Upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin-12

OBJECTIVE: Injection of dendritic cells (DC) engineered with recombinant adenoviral vectors to produce interleukin-12 (IL-12) inside experimental murine tumors frequently achieves complete regressions. In such a system the function of CD8(+) T cells has been shown to be an absolute requirement, in contrast to observations made upon depletion of CD4(+) T cells, which minimally affected the outcome. The aim of this work was to study the possible involvement of natural killer (NK) cells in this setting. MATERIALS, METHODS, AND RESULTS: Depletions with anti-AsialoGM1 antiserum showed only a small decrease in the proportion of complete regressions obtained that correlated with induction of NK activities in lymphatic tissues into which DC migrate, whereas combined depletions of CD4(+) and NK cells completely eliminated the antitumor effects. Likewise in vivo neutralization of interferon-gamma (IFN-gamma) also eliminated those therapeutic effects. Trying to define the cellular role played by NK cells in vivo, it was observed that injection of cultured DC inside the spleen of T- and B-cell-deficient (Rag1(-/-)) mice induced upregulation of NK activity only if DC had been adenovirally engineered to produce IL-12. In addition, identically transfected fibroblasts also activated NK cells, indicating that IL-12 transfection was the unique requirement. Equivalent human DC only activated in vitro the cytolytic and cytokine-secreting functions of autologous NK cells if transfected to express human IL-12. CONCLUSIONS: Overall, these results point out an important role played by NK cell activation in the potent immunotherapeutic effects elicited by intratumoral injection of IL-12--secreting DC and that NK activation under these conditions is mainly, if not only, dependent on IL-12

Thrombopenic purpura induced by a monoclonal antibody directed to a 35-kilodalton surface protein (p35) expressed on murine platelets and endothelial cells

OBJECTIVE: With the aim of obtaining monoclonal antibodies (mAbs) against mouse endothelial surface antigens, immunization of rats with a mouse-derived endothelial cell line (PY4.1) and subsequent hybridoma production were performed. MATERIALS AND METHODS: One of the mAbs produced by hybridoma EOL5F5 was selected for its surface binding to endothelial cell lines, and identification of the mAb-recognized antigen was performed by immunoprecipitation. Experiments were performed to analyze the effects of EOL5F5 on systemic administration to mice. RESULTS: EOL5F5-recognized antigen was a single band of 35 kDa under reducing and nonreducing conditions, features that do not match other known differentiation antigens with comparable tissue distribution. In vivo administration of purified EOL5F5 mAb to mice (n = 20) induced intense cutaneous purpura as well as severe but transient thrombocytopenia. Expression of EOL5F5-recognized antigen was detected on platelets from which it immunoprecipitated a moiety of identical electrophoretic pattern in SDS-PAGE, as the one recognized on endothelial cells. Immunohistochemically, EOL5F5-recognized antigen (p35) also was expressed on dermal capillaries, suggesting that, in addition to thrombocytopenia, damaging effects of the antibody on endothelial cells also might cause the observed purpura. CONCLUSIONS: Our results show induction of thrombocytopenic purpura in mice with an mAb against a single antigenic determinant expressed on both platelets and endothelium. EOL5F5 mAb injection sets the stage for useful experimental models that resemble immune thrombocytopenic purpura

Intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas

Stimulation of the antitumor immune response by dendritic cells (DC) is critically dependent on their tightly regulated ability to produce interleukin-12 (IL-12). To enhance this effect artificially, bone marrow (BM)-derived DC were genetically engineered to produce high levels of functional IL-12 by ex vivo infection with a recombinant defective adenovirus (AdCMVIL-12). DC-expressing IL-12 injected into the malignant tissue eradicated 50-100% well established malignant nodules derived from the injection of two murine colon adenocarcinoma cell lines. Successful therapy was dependent on IL-12 transfection and was mediated only by syngeneic, but not allogeneic BM-derived DC, indicating that compatible antigen-presenting molecules were required. The antitumor effect was inhibited by in vivo depletion of CD8+ T cells and completely abrogated by simultaneous depletion with anti-CD4 and anti-CD8 mAbs. Mice which had undergone tumor regression remained immune to a rechallenge with tumor cells, showing the achievement of long-lasting systemic immunity that also was able to reject simultaneously induced concomitant untreated tumors. Tumor regression was associated with a detectable CTL response directed against tumor-specific antigens probably captured by DC artificially released inside tumor nodules. Our results open the possibility of similarly treating the corresponding human malignancies

Improving efficacy of interleukin-12-transfected dendritic cells injected into murine colon cancer with anti-CD137 monoclonal antibodies and alloantigens

