Acute aerobic exercise-conditioned serum reduces colon cancer cell proliferation in vitro through interleukin-6-induced regulation of DNA damage.

Epidemiological evidence shows that regular physical activity is associated with reduced risk of primary and recurrent colon cancer. However, the underlying mechanisms of action are poorly understood. We evaluated the effects of stimulating a human colon cancer cell line (LoVo) with human serum collected before and after an acute exercise bout vs nonexercise control serum on cancer cell proliferation. We also measured exercise-induced changes in serum cytokines and intracellular protein expression to explore potential biological mechanisms. Blood samples were collected from 16 men with lifestyle risk factors for colon cancer (age ≥50 years; body mass index ≥25 kg/m2 ; physically inactive) before and immediately after an acute bout of moderate-intensity aerobic interval exercise (6 × 5 minutes intervals at 60% heart rate reserve) and a nonexercise control condition. Stimulating LoVo cells with serum obtained immediately after exercise reduced cancer cell proliferation compared to control (-5.7%; P = .002). This was accompanied by a decrease in LoVo cell γ-H2AX expression (-24.6%; P = .029), indicating a reduction in DNA damage. Acute exercise also increased serum IL-6 (24.6%, P = .002). Furthermore, stimulating LoVo cells with recombinant IL-6 reduced γ-H2AX expression (ß = -22.7%; P < .001) and cell proliferation (ß = -5.3%; P < .001) in a linear dose-dependent manner, mimicking the effect of exercise. These findings suggest that the systemic responses to acute aerobic exercise inhibit colon cancer cell proliferation in vitro, and this may be driven by IL-6-induced regulation of DNA damage and repair. This mechanism of action may partly underlie epidemiological associations linking regular physical activity with reduced colon cancer risk.

Anticancer activity of a novel methylated analogue of L-mimosine against an in vitro model of human malignant melanoma.

The anticancer activity of a series of novel synthesized, hydroxypyridone-based metal chelators (analogues of L-mimosine) was evaluated in an in vitro model of melanoma consisting of malignant melanoma (A375), non-melanoma epidermoid carcinoma (A431) and immortalized non-malignant keratinocyte (HaCaT) cells. More specifically, we have demonstrated that the L-enantiomer of a methylated analogue of L-mimosine (compound 22) can exert a potent anticancer effect in A375 cells when compared to either A431 or HaCaT cells. Moreover, we have demonstrated that this analogue has the ability to i) promote increased generation of reactive oxygen species (ROS), ii) activate both intrinsic and extrinsic apoptosis and iii) induce perturbations in cell cycle growth arrest. Our data highlights the potential of compound 22 to act as a promising therapeutic agent against an in vitro model of human malignant melanoma.

Anti-bacterial antibody and T cell responses in bronchiectasis are differentially associated with lung colonization and disease.

BACKGROUND: As a way to determine markers of infection or disease informing disease management, and to reveal disease-associated immune mechanisms, this study sought to measure antibody and T cell responses against key lung pathogens and to relate these to patients' microbial colonization status, exacerbation history and lung function, in Bronchiectasis (BR) and Chronic Obstructive Pulmonary Disease (COPD). METHODS: One hundred nineteen patients with stable BR, 58 with COPD and 28 healthy volunteers were recruited and spirometry was performed. Bacterial lysates were used to measure specific antibody responses by ELISA and T cells by ELIspot. Cytokine secretion by lysate-stimulated T cells was measured by multiplex cytokine assay whilst activation phenotype was measured by flow cytometry. RESULTS: Typical colonization profiles were observed in BR and COPD, dominated by P.aeruginosa, H.influenzae, S.pneumoniae and M.catarrhalis. Colonization frequency was greater in BR, showing association with increased antibody responses against P.aeruginosa compared to COPD and HV, and with sensitivity of 73% and specificity of 95%. Interferon-gamma T cell responses against P.aeruginosa and S.pneumoniae were reduced in BR and COPD, whilst reactive T cells in BR had similar markers of homing and senescence compared to healthy volunteers. Exacerbation frequency in BR was associated with increased antibodies against P. aeruginosa, M.catarrhalis and S.maltophilia. T cell responses against H.influenzae showed positive correlation with FEV1% (r = 0.201, p = 0.033) and negative correlation with Bronchiectasis Severity Index (r = - 0.287, p = 0.0035). CONCLUSION: Our findings suggest a difference in antibody and T cell immunity in BR, with antibody being a marker of exposure and disease in BR for P.aeruginosa, M.catarrhalis and H.influenzae, and T cells a marker of reduced disease for H.influenzae.

Reduction of Glucocorticoid Receptor Function in Chronic Fatigue Syndrome.

