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The presence of an immunosuppressive tumour microenvironment in oesophageal adenocarcinoma (OAC) is a major contributor to poor responses. Novel treatment strategies are required to supplement current regimens and improve patient survival. This study examined the immunomodulatory effects that radiation therapy and chemokine receptor antagonism impose on T cell phenotypes in OAC with a primary goal of identifying potential therapeutic targets to combine with radiation to improve anti-tumour responses. Compared with healthy controls, anti-tumour T cell function was impaired in OAC patients, demonstrated by lower IFN-γ production by CD4 + T helper cells and lower CD8 + T cell cytotoxic potential. Such diminished T cell effector functions were enhanced following treatment with clinically relevant doses of irradiation. Interestingly, CCR5 + T cells were significantly more abundant in OAC patient blood compared with healthy controls, and CCR5 surface expression by T cells was further enhanced by clinically relevant doses of irradiation. Moreover, irradiation enhanced T cell migration towards OAC patient-derived tumour-conditioned media (TCM). In vitro treatment with the CCR5 antagonist Maraviroc enhanced IFN-γ production by CD4 + T cells and increased the migration of irradiated CD8 + T cells towards irradiated TCM, suggesting its synergistic therapeutic potential in combination with irradiation. Overall, this study highlights the immunostimulatory properties of radiation in promoting anti-tumour T cell responses in OAC and increasing T cell migration towards chemotactic cues in the tumour. Importantly, the CCR5 antagonist Maraviroc holds promise to be repurposed in combination with radiotherapy to promote anti-tumour T cell responses in OAC.

Aim: Visceral obesity is a key risk factor in the development of oesophagogastric junctional adenocarcinoma (OGJ), predominantly via generation of systemic low grade inflammation. Obesity-induced inflammation promotes resistance to current standards of care, enhancing tumour cell growth and survival. This study investigates the effect of the visceral adipose tissue secretome from OGJ patients with early versus advanced tumours on T-cell immunity and the role of immune checkpoint blockade in enhancing anti-tumour immunity., Methods and Results: Visceral adipose conditioned media (ACM) from both early and late-stage OGJ patients significantly altered T cell activation status, upregulating co-stimulatory marker CD27 on T cells. ACM from both early and late-stage OGJ patients significantly altered immune checkpoint expression profiles downregulating immune checkpoints (ICs) on the surface of dual Th1/17-like and Th17-like cells and upregulating ICs on the surface of Th1-like cells and Treg cells. ACM derived from early-stage OGJ patients but not late-stage OGJ patients increased IFN-γ production by T cells. The addition of immune checkpoint blockers (ICBs) did not increase IFN-γ production by T cells in the presence of late-stage ACM, collectively highlighting the dichotomous immunostimulatory effect of early-stage ACM and immune-inhibitory effect of late-stage ACM. Interestingly, ACM from early-stage OGJ patients was more pro-inflammatory than ACM from late-stage patients, reflected by decreased levels of IL-17A/F, TNF-α, IL-1RA and IL-5., Conclusion: The ACM-induced upregulation of ICs on T cells highlights a therapeutic vulnerability that could be exploited by ICBs to harness anti-cancer immunity and improve clinical outcomes for OGJ patients. Schematic workflow - (A) visceral adipose tissue was taken from OAC patients at time of surgery and cultured for 72 h in media. (B) The harvested ACM was co-cultured with healthy donor PBMCs that were concurrently activated with anti-CD3/28 for 48 h and T cell immunophenotyping was carried out by flow cytometry. Key findings - (A) Early and late stage ACM enhanced a Th1-like phenotype and upregulated CTLA-4 on Th1-like cells. A Th17-like phenotype was also enhanced in addition with a Treg-like phenotype. CTLA-4 and PD-L1 were upregulated on the surface of Treg-like cells. (B) ICB-attenuated IL-17 production by T cells. However, ACM attenuated ICB-mediated reduction in IL-10 production by T cells. Higher levels of pro-inflammatory factors were found in early stage ACM compared with late stage ACM., (© 2023. The Author(s).)

