Maturation of monocyte-derived dendritic cells with Toll-like receptor 3 and 7/8 ligands combined with prostaglandin E2 results in high interleukin-12 production and cell migration

Dendritic cells (DC) are professional antigen-presenting cells of the immune system that play a key role in regulating T cell-based immunity. In vivo, the capacity of DC to activate T cells depends on their ability to migrate to the T cell areas of lymph nodes as well as on their maturation state. Depending on their cytokine-secreting profile, DC are able to skew the immune response in a specific direction. In particular, IL-12p70 producing DC drive T cells towards a T helper 1 type response. A serious disadvantage of current clinical grade ex vivo generated monocyte-derived DC is the poor IL-12p70 production. We have investigated the effects of Toll-like receptor (TLR)-mediated maturation on ex vivo generated human monocyte-derived DC. We demonstrate that in contrast to cytokine-matured DC, DC matured with poly(I:C) (TLR3 ligand) and/or R848 (TLR7/8 ligand) are able to produce vast amounts of IL-12p70, but exhibit a reduced migratory capacity. The addition of prostaglandin E2 (PGE2) improved the migratory capacity of TLR-ligand matured DC while maintaining their IL-12p70 production upon T cell encounter. We propose a novel clinical grade maturation protocol in which TLR ligands poly(I:C) and R848 are combined with PGE2 to generate DC with both high migratory capacity and IL-12p70 production upon T cell encounter

Ports and Waterways Design videos -Functional Design of Marinas

Video material for a masters level course on functional design and capacity planning of the various elements of ports and waterways, such as terminals and locks. The learning in this series of videos is essential for planning and designing the overall system

Immunogenicity of dendritic cells pulsed with CEA peptide or transfected with CEA mRNA for vaccination of colorectal cancer patients

Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. We have demonstrated that vaccination of autologous ex vivo cultured DCs results in the induction of tumor-specific immune responses in cancer patients, which correlates with clinical response. Optimization of antigen loading is one of the possibilities for further improving the efficacy of DC vaccination. Theoretically, transfection of DCs with RNA encoding a tumor-specific antigen may induce a broader immune response as compared to the most widely used technique of peptide pulsing. In this clinical study, RNA transfection was compared with peptide pulsing as an antigen loading strategy for DC vaccination. Patients with resectable liver metastases of colorectal cancer were vaccinated intravenously and intradermally 3 times weekly with either carcinoembryogenic antigen (CEA)-derived HLA-A2 binding peptide-loaded or CEA mRNA electroporated DCs prior to surgical resection of the metastases. All DCs were loaded with keyhole limpet hemocyanin (KLH) as a control protein. Evaluation of vaccine-induced immune reactivity consisted of T-cell proliferative responses and B-cell antibody responses against KLH in peripheral blood. CEA reactivity was determined in T-cell cultures of biopsies of post-treatment delayed type hypersensitivity skin tests. Sixteen patients were included. All patients showed T-cell responses against KLH upon vaccination. CEA peptide-specific T-cells were detected in 8 out of 11 patients in the peptide group, but in none of the 5 patients in the RNA group. In our study, DC CEA mRNA transfection was not superior to DC CEA peptide pulsing in the induction of a tumor-specific immune response in colorectal cancer patient