Glucocorticoid receptor (GR) function may have aetiopathogenic significance in chronic fatigue syndrome (CFS), via its essential role in mediating inflammatory responses as well as in hypothalamic-pituitary-adrenal axis regulation. GR function can be estimated ex vivo by measuring dexamethasone (dex) modulation of cytokine response to lipopolysaccharide (LPS), and in vivo using the impact of dex on cortisol levels. This study aimed to compare the GR function between CFS (n = 48), primary Sjögren's syndrome (a disease group control) (n = 27), and sedentary healthy controls (HCs) (n = 20), and to investigate its relationship with clinical measures. In the GR ex vivo response assay, whole blood was diluted and incubated with LPS (to stimulate cytokine production), with or without 10 or 100 nanomolar concentrations of dex. Cytometric bead array (CBA) and flow cytometry enabled quantification of cytokine levels (TNFα, interleukin- (IL-) 6, and IL-10) in the supernatants. In the in vivo response assay, five plasma samples were taken for determination of total cortisol concentration using ELISA at half-hourly intervals on two consecutive mornings separated by ingestion of 0.5 mg of dex at 11 pm. The association of the data from the in vivo and ex vivo analyses with reported childhood adversity was also examined. CFS patients had reduced LPS-induced IL-6 and TNFα production compared to both control groups and reduced suppression of TNFα by the higher dose of dex compared to HCs. Cortisol levels, before or after dex, did not differ between CFS and HCs. Cortisol levels were more variable in CFS than HCs. In the combined group (CFS plus HC), cortisol concentrations positively and ex vivo GR function (determined by dex-mediated suppression of IL-10) negatively correlated with childhood adversity score. The results do not support the hypothesis that GR dysregulation is aetiopathogenic in CFS and suggest that current and future endocrine cross-sectional studies in CFS may be vulnerable to the confounding influence of childhood trauma which is likely increased by comorbid depression.

MicroRNAs in the skin: role in development, homoeostasis and regeneration.

The skin is the largest organ of the integumentary system and possesses a vast number of functions. Due to the distinct layers of the skin and the variety of cells which populate each, a tightly regulated network of molecular signals control development and regeneration, whether due to programmed cell termination or injury. MicroRNAs (miRs) are a relatively recent discovery; they are a class of small non-coding RNAs which possess a multitude of biological functions due to their ability to regulate gene expression via post-transcriptional gene silencing. Of interest, is that a plethora of data demonstrates that a number of miRs are highly expressed within the skin, and are evidently key regulators of numerous vital processes to maintain non-aberrant functioning. Recently, miRs have been targeted as therapeutic interventions due to the ability of synthetic 'antagomiRs' to down-regulate abnormal miR expression, thereby potentiating wound healing and attenuating fibrotic processes which can contribute to disease such as systemic sclerosis (SSc). This review will provide an introduction to the structure and function of the skin and miR biogenesis, before summarizing the literature pertaining to the role of miRs. Finally, miR therapies will also be discussed, highlighting important future areas of research.

Myocardial ischemia and reperfusion leads to transient CD8 immune deficiency and accelerated immunosenescence in CMV-seropositive patients.

RATIONALE: There is mounting evidence of a higher incidence of coronary heart disease in cytomegalovirus-seropositive individuals. OBJECTIVE: The aim of this study was to investigate whether acute myocardial infarction triggers an inflammatory T-cell response that might lead to accelerated immunosenescence in cytomegalovirus-seropositive patients. METHODS AND RESULTS: Thirty-four patients with acute myocardial infarction undergoing primary percutaneous coronary intervention were longitudinally studied within 3 months after reperfusion (Cohort A). In addition, 54 patients with acute myocardial infarction and chronic myocardial infarction were analyzed in a cross-sectional study (Cohort B). Cytomegalovirus-seropositive patients demonstrated a greater fall in the concentration of terminally differentiated CD8 effector memory T cells (TEMRA) in peripheral blood during the first 30 minutes of reperfusion compared with cytomegalovirus-seronegative patients (-192 versus -63 cells/µL; P=0.008), correlating with the expression of programmed cell death-1 before primary percutaneous coronary intervention (r=0.8; P=0.0002). A significant proportion of TEMRA cells remained depleted for ≥3 months in cytomegalovirus-seropositive patients. Using high-throughput 13-parameter flow cytometry and human leukocyte antigen class I cytomegalovirus-specific dextramers, we confirmed an acute and persistent depletion of terminally differentiated TEMRA and cytomegalovirus-specific CD8(+) cells in cytomegalovirus-seropositive patients. Long-term reconstitution of the TEMRA pool in chronic cytomegalovirus-seropositive postmyocardial infarction patients was associated with signs of terminal differentiation including an increase in killer cell lectin-like receptor subfamily G member 1 and shorter telomere length in CD8(+) T cells (2225 versus 3397 bp; P<0.001). CONCLUSIONS: Myocardial ischemia and reperfusion in cytomegalovirus-seropositive patients undergoing primary percutaneous coronary intervention leads to acute loss of antigen-specific, terminally differentiated CD8 T cells, possibly through programmed cell death-1-dependent programmed cell death. Our results suggest that acute myocardial infarction and reperfusion accelerate immunosenescence in cytomegalovirus-seropositive patients.

