I molecules nor does it generate an in vivo IFN-γ response in C57BL/6 mice. Moreover, we clearly define the uptake and intracellular processing pathways of AntpMAPOVACD8 SS and SC revealing the majority of AntpMAPOVACD8 is taken up by DC via an endocytic, proteasome and tapasin independent mechanism. We also show that the uptake mechanism of AntpMAPOVACD8 is dose dependent and uptake or intracellular processing is not altered by the type of chemical linkage.

Cell-penetrating peptides: application in vaccine delivery.

Recent years have seen a surge in interest in cell-penetrating peptides (CPP) as an efficient means for delivering therapeutic targets into cellular compartments. The cell membrane is impermeable to hydrophilic substances yet linking to CPP can facilitate delivery into cells. Thus the unique translocatory property of CPP ensures they remain an attractive carrier, with the capacity to deliver cargoes in an efficient manner having applications in drug delivery, gene transfer and DNA vaccination. Fundamental for an effective vaccine is the delivery of antigen epitopes to antigen-presenting cells, ensuing processing and presentation and induction of an immune response. Vaccination with proteins or synthetic peptides incorporating CTL epitopes have proven limited due to the failure for exogenous antigens to be presented efficiently to T cells. Linking of antigens to CPP overcomes such obstacles by facilitating cellular uptake, processing and presentation of exogenous antigen for the induction of potent immune responses. This review will encompass the various strategies for the delivery of whole proteins, T cell epitopes and preclinical studies utilizing CPP for cancer vaccines.

Whole protein and defined CD8(+) and CD4(+) peptides linked to penetratin targets both MHC class I and II antigen presentation pathways.

Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4(+) and/or CD8(+) T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8(+) or CD4(+) T-cell responses in vitro. Furthermore, interferon (IFN)-γ-secreting CD4(+) and CD8(+) T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8(+) T-cell-dependent mechanism and did not require CD4(+) help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4(+) help enhanced antigen-specific IFN-γ-secreting CD8(+) T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model.

Explanting is an ex vivo model of renal epithelial-mesenchymal transition.

Recognised by their de novo expression of alpha-smooth muscle actin (SMA), recruitment of myofibroblasts is key to the pathogenesis of fibrosis in chronic kidney disease. Increasingly, we realise that epithelial-mesenchymal transition (EMT) may be an important source of these cells. In this study we describe a novel model of renal EMT. Rat kidney explants were finely diced on gelatin-coated Petri dishes and cultured in serum-supplemented media. Morphology and immunocytochemistry were used to identify mesenchymal (vimentin+, α-smooth muscle actin (SMA)+, desmin+), epithelial (cytokeratin+), and endothelial (RECA+) cells at various time points. Cell outgrowths were all epithelial in origin (cytokeratin+) at day 3. By day 10, 50 ± 12% (mean ± SE) of cytokeratin+ cells double-labelled for SMA, indicating EMT. Lectin staining established a proximal tubule origin. By day 17, cultures consisted only of myofibroblasts (SMA+/cytokeratin-). Explanting is a reproducible ex vivo model of EMT. The ability to modify this change in phenotype provides a useful tool to study the regulation and mechanisms of renal tubulointerstitial fibrosis.

The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells.

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24(high) (conventional CD8(+) equivalent) and CD11b(high) (CD8(-) equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA(+) plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-beta, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.

Immunomodulation in endometrial cancer.

Endometrial cancer is the most frequent gynecological cancer and the fourth most common cancer in women in the developed world. Over the last decade, immunotherapy has been the focus of intense investigation as a form of cancer treatment whereby the treatment initiates a host immune response ultimately eradicating the tumor. It has been suggested that in endometrial cancer and many other forms of cancer, immunosuppression poses a significant obstacle at inducing antitumor immunity by immunotherapy. This review will look at the different studies that have identified immunomodulation of T cells, cytokines and macrophages, and regulation of apoptotic and angiogenic factors in endometrial cancer patients that may contribute to the inefficiency of immunotherapy.

A Synthetic Nanoparticle Based Vaccine Approach Targeting MSP4/5 Is Immunogenic and Induces Moderate Protection Against Murine Blood-Stage Malaria.