Aim: Use of immune checkpoint blockade to enhance T cell-mediated immunity within the hostile tumour microenvironment (TME) is an attractive approach in oesophageal adenocarcinoma (OAC). This study explored the effects of the hostile TME, including nutrient deprivation and hypoxia, on immune checkpoint (IC) expression and T cell phenotypes, and the potential use of nivolumab to enhance T cell function under such conditions., Methods and Results: ICs were upregulated on stromal immune cells within the tumour including PD-L2, CTLA-4 and TIGIT. OAC patient-derived PBMCs co-cultured with OE33 OAC cells upregulated LAG-3 and downregulated the co-stimulatory marker CD27 on T cells, highlighting the direct immunosuppressive effects of tumour cells on T cells. Hypoxia and nutrient deprivation altered the secretome of OAC patient-derived PBMCs, which induced upregulation of PD-L1 and PD-L2 on OE33 OAC cells thus enhancing an immune-resistant phenotype. Importantly, culturing OAC patient-derived PBMCs under dual hypoxia and glucose deprivation, reflective of the conditions within the hostile TME, upregulated an array of ICs on the surface of T cells including PD-1, CTLA-4, A2aR, PD-L1 and PD-L2 and decreased expression of IFN-γ by T cells. Addition of nivolumab under these hostile conditions decreased the production of pro-tumorigenic cytokine IL-10., Conclusion: Collectively, these findings highlight the immunosuppressive crosstalk between tumour cells and T cells within the OAC TME. The ability of nivolumab to suppress pro-tumorigenic T cell phenotypes within the hostile TME supports a rationale for the use of immune checkpoint blockade to promote anti-tumour immunity in OAC. Study schematic: (A) IC expression profiles were assessed on CD45 + cells in peripheral whole blood and infiltrating tumour tissue from OAC patients in the treatment-naïve setting. (B) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then co-cultured for 48 h with OE33 cells. T cell phenotypes were then assessed by flow cytometry. (C) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then further cultured under conditions of nutrient deprivation or hypoxia for 48 h and T cell phenotypes were then assessed by flow cytometry., Key Findings: (A) TIGIT, CTLA-4 and PD-L2 were upregulated on CD45 + immune cells and CTLA-4 expression on CD45 + cells correlated with a subsequent decreased response to neoadjuvant regimen. (B) Following a 48 h co-culture with OE33 cells, T cells upregulated LAG-3 and decreased CD27 co-stimulatory marker. (C) Nutrient deprivation and hypoxia upregulated a range of ICs on T cells and decreased IFN-γ production by T cells. Nivolumab decreased IL-10 production by T cells under nutrient deprivation-hypoxic conditions., (© 2022. The Author(s).)

Introduction: This timely study assesses the immunosuppressive effects of surgery on cytotoxic Th1-like immunity and investigates if immune checkpoint blockade (ICB) can boost Th1-like immunity in the perioperative window in upper gastrointestinal cancer (UGI) patients., Methods: PBMCs were isolated from 11 UGI patients undergoing tumour resection on post-operative days (POD) 0, 1, 7 and 42 and expanded ex vivo using anti-CD3/28 and IL-2 for 5 days in the absence/presence of nivolumab or ipilimumab. T cells were subsequently immunophenotyped via flow cytometry to determine the frequency of T helper (Th)1-like, Th1/17-like, Th17-like and regulatory T cell (Tregs) subsets and their immune checkpoint expression profile. Lymphocyte secretions were also assessed via multiplex ELISA (IFN-γ, granzyme B, IL-17 and IL-10). The 48h cytotoxic ability of vehicle-, nivolumab- and ipilimumab-expanded PBMCs isolated on POD 0, 1, 7 and 42 against radiosensitive and radioresistant oesophageal adenocarcinoma tumour cells (OE33 P and OE33 R) was also examined using a cell counting kit-8 (CCK-8) assay to determine if surgery affected the killing ability of lymphocytes and whether the use of ICB could enhance cytotoxicity., Results: Th1-like immunity was suppressed in expanded PBMCs in the immediate post-operative setting. The frequency of expanded circulating Th1-like cells was significantly decreased post-operatively accompanied by a decrease in IFN-γ production and a concomitant increase in the frequency of expanded regulatory T cells with an increase in circulating levels of IL-10. Interestingly, PD-L1 and CTLA-4 immune checkpoint proteins were also upregulated on expanded Th1-like cells post-operatively. Additionally, the cytotoxic ability of expanded lymphocytes against oesophageal adenocarcinoma tumour cells was abrogated post-surgery. Of note, the addition of nivolumab or ipilimumab attenuated the surgery-mediated suppression of lymphocyte cytotoxicity, demonstrated by a significant increase in tumour cell killing and an increase in the frequency of Th1-like cells and Th1 cytokine production., Conclusion: These findings support the hypothesis of a surgery-mediated suppression in Th1-like cytotoxic immunity and highlights a rationale for the use of ICB within the perioperative setting to abrogate tumour-promoting effects of surgery and ameliorate the risk of recurrence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Davern, Gaughan, O’ Connell, Moran, Mylod, Sheppard, Ramjit, Yun-Tong Kung, Phelan, Davey, Ryan, Butler, Quinn, Howard, Tone, Phoenix, Butt, Lynam-Lennon, Maher, Ravi, Donohoe, Reynolds, Lysaght and Donlon.)

