EP-1349: Long term results of a phase I-II study of moderate hypofractionated IGRT in prostate cancer

Pregnant women diagnosed with gestational diabetes mellitus subjected to diet (GDMd) that do not reach normal glycaemia are passed to insulin therapy (GDMi). GDMd associates with increased human cationic amino acid transporter 1 (hCAT-1)-mediated transport of L-arginine and nitric oxide synthase (NOS) activity in foetoplacental vasculature, a phenomenon reversed by exogenous insulin. Whether insulin therapy results in reversal of the GDMd effect on the foetoplacental vasculature is unknown. We assayed whether insulin therapy normalizes GDMd-associated foetoplacental endothelial dysfunction. Primary cultures of human umbilical vein endothelial cells (HUVECs) from GDMi pregnancies were used to assay L-arginine transport kinetics, NOS activity, p44/42mapk and protein kinase B/Akt activation, and umbilical vein rings reactivity. HUVECs from GDMi or GDMd show increased hCAT-1 expression and maximal transport capacity, NOS activity, and eNOS, and p44/42mapk, but not Akt activator phosphorylation. Dilation in response to insulin or calcitonin-gene related peptide was impaired in umbilical vein rings from GDMi and GDMd pregnancies. Incubation of HUVECs in vitro with insulin (1 nmol/L) restored hCAT-1 and eNOS expression and activity, and eNOS and p44/42mapk activator phosphorylation. Thus, maternal insulin therapy does not seem to reverse GDMd-associated alterations in human foetoplacental vasculature.Fondo Nacional de Desarrollo Científico y Tecnológico Chileno 1150377 , 1150344 , 11150083Servicio de Salud de Medicina Oriente de Chile 1938–2016Marie Curie International Research 295185 - EULAMDIM

Use of a recombinant Salmonella enterica serovar Typhimurium strain expressing C-Raf for protection against C-Raf induced lung adenoma in mice

BACKGROUND: Serine-threonine kinases of the Raf family (A-Raf, B-Raf, C-Raf) are central players in cellular signal transduction, and thus often causally involved in the development of cancer when mutated or over-expressed. Therefore these proteins are potential targets for immunotherapy and a possible basis for vaccine development against tumors. In this study we analyzed the functionality of a new live C-Raf vaccine based on an attenuated Salmonella enterica serovar Typhimurium aroA strain in two Raf dependent lung tumor mouse models. METHODS: The antigen C-Raf has been fused to the C-terminal secretion signal of Escherichia coli α-hemolysin and expressed in secreted form by an attenuated aroA Salmonella enterica serovar Typhimurium strain via the α-hemolysin secretion pathway. The effect of the immunization with this recombinant C-Raf strain on wild-type C57BL/6 or lung tumor bearing transgenic BxB mice was analyzed using western blot and FACS analysis as well as specific tumor growth assays. RESULTS: C-Raf antigen was successfully expressed in secreted form by an attenuated Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. Immunization of wild-type C57BL/6 or tumor bearing mice provoked specific C-Raf antibody and T-cell responses. Most importantly, the vaccine strain significantly reduced tumor growth in two transgenic mouse models of Raf oncogene-induced lung adenomas. CONCLUSIONS: The combination of the C-Raf antigen, hemolysin secretion system and Salmonella enterica serovar Typhimurium could form the basis for a new generation of live bacterial vaccines for the treatment of Raf dependent human malignancies

Tick Extracellular Vesicles Enable Arthropod Feeding and Promote Distinct Outcomes of Bacterial Infection

Extracellular vesicles are thought to facilitate pathogen transmission from arthropods to humans and other animals. Here, we reveal that pathogen spreading from arthropods to the mammalian host is multifaceted. Extracellular vesicles from Ixodes scapularis enable tick feeding and promote infection of the mildly virulent rickettsial agent Anaplasma phagocytophilum through the SNARE proteins Vamp33 and Synaptobrevin 2 and dendritic epidermal T cells. However, extracellular vesicles from the tick Dermacentor andersoni mitigate microbial spreading caused by the lethal pathogen Francisella tularensis. Collectively, we establish that tick extracellular vesicles foster distinct outcomes of bacterial infection and assist in vector feeding by acting on skin immunity. Thus, the biology of arthropods should be taken into consideration when developing strategies to control vector-borne diseases

