Acute exercise-induced release of innate immune proteins via small extracellular vesicles changes with aerobic fitness and age.

AIM: Physical exercise triggers the secretion of small extracellular vesicles (sEVs) into the circulation in humans, enabling signalling crosstalk between tissues. Exercise-derived EVs and their cargo have been proposed to mediate adaptations to exercise; however, our understanding of how exercise-derived EV protein cargo is modulated by factors such as aerobic fitness and age of an individual is currently unknown. Here, we examined the circulating sEV proteome following aerobic exercise in healthy males of different ages and aerobic fitness to understand exercise-induced EV response during the aging process. METHODS: Twenty-eight healthy men completed a bout of 20-min cycling exercise at 70% estimated VO2peak . Small EVs were isolated from blood samples collected before and immediately after exercise, and then quantified using particle analysis and Western blotting. Small EV proteome was examined using quantitative proteomic analysis. RESULTS: We identified a significant increase in 13 proteins in small plasma EVs following moderate-to-vigorous intensity exercise. We observed distinct changes in sEV proteome after exercise in young, mature, unfit, and fit individuals, highlighting the impact of aerobic fitness and age on sEV protein secretion. Functional enrichment and pathway analysis identified that the majority of the significantly altered sEV proteins are associated with the innate immune system, including proteins known to be damage-associated molecular patterns (DAMPs). CONCLUSION: Together, our findings suggest that exercise-evoked acute stress can positively challenge the innate immune system through the release of signalling molecules such as DAMPs in sEVs, proposing a novel EV-based mechanism for moderate-to-vigorous intensity exercise in immune surveillance pathways.

Sex differences in the influence of type 2 diabetes (T2D)-related genes, parental history of T2D, and obesity on T2D development: a case-control study.

BACKGROUND: This study investigated the effect of sex and age at type 2 diabetes (T2D) diagnosis on the influence of T2D-related genes, parental history of T2D, and obesity on T2D development. METHODS: In this case-control study, 1012 T2D cases and 1008 healthy subjects were selected from the Diabetes in Mexico Study database. Participants were stratified by sex and age at T2D diagnosis (early, ≤ 45 years; late, ≥ 46 years). Sixty-nine T2D-associated single nucleotide polymorphisms were explored and the percentage contribution (R2) of T2D-related genes, parental history of T2D, and obesity (body mass index [BMI] and waist-hip ratio [WHR]) on T2D development was calculated using univariate and multivariate logistic regression models. RESULTS: T2D-related genes influenced T2D development most in males who were diagnosed early (R2 = 23.5%; females, R2 = 13.5%; males and females diagnosed late, R2 = 11.9% and R2 = 7.3%, respectively). With an early diagnosis, insulin production-related genes were more influential in males (76.0% of R2) while peripheral insulin resistance-associated genes were more influential in females (52.3% of R2). With a late diagnosis, insulin production-related genes from chromosome region 11p15.5 notably influenced males while peripheral insulin resistance and genes associated with inflammation and other processes notably influenced females. Influence of parental history was higher among those diagnosed early (males, 19.9%; females, 17.5%) versus late (males, 6.4%; females, 5,3%). Unilateral maternal T2D history was more influential than paternal T2D history. BMI influenced T2D development for all, while WHR exclusively influenced males. CONCLUSIONS: The influence of T2D-related genes, maternal T2D history, and fat distribution on T2D development was greater in males than females.

The prevalence of diabetes worldwide is slightly higher in men than in women, particularly in those aged 50 or younger (16.5% for men versus 13.5% for women). This suggests that hormonal differences could be critical in early development of Type 2 diabetes. Some known factors previously associated with T2D, such as genes, parental history of diabetes and obesity, could have a differential influence between both sexes for the development of T2D. We compared these factors between 1008 healthy individual and 1012 TD2 patients. In this comparison, we calculated the percentage of variability of the disease explained by each factor. As expected, the most noticeable differences between men and women were observed in T2D diagnoses before age 46. Genes had a greater effect in men than in women (23.5% vs. 13.5%). While genes involved in insulin production have a greater influence on men, genes involved in peripheric insulin resistance have a greater influence on women. The overall parental history of T2D influences similarly in males (19.9%) and females (17.5%), however, the unilateral genetic influence of the mother was much greater in males than in females. The influence of global and abdominal obesity played a greater role in men than in women. In T2D diagnoses after age of 45, the influence of genes and parental history of diabetes decreases markedly, and the relative influence of global obesity augments. However, while genes linked to insulin resistance and inflammation predominate in females, genes linked to insulin secretion predominate in males.