Analysis of dendritic cell trafficking using EGFP-transgenic mice

Dendritic cells (DCs) are professional antigen presenting cells, well equipped to initiate an immune response. For effective induction of an immune response, DC should migrate from the periphery to the lymph node to present the antigen to T lymphocytes. Currently, tumor-antigen loaded DCs are used in clinical vaccination trials in cancer patients. To investigate the migratory capacity of DC in vivo, a variety of fluorescent and radioactive labels have been used. Here we introduce a novel tool to study DC migration in vivo: DCs generated from enhanced green fluorescent protein (EGFP)-transgenic mice. DC from EGFP-transgenic mice display typical DC behavior and can be matured without affecting their autofluorescence in vitro. In addition, the continuously produced cytoplasmic EGFP in living cells functions as a viability marker, since EGFP released from dying cells does not stain DC from C57Bl/6 mice upon coculture. In vivo migration studies using EGFP-DC and indium-111-labeled DC were performed to determine the efficiency of i.d. versus s.c. administered DC to reach the draining lymph node. The analysis demonstrates that i.d. injection increases the amount of EGFP-DC/indium-111-labeled DC in the lymph node compared to s.c. injection. Subsequent quantitative, phenotypical and ultrastuctural analysis demonstrate that DC generated from EGFP-transgenic mice are well suited to study the migratory behavior, distribution and phenotype of DC in viv

Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice

Tumor microenvironments feature immune inhibitory mechanisms that prevent T cells from generating effective antitumor immune responses. Therapeutic interventions aimed at disrupting these inhibitory mechanisms have been shown to enhance antitumor immunity, but they lack direct cytotoxic effects. Here, we investigated the effect of cytotoxic cancer chemotherapeutics on immune inhibitory pathways. We observed that exposure to platinum-based chemotherapeutics markedly reduced expression of the T cell inhibitory molecule programmed death receptor-ligand 2 (PD-L2) on both human DCs and human tumor cells. Downregulation of PD-L2 resulted in enhanced antigen-specific proliferation and Th1 cytokine secretion as well as enhanced recognition of tumor cells by T cells. Further analysis revealed that STAT6 controlled downregulation of PD-L2. Consistent with these data, patients with STAT6-expressing head and neck cancer displayed enhanced recurrence-free survival upon treatment with cisplatin-based chemoradiation compared with patients with STAT6-negative tumors, demonstrating the clinical relevance of platinum-induced STAT6 modulation. We therefore conclude that platinum-based anticancer drugs can enhance the immunostimulatory potential of DCs and decrease the immunosuppressive capability of tumor cells. This dual action of platinum compounds may extend their therapeutic application in cancer patients and provides a rationale for their use in combination with immunostimulatory compounds

Route of administration modulates the induction of dendritic cell vaccine-induced antigen-specific T cells in advanced melanoma patients

Contains fulltext : 96310.pdf (publisher's version ) (Closed access)PURPOSE: It is unknown whether the route of administration influences dendritic cell (DC)-based immunotherapy. We compared the effect of intradermal versus intranodal administration of a DC vaccine on induction of immunologic responses in melanoma patients and examined whether concomitant administration of interleukin (IL)-2 increases the efficacy of the DC vaccine. EXPERIMENTAL DESIGN: HLA-A2.1(+) melanoma patients scheduled for regional lymph node dissection were vaccinated four times biweekly via intradermal or intranodal injection with 12 x 10 to 17 x 10 mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (KLH). Half of the patients also received low-dose IL-2 (9 MIU daily for 7 days starting 3 days after each vaccination). KLH-specific B- and T-cell responses were monitored in blood. gp100- and tyrosinase-specific T-cell responses were monitored in blood by tetramer analysis and in biopsies from delayed-type hypersensitivity (DTH) skin tests by tetramer and functional analyses with (51)Cr release assays or IFNgamma release, following coculture with peptide-pulsed T2 cells or gp100- or tyrosinase-expressing tumor cells. RESULTS: In 19 of 43 vaccinated patients, functional tumor antigen-specific T cells could be detected. Although significantly more DCs migrated to adjacent lymph nodes upon intranodal vaccination, this was also highly variable with a complete absence of migration in 7 of 24 intranodally vaccinated patients. Intradermal vaccinations proved superior in inducing functional tumor antigen-specific T cells. Coadministration of IL-2 did not further augment the antigen-specific T-cell response but did result in higher regulatory T-cell frequencies. CONCLUSION: Intradermal vaccination resulted in superior antitumor T-cell induction when compared with intranodal vaccination. No advantage of additional IL-2 treatment could be shown