Tumour acidosis contributes to cancer progression by inhibiting anti-tumour immunity. However, the effect of acidosis on anti-tumour T cell phenotypes in oesophageal adenocarcinoma (OAC) is unknown. Therefore, this study investigated the effect of acidosis on anti-tumour T cell profiles and if immune checkpoint blockade (ICB) could enhance anti-tumour T cell immunity under acidosis. Acidic conditions substantially altered immune checkpoint expression profiles of OAC patient-derived T cells, upregulating TIM-3, LAG-3 and CTLA-4. Severe acidosis (pH 5.5) significantly decreased the percentage of central memory CD4 + T cells, an effect that was attenuated by ICB treatment. ICB increased T cell production of IFN-γ under moderate acidosis (pH 6.6) but not severe acidosis (pH 5.5) and decreased IL-10 production by T cells under severe acidic conditions only. A link between lactate and metastasis was also depicted; patients with nodal metastasis had higher serum lactate levels (p = 0.07) which also positively correlated with circulating levels of pro-angiogenic factor Tie-2. Our findings establish that acidosis-induced upregulation of immune checkpoints on T cells may potentially contribute to immune evasion and disease progression in OAC. However, acidic conditions curtailed ICB efficacy, supporting a rationale for utilizing systemic oral buffers to neutralize tumour acidity to improve ICB efficacy. Study schematic-PBMCs were isolated from OAC patients (A) and expanded ex vivo for 7 days using anti-CD3/28 +IL-2 T cell activation protocol (B) and further cultured for 48 h under increasing acidic conditions in the absence or presence of immune checkpoint blockade (nivolumab, ipilimumab or dual nivolumab + ipilimumab) (C). Immunophenotyping was then carried out to assess immune checkpoint expression profiles and anti-tumour T cell phenotypes (D). Serum lactate was assessed in OAC patients (E-F) and levels were correlated with patient demographics (G) and the levels of circulating immune/pro-angiogenic cytokines that were determined by multiplex ELISA (H). Key Findings-severe acidic conditions upregulated multiple immune checkpoints on T cells (I). Efficacy of ICB was curtailed under severe acidic conditions (J). Circulating lactate levels positively correlated with circulating levels of pro-angiogenic factor tie-2 and higher serum lactate levels were found in patients who had nodal metastasis (K)., (© 2022. The Author(s).)

The perioperative period theoretically is a critical time of opportunity for the progression of pre-existing tumour micrometastasis. Therefore,the timing of introducing cancer therapies including chemotherapy, radiation therapy and immunotherapies in the postoperative period is important. A thorough exploration of the perioperative immune events at a cellular level in combination with an intricate review of available clinical rials was conducted to extrapolate the effects of oncological surgery on the perioperative immune milieu.This is timely in view of the recently published Checkmate-577 trial which demonstrated significant disease-free survival in carcinoma of the gastroesophageal junction with the use of adjuvant anti-programmed cell deathprotein 1(PD-1) immunotherapy.This review focusing in particular on perioperative immunosuppression, identifies potential modifiable factors, the effects of perioperative conditioning and optimisation, the most recent trials in the curative setting for Gastrointestinal malignancies and the new treatment avenues possible in the context of the combination of immunotherapy and major oncological gastrointestinal surgery., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Response rates to immune checkpoint blockade (ICB) remain low in oesophageal adenocarcinoma (OAC). Combining ICB with immunostimulatory chemotherapies to boost response rates is an attractive approach for converting 'cold' tumours into 'hot' tumours. This study profiled immune checkpoint (IC) expression on circulating and tumour-infiltrating T cells in OAC patients and correlated these findings with clinical characteristics. The effect of first-line chemotherapy regimens (FLOT and CROSS) on anti-tumour T cell immunity was assessed to help guide design of ICB and chemotherapy combinations in the first-line setting. The ability of ICB to enhance lymphocyte-mediated cytolysis of OAC cells in the absence and presence of post-FLOT and post-CROSS chemotherapy tumour cell secretome was assessed by a CCK-8 assay. Expression of ICs on T cells positively correlated with higher grade tumours and a subsequent poor response to neoadjuvant treatment. First-line chemotherapy regimens substantially altered IC expression profiles of T cells increasing PD-1, A2aR, KLRG-1, PD-L1, PD-L2 and CD160 and decreasing TIM-3 and LAG-3. In addition, pro-inflammatory T cell cytokine profiles were enhanced by first-line chemotherapy regimens. T cell activation status was significantly altered; both chemotherapy regimens upregulated co-stimulatory markers ICOS and CD69 yet downregulated co-stimulatory marker CD27. However, ICB attenuated chemotherapy-induced downregulation of CD27 on T cells and promoted differentiation of effector memory T cells into a terminally differentiated state. Importantly, dual nivolumab-ipilimumab treatment increased lymphocyte-mediated cytolysis of OAC cells, an effect further enhanced in the presence of post-FLOT tumour cell secretome. These findings justify a rationale to administer ICBs concurrently with first-line chemotherapies., (Copyright © 2022. Published by Elsevier Inc.)