First-in-human clinical trial of allogeneic, platelet-derived extracellular vesicles as a potential therapeutic for delayed wound healing.

The release of growth factors, cytokines and extracellular matrix modifiers by activated platelets is an important step in the process of healthy wound healing. Extracellular vesicles (EVs) released by activated platelets carry this bioactive cargo in an enriched form, and may therefore represent a potential therapeutic for the treatment of delayed wound healing, such as chronic wounds. While EVs show great promise in regenerative medicine, their production at clinical scale remains a critical challenge and their tolerability in humans is still to be fully established. In this work, we demonstrate that Ligand-based Exosome Affinity Purification (LEAP) chromatography can successfully isolate platelet EVs (pEVs) of clinical grade from activated platelets, which retain the regenerative properties of the parent cell. LEAP-isolated pEVs display the expected biophysical features of EV populations and transport essential proteins in wound healing processes, including insulin growth factor (IGF) and transforming growth factor beta (TGF-ß). In vitro studies show that pEVs induce proliferation and migration of dermal fibroblasts and increase dermal endothelial cells' angiogenic potential, demonstrating their wound healing potential. pEV treatment activates the ERK and Akt signalling pathways within recipient cells. In a first-in-human, double-blind, placebo-controlled, phase I clinical trial of healthy volunteer adults, designed primarily to assess safety in the context of wound healing, we demonstrate that injections of LEAP-purified pEVs in formulation buffer are safe and well tolerated (Plexoval II study, ACTRN12620000944932). As a secondary objective, biological activity in the context of wound healing rate was assessed. In this cohort of healthy participants, in which the wound bed would not be expected to be deficient in the bioactive cargo that pEVs carry, all wounds healed rapidly and completely and no difference in time to wound closure of the treated and untreated wounds was observed at the single dose tested. The outcomes of this study evidence that pEVs manufactured through the LEAP process can be injected safely in humans as a potential wound healing treatment, and warrant further study in clinical trials designed expressly to assess therapeutic efficacy in patients with delayed or disrupted wound healing.

Exercise increases the release of NAMPT in extracellular vesicles and alters NAD+ activity in recipient cells.

Aging is associated with a loss of metabolic homeostasis, with cofactors such as nicotinamide adenine dinucleotide (NAD+ ) declining over time. The decrease in NAD+ production has been linked to the age-related loss of circulating extracellular nicotinamide phosphoribosyltransferase (eNAMPT), the rate-limiting enzyme in the NAD+ biosynthetic pathway. eNAMPT is found almost exclusively in extracellular vesicles (EVs), providing a mechanism for the distribution of the enzyme in different tissues. Currently, the physiological cause for the release of eNAMPT is unknown, and how it may be affected by age and physical exercise. Here, we show that release of small EVs into the bloodstream is stimulated following moderate intensity exercise in humans. Exercise also increased the eNAMPT content in EVs, most prominently in young individuals with higher aerobic fitness. Both mature fit and young unfit individuals exhibited a limited increase in EV-eNAMPT release following exercise, indicating that this mechanism is related to both the age and physical fitness of a person. Notably, unfit mature individuals were unable to increase the release of eNAMPT in EVs after exercise, suggesting that lower fitness levels and aging attenuate this important signalling mechanism in the body. EVs isolated from exercising humans containing eNAMPT were able to alter the abundance of NAD+ and SIRT1 activity in recipient cells compared to pre-exercise EVs, indicating a pathway for inter-tissue signalling promoted through exercise. Our results suggest a mechanism to limit age-related NAD+ decline, through the systemic delivery of eNAMPT via EVs released during exercise.

Therapeutic ISCOMATRIX™ adjuvant vaccine elicits effective anti-tumor immunity in the TRAMP-C1 mouse model of prostate cancer.

Cancer vaccine development has proven challenging with the exception of some virally induced cancers for which prophylactic vaccines exist. Currently, there is only one FDA approved vaccine for the treatment of prostate cancer and as such prostate cancer continues to present a significant unmet medical need. In this study, we examine the effectiveness of a therapeutic cancer vaccine that combines the ISCOMATRIX™ adjuvant (ISCOMATRIX) with the Toll-like receptor 3 agonist, polyinosinic-polycytidylic acid (Poly I:C), and Flt3L, FMS-like tyrosine kinase 3 ligand. We employed the TRAMP-C1 (transgenic adenocarcinoma of the mouse prostate) model of prostate cancer and the self-protein mPAP (prostatic acid phosphatase) as the tumor antigen. ISCOMATRIX™-mPAP-Poly I:C-Flt3L was delivered in a therapeutic prime-boost regime that was consistently able to achieve complete tumor regression in 60% of animals treated and these tumor-free animals were protected upon rechallenge. Investigations into the underlying immunological mechanisms contributing to the effectiveness of this vaccine identified that both innate and adaptive responses are elicited and required. NK cells, CD4+ T cells and interferon-γ were all found to be critical for tumor control while tumor infiltrating CD8+ T cells became disabled by an immunosuppressive microenvironment. There is potential for broader application of this cancer vaccine, as we have been able to demonstrate effectiveness in two additional cancer models; melanoma (B16-OVA) and a model of B cell lymphoma (Eµ-myc-GFP-OVA).