Prophylactic vaccines are potent activators of monocyte-derived dendritic cells and drive effective anti-tumor responses in melanoma patients at the cost of toxicity

Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients, predominantly with DC matured with pro-inflammatory cytokines and prostaglandin E2. We studied the safety and efficacy of vaccination with monocyte-derived DC matured with a cocktail of prophylactic vaccines that contain clinical-grade Toll-like receptor ligands (BCG, Typhim, Act-HIB) and prostaglandin E2 (VAC-DC). Stage III and IV melanoma patients were vaccinated via intranodal injection (12 patients) or combined intradermal/intravenous injection (16 patients) with VAC-DC loaded with keyhole limpet hemocyanin (KLH) and mRNA encoding tumor antigens gp100 and tyrosinase. Tumor antigen-specific T cell responses were monitored in blood and skin-test infiltrating-lymphocyte cultures. Almost all patients mounted prophylactic vaccine- or KLH-specific immune responses. Both after intranodal injection and after intradermal/intravenous injection, tumor antigen-specific immune responses were detected, which coincide with longer overall survival in stage IV melanoma patients. VAC-DC induce local and systemic CTC grade 2 and 3 toxicity, which is most likely caused by BCG in the maturation cocktail. The side effects were self-limiting or resolved upon a short period of systemic steroid therapy. We conclude that VAC-DC can induce functional tumor-specific responses. Unfortunately, toxicity observed after vaccination precludes the general application of VAC-DC, since in DC maturated with prophylactic vaccines BCG appears to be essential in the maturation cocktai

Cardiopulmonary resuscitation in adults over 80 : outcome and the perception of appropriateness by clinicians

OBJECTIVES: To determine the prevalence of clinician perception of inappropriate cardiopulmonary resuscitation (CPR) regarding the last out‐of‐hospital cardiac arrest (OHCA) encountered in an adult 80 years or older and its relationship to patient outcome. DESIGN: Subanalysis of an international multicenter cross‐sectional survey (REAPPROPRIATE). SETTING: Out‐of‐hospital CPR attempts registered in Europe, Israel, Japan, and the United States in adults 80 years or older. PARTICIPANTS: A total of 611 clinicians of whom 176 (28.8%) were doctors, 123 (20.1%) were nurses, and 312 (51.1%) were emergency medical technicians/paramedics. RESULTS AND MEASUREMENTS: The last CPR attempt among patients 80 years or older was perceived as appropriate by 320 (52.4%) of the clinicians; 178 (29.1%) were uncertain about the appropriateness, and 113 (18.5%) perceived the CPR attempt as inappropriate. The survival to hospital discharge for the “appropriate” subgroup was 8 of 265 (3.0%), 1 of 164 (.6%) in the “uncertain” subgroup, and 2 of 107 (1.9%) in the “inappropriate” subgroup (P = .23); 503 of 564 (89.2%) CPR attempts involved non‐shockable rhythms. CPR attempts in nursing homes accounted for 124 of 590 (21.0%) of the patients and were perceived as appropriate by 44 (35.5%) of the clinicians; 45 (36.3%) were uncertain about the appropriateness; and 35 (28.2%) perceived the CPR attempt as inappropriate. The survival to hospital discharge for the nursing home patients was 0 of 107 (0%); 104 of 111 (93.7%) CPR attempts involved non‐shockable rhythms. Overall, 36 of 543 (6.6%) CPR attempts were undertaken despite a known written do not attempt resuscitation decision; 14 of 36 (38.9%) clinicians considered this appropriate, 9 of 36 (25.0%) were uncertain about its appropriateness, and 13 of 36 (36.1%) considered this inappropriate. CONCLUSION: Our findings show that despite generally poor outcomes for older patients undergoing CPR, many emergency clinicians do not consider these attempts at resuscitation to be inappropriate. A professional and societal debate is urgently needed to ensure that first we do not harm older patients by futile CPR attempts. J Am Geriatr Soc 68:39–45, 201