Antibody and T-cell responses associated with experimental human malaria infection or vaccination show limited relationships.

This study examined specific antibody and T-cell responses associated with experimental malaria infection or malaria vaccination, in malaria-naive human volunteers within phase I/IIa vaccine trials, with a view to investigating inter-relationships between these types of response. Malaria infection was via five bites of Plasmodium falciparum-infected mosquitoes, with individuals reaching patent infection by 11-12 days, having harboured four or five blood-stage cycles before drug clearance. Infection elicited a robust antibody response against merozoite surface protein-119 , correlating with parasite load. Classical class switching was seen from an early IgM to an IgG1-dominant response of increasing affinity. Malaria-specific T-cell responses were detected in the form of interferon-γ and interleukin-4 (IL-4) ELIspot, but their magnitude did not correlate with the magnitude of antibody or its avidity, or with parasite load. Different individuals who were immunized with a virosome vaccine comprising influenza antigens combined with P. falciparum antigens, demonstrated pre-existing interferon-γ, IL-2 and IL-5 ELIspot responses against the influenza antigens, and showed boosting of anti-influenza T-cell responses only for IL-5. The large IgG1-dominated anti-parasite responses showed limited correlation with T-cell responses for magnitude or avidity, both parameters being only negatively correlated for IL-5 secretion versus anti-apical membrane antigen-1 antibody titres. Overall, these findings suggest that cognate T-cell responses across a range of magnitudes contribute towards driving potentially effective antibody responses in infection-induced and vaccine-induced immunity against malaria, and their existence during immunization is beneficial, but magnitudes are mostly not inter-related.

The Bisdioxopiperazine ICRF-193 Attenuates LPS-induced IL-1ß Secretion by Macrophages.

Inhibiting pathological secretion of Interleukin-1ß has shown beneficial effects in disease models and in the clinic and thus there is interest in finding inhibitors that can reduce its release from macrophages in response to their activation by foreign pathogens. We used an in vitro human macrophage model to investigate whether ICRF-193, a Topoisomerase II inhibitor could modulate IL1B mRNA expression and IL-1ß secretion. These macrophage-like cells readily secrete IL-1ß in response to Lipopolysaccharide (LPS). Upon exposure to a non-toxic dose of ICRF-193, IL-1ß secretion was diminished by ~ 40%; however, level of transcription of IL1B was unaffected. We show that there was no Topoisomerase 2B (TOP2B) binding to several IL1B gene sites, which may explain why ICRF-193 does not alter IL1B mRNA levels. Hence, we show for the first time that ICRF-193 can reduce IL-1ß secretion. Its low cost and the development of water-soluble prodrugs of ICRF-193 warrants its further investigation in the modulation of pathological secretion of this cytokine for the treatment of inflammatory disorders. (165 words).

Tracking Circulating HLA-Specific IgG-Producing Memory B Cells with the B-Cell ImmunoSpot Assay.

Donor-specific antibodies (DSA) against human leukocyte antigen (HLA) molecules are a major risk factor for rejection of transplanted organs (in antibody-mediated rejection [ABMR]), particularly in patients who have prior sensitization or receive insufficient immunosuppression through minimization or noncompliance. These DSA are measured routinely in the serum of patients prior to transplantation mainly using bead-based technologies or cell-based assays. However, the absence of detectable serum DSA does not always reflect the absence of sensitization or histologically defined ABMR, and so it has been proposed that the detection and measurement of memory B cells capable of secreting antibodies against donor HLA antigens could be carried out using B-cell ImmunoSpot, to better inform the degree of immune sensitization of transplant patients prior to as well as after transplantation. Such an assay is described here.

Heat-shock proteins as dendritic cell-targeting vaccines--getting warmer.