Immunization Strategies Producing a Humoral IgG Immune Response against Devil Facial Tumor Disease in the Majority of Tasmanian Devils Destined for Wild Release.

Devil facial tumor disease (DFTD) is renowned for its successful evasion of the host immune system. Down regulation of the major histocompatabilty complex class I molecule (MHC-I) on the DFTD cells is a primary mechanism of immune escape. Immunization trials on captive Tasmanian devils have previously demonstrated that an immune response against DFTD can be induced, and that immune-mediated tumor regression can occur. However, these trials were limited by their small sample sizes. Here, we describe the results of two DFTD immunization trials on cohorts of devils prior to their wild release as part of the Tasmanian Government's Wild Devil Recovery project. 95% of the devils developed anti-DFTD antibody responses. Given the relatively large sample sizes of the trials (N = 19 and N = 33), these responses are likely to reflect those of the general devil population. DFTD cells manipulated to express MHC-I were used as the antigenic basis of the immunizations in both trials. Although the adjuvant composition and number of immunizations differed between trials, similar anti-DFTD antibody levels were obtained. The first trial comprised DFTD cells and the adjuvant combination of ISCOMATRIX™, polyIC, and CpG with up to four immunizations given at monthly intervals. This compared to the second trial whereby two immunizations comprising DFTD cells and the adjuvant combination ISCOMATRIX™, polyICLC (Hiltonol®) and imiquimod were given a month apart, providing a shorter and, therefore, more practical protocol. Both trials incorporated a booster immunization given up to 5 months after the primary course. A key finding was that devils in the second trial responded more quickly and maintained their antibody levels for longer compared to devils in the first trial. The different adjuvant combination incorporating the RNAase resistant polyICLC and imiquimod used in the second trial is likely to be responsible. The seroconversion in the majority of devils in these anti-DFTD immunization trials was remarkable, especially as DFTD is hallmarked by its immune evasion mechanisms. Microsatellite analyzes of MHC revealed that some MHC-I microsatellites correlated to stronger immune responses. These trials signify the first step in the long-term objective of releasing devils with immunity to DFTD into the wild.

Regression of devil facial tumour disease following immunotherapy in immunised Tasmanian devils.

Devil facial tumour disease (DFTD) is a transmissible cancer devastating the Tasmanian devil (Sarcophilus harrisii) population. The cancer cell is the 'infectious' agent transmitted as an allograft by biting. Animals usually die within a few months with no evidence of antibody or immune cell responses against the DFTD allograft. This lack of anti-tumour immunity is attributed to an absence of cell surface major histocompatibility complex (MHC)-I molecule expression. While the endangerment of the devil population precludes experimentation on large experimental groups, those examined in our study indicated that immunisation and immunotherapy with DFTD cells expressing surface MHC-I corresponded with effective anti-tumour responses. Tumour engraftment did not occur in one of the five immunised Tasmanian devils, and regression followed therapy of experimentally induced DFTD tumours in three Tasmanian devils. Regression correlated with immune cell infiltration and antibody responses against DFTD cells. These data support the concept that immunisation of devils with DFTD cancer cells can successfully induce humoral responses against DFTD and trigger immune-mediated regression of established tumours. Our findings support the feasibility of a protective DFTD vaccine and ultimately the preservation of the species.

The combination of ISCOMATRIX adjuvant and TLR agonists induces regression of established solid tumors in vivo.