Intralesional administration of cultured dendritic cells (DCs) engineered to produce IL-12 by in vitro infection with recombinant adenovirus frequently displays eradicating efficacy against established subcutaneous tumors derived from the CT26 murine colon carcinoma cell line. The elicited response is mainly mediated by cytolytic T lymphocytes. In order to search for strategies that would enhance the efficacy of the therapeutic procedure against less immunogenic tumors, we moved onto malignancies derived from the inoculation of MC38 colon cancer cells that are less prone to undergo complete regression upon a single intratumoral injection of IL-12-secreting DCs. In this model, we found that repeated injections of such DCs, as opposed to a single injection, achieved better efficacy against both the injected and a distantly implanted tumor; that the use of semiallogeneic DCs that are mismatched in one MHC haplotype with the tumor host showed slightly better efficacy; and that the combination of this treatment with systemic injections of immunostimulatory anti-CD137 (4-1BB) monoclonal antibody achieved potent combined effects that correlated with the antitumor immune response measured in IFN-gamma ELISPOT assays. The elicited systemic immune response eradicates concomitant untreated lesions in most cases. Curative efficacy was also found against some tumors established for 2 weeks when these strategies were used in combination. These are preclinical pieces of evidence to be considered in order to enhance the therapeutic benefit of a strategy that is currently being tested in clinical trials. Supplementary Material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html

Export and turnover of transparent exopolymer particles into the deep ocean

2nd Meeting of the Iberian Ecological Society (SIBECOL), XXI conference of the Iberian Association of Limnology (AIL) and 21st National Congress of the Portuguese Ecological Society (SPECO), 3-8 July 2022, AveiroAcidic polysaccharides released by phytoplankton and prokaryotic heterotrophs promote the formation of gel-like transparent exopolymer particles (TEPs). TEPs play a key role in the biological carbon pump due to their carbon-rich composition and their ability to coagulate and sink towards the deep ocean. Yet, very little is known about TEP distribution, export, and turnover at a global scale, particularly at deep ocean depths. We provide the first inventory of TEP from the surface up to 4000 m depth in the Atlantic, Indian, and Pacific Oceans and have assessed their contribution to carbon export into the deep ocean. Primary production determines TEP concentration above the deep chlorophyll maximum, and prokaryotic biomass also contributes in deeper waters. In the deep ocean waters, TEP concentrations are lower and mirror the concentrations in the surface, evidencing the importance of TEP sinking both at the export depth (200 m) with a global value of 2.9 Pg C year-1 and at the sequestration depth (1000 m) of 0.9 Pg C year-1 of particulate carbon. However, incubation experiments across ocean basins depicted rapid TEP turnover rates of 71 and 333 days (on average) within the export and sequestration depths, respectively. These findings reveal that the export of carbon by TEP sinking towards deep oceans escapes from long-term paths of the global carbon cycleN

Seroprevalence and Risk Factors Associated to Mycobacterium bovis in Wild Artiodactyl Species from Southern Spain, 2006–2010

The control of bovine tuberculosis (bTB) is at a critical point in the last stage of eradication in livestock. Wildlife species recently have emerged infected with TB in Europe, particularly ungulates in the Iberian Peninsula. Epidemiological information regarding TB in wild ungulates including affected species, prevalence, associated risk factors and appropriate diagnostic methods need to be obtained in these countries

Large carbon export, but short residence times, of transparent exopolymer particles in the global ocean

ASLO Aquatic Sciences Meeting 2023, Resilience and Recovery in Aquatic Systems, 4–9 June 2023, Palma de Mallorca, SpainAcidic polysaccharides released by phytoplankton and prokaryotic heterotrophs promote the formation of gel-like transparent exopolymer particles (TEPs). TEPs can have a relevant contribution to the biological carbon pump due to their carbon-rich composition and their ability to coagulate and sink towards the deep ocean. However, little is known about TEPs distribution, carbon export, and residence times below the export (200 m) and sequestration (1000 m) depths. We provide the first comprehensive inventory of TEP from the ocean surface to a depth of 4000 meters in the tropical and subtropical Atlantic, Indian, and Pacific Oceans, evaluating its contribution to carbon export and sequestration into the deep ocean. Results indicate that TEP concentration is primarily determined by primary production, with higher concentrations located above the deep chlorophyll maxima. In the deep ocean, TEP concentrations are lower yet mirror the concentrations in the surface, demonstrating the significance of TEP sinking below both the export compartment (2.8 Pg C yr-1; 27% of total POC flux at 200 m) and the sequestration compartment (0.8 Pg C yr-1; 36% of total POC flux at 1000 m). In situ incubation experiments conducted across ocean basins indicate short TEP residence times, averaging 27 and 333 days in the export and sequestration compartments, respectively. These findings reveal that the export and subsequent sequestration of carbon by TEP sinking into the deep ocean diverts it from the long times observed for the dissolved carbon fraction (i.e. centuries) in the global carbon cycleN

Insulin and IGF1 signalling pathways in human astrocytes <i>in vitro</i> and <i>in vivo</i>; characterisation, subcellular localisation and modulation of the receptors.