Malaria remains a significant health problem in many tropical and sub-tropical regions. The development of vaccines against the clinically active blood-stage of infection needs to consider variability and polymorphism in target antigens, and an adjuvant system able to induce broad spectrum immunity comprising both antibodies and helper T cells. Moreover, recent studies have shown some conventional pro-inflammatory adjuvants can also promote expansion of immunosuppressive regulatory T cells (Treg) and myeloid derived suppressor cells (MDSC), both of which could negatively impact malaria disease progression. Herein, we explore the ability of a model nanoparticle delivery system (polystyrene nanoparticles; PSNPs), previously proven to not induce conventional inflammation, Treg or MDSC, to induce immunity to MSP4/5 from Plasmodium yoelii, a member of the MSP4 and MSP5 family of proteins which are highly conserved across diverse malaria species including P. falciparum. The results show PSNPs-MSP4/5 conjugates are highly immunogenic, inducing immune responses comprising both T helper 1 (Th1) and Th2 cellular immunity, and a spectrum of antibody subclasses including IgG1, IgG2a, and IgG2b. Benchmarked against Alum and Complete Freund's Adjuvant (CFA), the immune responses that were induced were of comparable or higher magnitude, for both T cell frequencies by ELISpot and antibody responses in terms of ELISA end titer. Importantly, immunization with PSNPs-MSP4/5 induced partial protection against malaria blood-stage infection (50-80%) shown to be mechanistically dependent on interferon gamma (IFN-γ) production. These results expand the scope of adjuvants considered for malaria blood-stage vaccine development to those that do not use conventional adjuvant pathways and emphasizes the critical role of cellular immunity and specifically IFN-γ producing cells in providing moderate protection against blood-stage malaria comparable to Freunds adjuvant.

Pilot phase III immunotherapy study in early-stage breast cancer patients using oxidized mannan-MUC1 [ISRCTN71711835].

INTRODUCTION: Mucin 1 (MUC1) is a high molecular weight glycoprotein overexpressed on adenocarcinoma cells and is a target for immunotherapy protocols. To date, clinical trials against MUC1 have included advanced cancer patients. Herein, we report a trial using early stage breast cancer patients and injection of oxidized mannan-MUC1. METHOD: In a randomized, double-blind study, 31 patients with stage II breast cancer and with no evidence of disease received subcutaneous injections of either placebo or oxidized mannan-MUC1, to immunize against MUC1 and prevent cancer reoccurrence/metastases. Twenty-eight patients received the full course of injections of either oxidized mannan-MUC1 or placebo. Survival and immunological assays were assessed. RESULTS: After more than 5.5 years had elapsed since the last patient began treatment (8.5 years from the start of treatment of the first patient), the recurrence rate in patients receiving the placebo was 27% (4/15; the expected rate of recurrence in stage II breast cancer); those receiving immunotherapy had no recurrences (0/16), and this finding was statistically significant (P = 0.0292). Of the patients receiving oxidized mannan-MUC1, nine out of 13 had measurable antibodies to MUC1 and four out of 10 had MUC1-specific T cell responses; none of the placebo-treated patients exhibited an immune response to MUC1. CONCLUSION: The results suggest that, in early breast cancer, MUC1 immunotherapy is beneficial, and that a larger phase III study should be undertaken.

Vaccine delivery by penetratin: mechanism of antigen presentation by dendritic cells.

Cell-penetrating peptides (CPP) or membrane-translocating peptides such as penetratin from Antennapedia homeodomain or TAT from human immunodeficiency virus are useful vectors for the delivery of protein antigens or their cytotoxic (Tc) or helper (Th) T cell epitopes to antigen-presenting cells. Mice immunized with CPP containing immunogens elicit antigen-specific Tc and/or Th responses and could be protected from tumor challenges. In the present paper, we investigate the mechanism of class I and class II antigen presentation of ovalbumin covalently linked to penetratin (AntpOVA) by bone marrow-derived dendritic cells with the use of biochemical inhibitors of various pathways of antigen processing and presentation. Results from our study suggested that uptake of AntpOVA is via a combination of energy-independent (membrane fusion) and energy-dependent pathways (endocytosis). Once internalized by either mechanism, multiple tap-dependent or independent antigen presentation pathways are accessed while not completely dependent on proteasomal processing but involving proteolytic trimming in the ER and Golgi compartments. Our study provides an understanding on the mechanism of antigen presentation mediated by CPP and leads to greater insights into future development of vaccine formulations.

Quantitative proteomics of bronchoalveolar lavage fluid in lung adenocarcinoma.

BACKGROUND: The most commonly reported primary lung cancer subtype is adenocarcinoma, which is associated with a poor prognosis and short survival. Proteomic studies on human body fluids such as bronchoalveolar lavage fluid (BALF) have become essential methods for biomarker discovery, examination of tumor pathways and investigation of potential treatments. AIM: This study used quantitative proteomics to investigate the up-regulation of novel proteins in BALF from patients with primary lung adenocarcinoma in order to identify potential biomarkers. MATERIALS AND METHODS: BALF samples from individuals with and without primary lung adenocarcinoma were analyzed using liquid chromatography-mass spectrometry. RESULTS: One thousand and one hundred proteins were identified, 33 of which were found to be consistently overexpressed in all lung adenocarcinoma samples compared to non-cancer controls. A number of overexpressed proteins have been previously shown to be related to lung cancer progression including S100-A8, annexin A1, annexin A2, thymidine phosphorylase and transglutaminase 2. CONCLUSION: The overexpression of a number of specific proteins in BALF from patients with primary lung adenocarcinoma may be used as a potential biomarker for lung adenocarcinoma.