The development of therapeutic vaccines for treatment of established cancer has proven challenging. Cancer vaccines not only need to induce a robust tumor Ag-specific immune response but also need to overcome the tolerogenic and immunosuppressive microenvironments that exist within many solid cancers. ISCOMATRIX adjuvant (ISCOMATRIX) is able to induce both tumor Ag-specific cellular and Ab responses to protect mice against tumor challenge, but this is insufficient to result in regression of established solid tumors. In the current study, we have used B16-OVA melanoma, Panc-OVA pancreatic, and TRAMP-C1 prostate cancer mouse tumor models to test therapeutic efficacy of ISCOMATRIX vaccines combined with other immune modulators. The coadministration of an ISCOMATRIX vaccine with the TLR3 agonist, polyinosinic-polycytidylic acid, and TLR9 agonist, CpG, reduced tumor growth in all tumor models and the presence of ISCOMATRIX in the formulation was critical for the therapeutic efficacy of the vaccine. This vaccine combination induced a robust and multifunctional CD8(+) T cell response. Therapeutic protection required IFN-γ and CD8(+) T cells, whereas NK and CD4(+) T cells were found to be redundant. ISCOMATRIX vaccines combined with TLR3 and TLR9 agonists represent a promising cancer immunotherapy strategy.

Combination of adjuvants: the future of vaccine design.

It is thought that the development of vaccines for the treatment of infectious diseases and cancer is likely to be achieved in the coming decades. This is partially due to a better understanding of the regulatory networks connecting innate with adaptive immune responses. The innate immune response is triggered by the recognition of conserved pathogen-associated molecular patterns by germ line-coded pattern recognition receptors. Several families of pattern recognition receptors have been characterized, including Toll-like receptors and nucleotide-binding domain receptors. The identification of their ligands has driven the development of novel adjuvants many of which have been tested in vaccine clinical trials. Here, the authors review recent preclinical data and clinical trial results supporting the view that combinations of adjuvants are the way forward in vaccine design. Multiadjuvanted vaccines can stimulate the broad and robust protective immune responses required to fight chronic infectious diseases and cancer.

ISCOMATRIX: a novel adjuvant for use in prophylactic and therapeutic vaccines against infectious diseases.

The ISCOMATRIX adjuvant has antigen delivery and presentation properties as well as immunomodulatory capabilities, which combine to provide enhanced and accelerated immune responses. The responses are broad, including a range of subclasses of antibodies as well as CD4(+) and CD8(+) T-cells. A range of ISCOMATRIX vaccines (ISCOMATRIX adjuvant combined with antigen) have now been tested in clinical trials and have been shown to be generally safe and well tolerated as well as immunogenic, generating both antibody (Ab) and T-cell responses. The mechanisms by which ISCOMATRIX adjuvant facilitates its immune effects are the scope of significant study and indicate that ISCOMATRIX adjuvant (i) rapidly traffics antigen into the cytosol of multiple dendritic cell subsets, (ii) induces the induction of an array of cytokines and chemokines and (iii) links the innate and adaptive immune responses in vivo in a Toll-like-receptor-independent but MyD88-dependent manner. These data highlight the clinical utility of ISCOMATRIX adjuvant in the development of prophylactic and therapeutic vaccines for infectious disease.

Application of CD27 as a marker for distinguishing human NK cell subsets.

It has long been recognized that human NK cells can be divided into two phenotypically and functionally distinct subsets, based on their levels of expression of CD56. We recently found that CD27 distinguishes subsets of mature mouse NK cells. Here we report that CD27 can be used as a marker to discriminate human NK cell subsets. The majority of peripheral blood human NK cells were CD27(lo)/CD56(dim) NK cells, whereas the minor CD27(hi) NK cell population correspondingly displayed a CD56(bright) phenotype. Distinctions between CD27(lo) and CD27(hi) NK cells in their receptor expression and typical NK cell functions such as cytotoxicity and cytokine production can be easily delineated. Therefore, we propose the dual use of CD27 and CD56 as maturation/subset markers for human NK cells. The identification of CD27 subsets in both mice and humans will allow more accurate projections of the role of NK cell subsets in murine models of human pathologies where NK cells are involved.

Demonstration of inflammation-induced cancer and cancer immunoediting during primary tumorigenesis.

Here we report the effects of loss of the Toll-like receptor-associated signaling adaptor myeloid-differentiation factor 88 (MyD88) on tumor induction in two distinct mouse models of carcinogenesis. The 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA)-induced skin papilloma model depends on proinflammatory processes, whereas the 3'-methylcholanthrene (MCA) induction of fibrosarcoma has been used by tumor immunologists to illustrate innate and adaptive immune surveillance of cancer. When exposed to a combination of DMBA/TPA, mice lacking MyD88 formed fewer skin papillomas than genetically matched WT controls treated in a similar manner. Unexpectedly, however, fewer MyD88-/- mice formed sarcomas than WT controls when exposed to MCA. In contrast, MyD88-deficient mice did not show a defective ability to reject highly immunogenic transplanted tumors, including MCA sarcomas. Despite the reported role of TNF in chronic inflammation, TNF-deficient mice were significantly more susceptible to MCA-induced sarcoma than WT mice. Overall, these data not only confirm the key role that MyD88 plays in promoting tumor development but also demonstrate that inflammation-induced carcinogenesis and cancer immunoediting can indeed occur in the same mouse tumor model.