The Ocular Conjunctiva as a Mucosal Immunization Route: A Profile of the Immune Response to the Model Antigen Tetanus Toxoid

Background: In a quest for a needle-free vaccine administration strategy, we evaluated the ocular conjunctiva as an alternative mucosal immunization route by profiling and comparing the local and systemic immune responses to the subcutaneous or conjunctival administration of tetanus toxoid (TTd), a model antigen. Materials and methods: BALB/c and C57BL/6 mice were immunized either subcutaneously with TTd alone or via the conjunctiva with TTd alone, TTd mixed with 2% glycerol or TTd with merthiolate-inactivated whole-cell B. pertussis (wBP) as adjuvants. Mice were immunized on days 0, 7 and 14 via both routes, and an evaluation of the local and systemic immune responses was performed two weeks after the last immunization. Four weeks after the last immunization, the mice were challenged with a lethal dose (2 x LD50) of tetanus toxin. Results: The conjunctival application of TTd in BALB/c mice induced TTd-specific secretory IgA production and skewed the TTd-specific immune response toward a Th1/Th17 profile, as determined by the stimulation of IFN gamma and IL-17A secretion and/or the concurrent pronounced reduction of IL-4 secretion, irrespective of the adjuvant. In conjunctivaly immunized C57BL/6 mice, only TTd administered with wBP promoted the establishment of a mixed Th1/Th17 TTd-specific immune response, whereas TTd alone or TTd in conjunction with glycerol initiated a dominant Th1 response against TTd. Immunization via the conjunctiva with TTd plus wBP adjuvant resulted in a 33% survival rate of challenged mice compared to a 0% survival rate in non-immunized animals (p lt 0.05). Conclusion: Conjunctival immunization with TTd alone or with various adjuvants induced TTd-specific local and systemic immune responses, predominantly of the Th1 type. The strongest immune responses developed in mice that received TTd together with wBP, which implies that this alternative route might tailor the immune response to fight intracellular bacteria or viruses more effectively

Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world's leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1-893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/cmice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocularmucosa was well tolerated without signs of inflammation. N-PmpC- specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFN gamma immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage

The Role of Research in Viral Disease Eradication and Elimination Programs: Lessons for Malaria Eradication

Using their experiences from, and analysis of, global campaigns to eradicate smallpox, poliomyelitis, and measles, Myron Levine and colleagues derive lessons for malaria eradication

Live Recombinant Salmonella Typhi Vaccines Constructed to Investigate the Role of rpoS in Eliciting Immunity to a Heterologous Antigen

We hypothesized that the immunogenicity of live Salmonella enterica serovar Typhi vaccines expressing heterologous antigens depends, at least in part, on its rpoS status. As part of our project to develop a recombinant attenuated S. Typhi vaccine (RASTyV) to prevent pneumococcal diseases in infants and children, we constructed three RASTyV strains synthesizing the Streptococcus pneumoniae surface protein PspA to test this hypothesis. Each vector strain carried ten engineered mutations designed to optimize safety and immunogenicity. Two S. Typhi vector strains (χ9639 and χ9640) were derived from the rpoS mutant strain Ty2 and one (χ9633) from the RpoS+ strain ISP1820. In χ9640, the nonfunctional rpoS gene was replaced with the functional rpoS gene from ISP1820. Plasmid pYA4088, encoding a secreted form of PspA, was moved into the three vector strains. The resulting RASTyV strains were evaluated for safety in vitro and for immunogenicity in mice. All three RASTyV strains were similar to the live attenuated typhoid vaccine Ty21a in their ability to survive in human blood and human monocytes. They were more sensitive to complement and were less able to survive and persist in sewage and surface water than their wild-type counterparts. Adult mice intranasally immunized with any of the RASTyV strains developed immune responses against PspA and Salmonella antigens. The RpoS+ vaccines induced a balanced Th1/Th2 immune response while the RpoS− strain χ9639(pYA4088) induced a strong Th2 immune response. Immunization with any RASTyV provided protection against S. pneumoniae challenge; the RpoS+ strain χ9640(pYA4088) provided significantly greater protection than the ISP1820 derivative, χ9633(pYA4088). In the pre-clinical setting, these strains exhibited a desirable balance between safety and immunogenicity and are currently being evaluated in a Phase 1 clinical trial to determine which of the three RASTyVs has the optimal safety and immunogenicity profile in human hosts