Background The insulin/IGF1 signalling (IIS) pathways are involved in longevity regulation and are dysregulated in neurons in Alzheimer’s disease (AD). We previously showed downregulation in IIS gene expression in astrocytes with AD-neuropathology progression, but IIS in astrocytes remains poorly understood. We therefore examined the IIS pathway in human astrocytes and developed models to reduce IIS at the level of the insulin or the IGF1 receptor (IGF1R). Results We determined IIS was present and functional in human astrocytes by immunoblotting and showed astrocytes express the insulin receptor (IR)-B isoform of Ir. Immunocytochemistry and cell fractionation followed by western blotting revealed the phosphorylation status of insulin receptor substrate (IRS1) affects its subcellular localisation. To validate IRS1 expression patterns observed in culture, expression of key pathway components was assessed on post-mortem AD and control tissue using immunohistochemistry. Insulin signalling was impaired in cultured astrocytes by treatment with insulin + fructose and resulted in decreased IR and Akt phosphorylation (pAkt S473). A monoclonal antibody against IGF1R (MAB391) induced degradation of IGF1R receptor with an associated decrease in downstream pAkt S473. Neither treatment affected cell growth or viability as measured by MTT and Cyquant® assays or GFAP immunoreactivity. Discussion IIS is functional in astrocytes. IR-B is expressed in astrocytes which differs from the pattern in neurons, and may be important in differential susceptibility of astrocytes and neurons to insulin resistance. The variable presence of IRS1 in the nucleus, dependent on phosphorylation pattern, suggests the function of signalling molecules is not confined to cytoplasmic cascades. Down-regulation of IR and IGF1R, achieved by insulin + fructose and monoclonal antibody treatments, results in decreased downstream signalling, though the lack of effect on viability suggests that astrocytes can compensate for changes in single pathways. Changes in signalling in astrocytes, as well as in neurons, may be important in ageing and neurodegeneration

Plastic accumulation in the Mediterranean Sea

Concentrations of floating plastic were measured throughout the Mediterranean Sea to assess whether this basin can be regarded as a great accumulation region of plastic debris. We found that the average density of plastic (1 item per 4 m2), as well as its frequency of occurrence (100% of the sites sampled), are comparable to the accumulation zones described for the five subtropical ocean gyres. Plastic debris in the Mediterranean surface waters was dominated by millimeter-sized fragments, but showed a higher proportion of large plastic objects than that present in oceanic gyres, reflecting the closer connection with pollution sources. The accumulation of floating plastic in the Mediterranean Sea (between 1,000 and 3,000 tons) is likely related to the high human pressure together with the hydrodynamics of this semi-enclosed basin, with outflow mainly occurring through a deep water layer. Given the biological richness and concentration of economic activities in the Mediterranean Sea, the affects of plastic pollution on marine and human life are expected to be particularly frequent in this plastic accumulation region

Experimental study of differentially rotating supersonic plasma flows produced by aluminium wire array Z-pinches

A novel approach to cylindrical wire array z-pinches has been developed in order to create a rotating plasma flow analogous to astrophysical accretion discs. The method involves subjecting the wire array to a cusp magnetic field (B_r) to create converging off axis ablation streams to form a rotating flow. The rotation is sustained by the ram pressure of the ablation streams in a quasi-equilibrium state for approximately 150 ns. This corresponds to one full rotation of the plasma about the axis. The rotating plasma is supersonic with Mach number ~2 and a radially constant rotation velocity between 60 and 75 km/s; the angular velocity therefore has an r^-1 dependence and the flow is differential. A Thomson scattering diagnostic is used to measure the electron and ion temperatures as Te ~30 eV and Ti >55 eV and the ionisation of the plasma (Z) between 6 and 8. These parameters are used to calculate the Reynolds number (10^5 to 10^6) and magnetic Reynolds numbers (20 to 100) which are large enough for viscous and resistive effects to be negligible on the large scale of the flow. These are of sufficient magnitude for the experiment to be scalable to astrophysical accretion discs. Further more the Reynolds number for the experiment is large enough for shear instabilities to manifest in the plasma. Some evidence for this can be seen in XUV images and Thomson spectra which indicate the development of perturbations and vorticity within the flow. Predictions for the growth rate of the Kelvin Helmholtz instability, 12 to 40 ns, agree reasonably well with the observed perturbation growth of ~30 ns. It is also possible that shear instabilities are driving hydrodynamic turbulence. Turbulent heating of the plasma could explain the approximately 500 eV increase in the ion temperature observed from some Thomson spectra. Further work is required however to prove the existence of shear flows and turbulence within the experiments.Open Acces