Projeção da dinâmica da floresta natural de Terra-firme, região de Manaus-AM, com o uso da cadeia de transição probabilística de Markov / Dynamics of the Terra-firme primary forest in Manaus-AM region using the Markov probabilistic transition matrix

Os fatores que envolvem os processos da dinâmica da floresta influenciam a sua biodiversidade e, portanto, a qualidade da floresta. A definição de estratégias que envolve a proteção e o uso adequado da floresta manejada e a recuperação de áreas já degradadas tornam-se possível com o estudo da estrutura e dinâmica da floresta primária por meio de informações como a mortalidade, o recrutamento e a permanência das árvores no sistema florestal. Este trabalho teve como objetivo avaliar a dinâmica de uma floresta não perturbada e fazer projeções da dinâmica florestal usando a matriz de transição probabilística (Cadeia de Markov). As taxas de recrutamento, mortalidade e incremento foram determinadas a partir de inventários florestais realizados em dois transectos, nos sentidos Norte-Sul e Leste-Oeste (20 x 2500 m cada, totalizando 10 ha), localizados no km 50 da BR 174, na estrada vicinal ZF-2, Manaus/AM, nos anos de 2000 e 2004. A floresta acumulou 8,34 t.ha-1.ano-1 de biomassa fresca acima do solo. De acordo com projeção para 2008, o número total de árvores diminuirá em 2,67 por cento (de 5987 indivíduos (2004) para 5827 (2008)) e a mortalidade será 15 por cento maior (de 264 (2004) para 311 (2008)). O teste Qui-quadrado mostrou que não há diferença significativa (1 por cento de probabilidade) entre as informações coletadas e projetadas. Esses resultados permitem concluir que a Cadeia de Markov é um eficiente instrumento para projetar a dinâmica da floresta natural, contribuindo para o planejamento em curto prazo das atividades que utilizam os recursos florestais.

To combine protection and utilization of forest resources in the tropics, the understanding of forest dynamics is essential. It is also important in the definition of strategies for rehabilitation of degraded areas. In Forestry, forest dynamics could be translated as the understanding of recruitment, mortality and biomass increment rates over time. For this study, these rates were estimated based on measurements carried out in 2000 and 2004 over two transects measuring 20 by 2500 m (5 hectares) each, in Manaus region. This paper deals with forest dynamics of a pristine forest based on the probabilistic transition matrix (the first-order Markov Chain) approach. The main objective is to report 4-year (2000 to 2004) changes in the forest structure. Diameter distribution and tree mortality will be projected ahead to 2008 (t+2), based upon a 4-year period of observations completed in 2004 (t+1) and its immediate past in 2000 (t). In terms of fresh aboveground biomass, this site accumulated 8.34 t.ha-1.ano-1. The chi ² test has shown no statistical difference (p = 0.01) between observed diameter frequency and the expected projected by Markov Chain. This result indicates that the Markov Chain approach is a reliable tool to project the forest dynamics on a short-term basis. In 2008, the total number of individuals will have a decrease of 2.7 percent, and the mortality rate will 15 percent higher than in 2004.

Suppressor of cytokine signaling (SOCS)-5 is a potential negative regulator of epidermal growth factor signaling.

The suppressors of cytokine signaling (SOCS) proteins are a family of SH2 domain-containing intracellular inhibitors of cytokine signal transduction that act by several different mechanisms. Recent evidence suggests that the action of the SOCS proteins may extend beyond the cytokine receptors to signaling initiated by members of the tyrosine kinase receptor family. In this study, the ability of SOCS-5 to negatively regulate signaling cascades downstream of the epidermal growth factor receptor (EGF-R) has been examined by using an EGF-responsive cell line engineered to constitutively express the EGF-R and SOCS-5 or SOCS-5 mutants. SOCS-5 associated with the EGF-R complex in an EGF-independent manner, and the mitogenic response to EGF of all SOCS-5-expressing cell lines was dramatically inhibited when compared with control cell lines. Furthermore, this effect was abrogated after deletion of the SOCS-5 SOCS box. This result suggests that the inhibition of signaling occurs through enhanced proteasomal degradation of the EGF-R through SOCS box recruitment of E3 ubiquitin ligase activity.