Investigating the architecture and internal structure of the TOI-561 system planets with CHEOPS, HARPS-N, and TESS

We present a precise characterization of the TOI-561 planetary system obtained by combining previously published data with TESS and CHEOPS photometry, and a new set of 62 HARPS-N radial velocities (RVs). Our joint analysis confirms the presence of four transiting planets, namely TOI-561 b (P = 0.45 d, R = 1.42 R-circle plus, M = 2.0 M-circle plus), c (P = 10.78 d, R = 2.91 R-circle plus, M = 5.4 M-circle plus), d (P = 25.7 d, R = 2.82 R-circle plus, M = 13.2 M-circle plus), and e (P = 77 d, R = 2.55 R-circle plus, M = 12.6 R-circle plus). Moreover, we identify an additional, long-period signal (>450 d) in the RVs, which could be due to either an external planetary companion or to stellar magnetic activity. The precise masses and radii obtained for the four planets allowed us to conduct interior structure and atmospheric escape modelling. TOI-561 b is confirmed to be the lowest density (rho(b) = 3.8 +/- 0.5 g cm(-3)) ultra-short period (USP) planet known to date, and the low metallicity of the host star makes it consistent with the general bulk density-stellar metallicity trend. According to our interior structure modelling, planet b has basically no gas envelope, and it could host a certain amount of water. In contrast, TOI-561 c, d, and e likely retained an H/He envelope, in addition to a possibly large water layer. The inferred planetary compositions suggest different atmospheric evolutionary paths, with planets b and c having experienced significant gas loss, and planets d and e showing an atmospheric content consistent with the original one. The uniqueness of the USP planet, the presence of the long-period planet TOI-561 e, and the complex architecture make this system an appealing target for follow-up studies

Salmonella Typhi-specific multifunctional CD8+ T cells play a dominant role in protection from typhoid fever in humans.

BACKGROUND: Typhoid fever, caused by the human-restricted organism Salmonella Typhi (S. Typhi), is a major public health problem worldwide. Development of novel vaccines remains imperative, but is hampered by an incomplete understanding of the immune responses that correlate with protection. METHODS: Recently, a controlled human infection model was re-established in which volunteers received ~10(3) cfu wild-type S. Typhi (Quailes strain) orally. Twenty-one volunteers were evaluated for their cell-mediated immune (CMI) responses. Ex vivo PBMC isolated before and up to 1 year after challenge were exposed to three S. Typhi-infected targets, i.e., autologous B lymphoblastoid cell-lines (B-LCL), autologous blasts and HLA-E restricted AEH B-LCL cells. CMI responses were evaluated using 14-color multiparametric flow cytometry to detect simultaneously five intracellular cytokines/chemokines (i.e., IL-17A, IL-2, IFN-g, TNF-a and MIP-1b) and a marker of degranulation/cytotoxic activity (CD107a). RESULTS: Herein we provide the first evidence that S. Typhi-specific CD8+ responses correlate with clinical outcome in humans challenged with wild-type S. Typhi. Higher multifunctional S. Typhi-specific CD8+ baseline responses were associated with protection against typhoid and delayed disease onset. Moreover, following challenge, development of typhoid fever was accompanied by decreases in circulating S. Typhi-specific CD8+ T effector/memory (TEM) with gut homing potential, suggesting migration to the site(s) of infection. In contrast, protection against disease was associated with low or no changes in circulating S. Typhi-specific TEM. CONCLUSIONS: These studies provide novel insights into the protective immune responses against typhoid disease that will aid in selection and development of new vaccine candidates