Heat-shock proteins (hsp) provide a natural link between innate and adaptive immune responses by combining the ideal properties of antigen carriage (chaperoning), targeting and activation of antigen-presenting cells (APC), including dendritic cells (DC). Targeting is achieved through binding of hsp to distinct cell surface receptors and is followed by antigen internalization, processing and presentation. An improved understanding of the interaction of hsp with DC has driven the development of numerous hsp-containing vaccines, designed to deliver antigens directly to DC. Studies in mice have shown that for cancers, such vaccines generate impressive immune responses and protection from tumour challenge. However, translation to human use, as for many experimental immunotherapies, has been slow partly because of the need to perform trials in patients with advanced cancers, where demonstration of efficacy is challenging. Recently, the properties of hsp have been used for development of prophylactic vaccines against infectious diseases including tuberculosis and meningitis. These hsp-based vaccines, in the form of pathogen-derived hsp-antigen complexes, or recombinant hsp combined with selected antigens in vitro, offer an innovative approach against challenging diseases where broad antigen coverage is critical.

Clinical assessment of a recombinant simian adenovirus ChAd63: a potent new vaccine vector.

BACKGROUND: Vaccine development in human Plasmodium falciparum malaria has been hampered by the exceptionally high levels of CD8(+) T cells required for efficacy. Use of potently immunogenic human adenoviruses as vaccine vectors could overcome this problem, but these are limited by preexisting immunity to human adenoviruses. METHODS: From 2007 to 2010, we undertook a phase I dose and route finding study of a new malaria vaccine, a replication-incompetent chimpanzee adenovirus 63 (ChAd63) encoding the preerythrocytic insert multiple epitope thrombospondin-related adhesion protein (ME-TRAP; n = 54 vaccinees) administered alone (n = 28) or with a modified vaccinia virus Ankara (MVA) ME-TRAP booster immunization 8 weeks later (n = 26). We observed an excellent safety profile. High levels of TRAP antigen-specific CD8(+) and CD4(+) T cells, as detected by interferon γ enzyme-linked immunospot assay and flow cytometry, were induced by intramuscular ChAd63 ME-TRAP immunization at doses of 5 × 10(10) viral particles and above. Subsequent administration of MVA ME-TRAP boosted responses to exceptionally high levels, and responses were maintained for up to 30 months postvaccination. CONCLUSIONS: The ChAd63 chimpanzee adenovirus vector appears safe and highly immunogenic, providing a viable alternative to human adenoviruses as vaccine vectors for human use. CLINICAL TRIALS REGISTRATION: NCT00890019.

Role of monocytes/macrophages in renin-angiotensin system-induced hypertension and end organ damage.

The renin-angiotensin system (RAS) is a central modulator of cardiovascular physiology. Pathophysiology of hypertension is commonly accompanied by hyper-activation of RAS. Angiotensin II receptor blockers (ARBs) and Angiotensin-converting enzyme (ACE) inhibitors are the gold standard treatment for hypertension. Recently, several studies highlighted the crucial role of immune system in hypertension. Angiotensin-II-induced hypertension is associated with low grade inflammation characterized by innate and adaptive immune system dysfunction. Throughout the progression of hypertension, monocyte/macrophage cells appear to have a crucial role in vascular inflammation and interaction with the arterial wall. Since myelomonocytic cells potentially play a key role in angiotensin-II-induced hypertension and organ damage, pharmacological targeting of RAS components in monocyte/macrophages may possibly present an innovative strategy for treatment of hypertension and related pathology.

The Effect of Menstrual-Cycle Phase on Immune Responses to a 5-km Cycling Time Trial: An Exploratory Study.

PURPOSE: Exercise has transient effects on the immune system that could influence infection risk and tissue recovery after exercise. Little is known about how the menstrual cycle interacts with the immune responses to acute exercise. This exploratory study sought to evaluate the effect of menstrual-cycle phase on peripheral blood mononuclear cell counts before and immediately after a bout of intense aerobic exercise. METHODS: Seven naturally menstruating women (age: 27 [3] y) completed three 5-km cycling time trials coinciding with the early-follicular, late-follicular, and mid-luteal stage, confirmed by hormonal measurement. Venous blood samples were taken and examined for the presence of immune cell types using flow cytometry. RESULTS: Reductions in circulating CCR7+CD45RA+ naïve CD4+ T cells, CD4+CD25+ regulatory T cells, and CD56+CD57+ natural killer cells observed during the early-follicular phase were attenuated when exercise was performed during the late-follicular phase. Similarly, reductions in circulating CD56+CD57+ natural killer cells and CD14+TLR4+ monocytes following exercise in the early-follicular phase were abolished when exercise was performed in the midluteal phase. CONCLUSIONS: These preliminary findings indicate that the effect of acute high-intensity exercise on immune-cell mobilization and activation varies across the menstrual cycle, potentially impacting the anti-inflammatory effects of regulatory T cells and the cell-mediated effects of both natural killer CD57+ cells and monocytes expressing TLR4.

Next generation organoid engineering to replace animals in cancer drug testing.

Cancer therapies have several clinical challenges associated with them, namely treatment toxicity, treatment resistance and relapse. Due to factors ranging from patient profiles to the tumour microenvironment (TME), there are several hurdles to overcome in developing effective treatments that have low toxicity that can mitigate emergence of resistance and occurrence of relapse. De novo cancer development has the highest drug attrition rates with only 1 in 10,000 preclinical candidates reaching the market. To alleviate this high attrition rate, more mimetic and sustainable preclinical models that can capture the disease biology as in the patient, are required. Organoids and next generation 3D tissue engineering is an emerging area that aims to address this problem. Advancement of three-dimensional (3D) in vitro cultures into complex organoid models incorporating multiple cell types alongside acellular aspects of tissue microenvironments can provide a system for therapeutic testing. Development of microfluidic technologies have furthermore increased the biomimetic nature of these models. Additionally, 3D bio-printing facilitates generation of tractable ex vivo models in a controlled, scalable and reproducible manner. In this review we highlight some of the traditional preclinical models used in cancer drug testing and debate how next generation organoids are being used to replace not only animal models, but also some of the more elementary in vitro approaches, such as cell lines. Examples of applications of the various models will be appraised alongside the future challenges that still need to be overcome.

Revisiting the Elusive Hepatitis C Vaccine.

The impactful discovery and subsequent characterisation of hepatitis C virus (HCV), an RNA virus of the flavivirus family, led to the awarding of the 2020 Nobel Prize in Physiology or Medicine to Harvey J. Alter, Michael Houghton and Charles M. Rice [...].

Malaria vaccine development: lessons from the field.

Studies and trials in the field are key to the development of a vaccine for malaria. Our limited knowledge of naturally acquired immunity and of transmission dynamics and disease causation in the field imposes limitations on our ability to predict the efficacy of candidate vaccinations, and the eventual outcome on deploying an efficacious vaccine at a population level.

Multiple functions of human T cells generated by experimental malaria challenge.

Protective immunity generated following malaria infection may be comprised of Ab or T cells against malaria Ag of different stages; however, the short-lived immunity that is observed suggests deficiency in immune memory or regulatory activity. In this study, cellular immune responses were investigated in individuals receiving Plasmodium falciparum sporozoite challenge by the natural (mosquito bite) route as part of a malaria vaccine efficacy trial. Parasitemia, monitored by blood film microscopy and PCR, was subsequently cleared with drugs. All individuals demonstrated stable IFN-gamma, IL-2 and IL-4 ex vivo ELISPOT effector responses against P. falciparum-infected RBC (iRBC) Ag, 28 and 90 days after challenge. However, infected RBC-specific central memory responses, as measured by IFN-gamma cultured ELISPOT, were low and unstable over time, despite CD4(+) T cells being highly proliferative by CFSE dilution, and showed an inverse relationship to parasite density. In support of the observation of poor memory, co-culture experiments showed reduced responses to common recall Ag, indicating malaria-specific regulatory activity. This activity could not be accounted for by the expression of IL-10, TGF-beta, FOXP3 or CTLA-4, but proliferating T cells expressed high levels of CD95, indicating a pro-apoptotic phenotype. Lastly, there was an inverse relationship between FOXP3 expression, when measured 10 days after challenge, and ex vivo IFN-gamma measured more than 100 days later. This study shows that malaria infection elicits specific Th1 and Th2 effector cells, but concomitant weak central memory and regulatory activity, which may help to explain the short-lived immunity observed.

Integrating microfluidics and biosensing on a single flexible acoustic device using hybrid modes.

Integration of microfluidics and biosensing functionalities on a single device holds promise in continuous health monitoring and disease diagnosis for point-of-care applications. However, the required functions of fluid handling and biomolecular sensing usually arise from different actuation mechanisms. In this work, we demonstrate that a single acoustofluidic device, based on a flexible thin film platform, is able to generate hybrid wave modes, which can be used for fluidic actuation (Lamb waves) and biosensing (thickness shear waves). On this integrated platform, we show multiple and sequential functions of mixing, transport and disposal of liquid volumes using Lamb waves, whilst the thickness bulk shear waves allow us to sense the chemotherapeutic Imatinib, using an aptamer-based strategy, as would be required for therapy monitoring. Upon binding, the conformation of the aptamer results in a change in coupled mass, which has been detected. This platform architecture has the potential to generate a wide range of simple sample-to-answer biosensing acoustofluidic devices.