Recent studies have demontrated that immune checkpoint receptors are expressed on the surface of oesophageal adenocarcinoma (OAC) cells and might confer a survival advantage. This study explores the role of PD-1 and TIGIT signalling in OAC cells in either promoting or inhibiting the survival of OAC cells under characteristic features of the tumour microenvironment including nutrient-deprivation and hypoxia. PD-1 and TIGIT are expressed in normal and pre-malignant oesophageal epithelial cells and this expression significantly decreases along the normal- Barrett's Oesophagus- OAC disease sequence. However, glucose-deprivation and hypoxia significantly upregulated PD-1 and TIGIT on the surface of OAC cells in vitro. PD-1 blockade decreased OAC cell proliferation under normoxia but enhanced proliferation and decreased cell death in OAC cells under hypoxia and glucose-deprivation. TIGIT blockade decreased proliferation and induced OAC cell death, an effect that was maintained under nutrient-deprivation and hypoxia. Basal respiration and glycolytic reserve were enhanced and GLUT1 was upregulated on the surface of a subpopulation of OAC cells following PD-1 blockade. In contrast, TIGIT blockade enhanced a glycolytic phenotype in OAC cells, yet decreased other metabolic parameters including oxidative phosphorylation and basal respiration. Interestingly, inhibition of oxidative phosphorylation significantly upregulated TIGIT expression and inhibition of oxidative phosphorylation and glycolysis significantly decreased PD-1 on the surface of a subpopulation of OAC cells in vitro. These findings suggest an immune-independent mechanism for PD-1 inhibitor resistance in hypoxic tumours and suggest that TIGIT might be a more effective therapeutic target in OAC compared with PD-1 for treating hypoxic tumours., (Copyright © 2022. Published by Elsevier Inc.)

Chemotherapy upregulates immune checkpoint (IC) expression on the surface of tumour cells and IC-intrinsic signalling confers a survival advantage against chemotherapy in several cancer-types including oesophageal adenocarcinoma (OAC). However, the signalling pathways mediating chemotherapy-induced IC upregulation and the mechanisms employed by ICs to protect OAC cells against chemotherapy remain unknown. Longitudinal profiling revealed that FLOT-induced IC upregulation on OE33 OAC cells was sustained for up to 3 weeks post-treatment, returning to baseline upon complete tumour cell recovery. Pro-survival MEK signalling mediated FLOT-induced upregulation of PD-L1, TIM-3, LAG-3 and A2aR on OAC cells promoting a more immune-resistant phenotype. Single agent PD-1, PD-L1 and A2aR blockade decreased OAC cell viability, proliferation and mediated apoptosis. Mechanistic insights demonstrated that blockade of the PD-1 axis decreased stem-like marker ALDH and expression of DNA repair genes. Importantly, combining single agent PD-1, PD-L1 and A2aR blockade with FLOT enhanced cytotoxicity in OAC cells. These findings reveal novel mechanistic insights into the immune-independent functions of IC-intrinsic signalling in OAC cells with important clinical implications for boosting the efficacy of the first-line FLOT chemotherapy regimen in OAC in combination with ICB, to not only boost anti-tumour immunity but also to suppress IC-mediated promotion of key hallmarks of cancer that drive tumour progression., (© 2022. The Author(s).)

Background: Immune checkpoint inhibitors (ICIs) are being investigated for their role as an adjunct in the multimodal treatment of esophageal adenocarcinoma (EAC). The most effective time to incorporate ICIs remains unknown. Our study profiles systemic anti-tumor immunity perioperatively to help inform the optimal timing of ICIs into current standards of care for EAC patients., Methods: Systemic immunity in 11 EAC patients was phenotyped immediately prior to esophagectomy (POD-0) and post-operatively (POD)-1, 3, 7 and week 6. Longitudinal serological profiling was conducted by ELISA. The frequency of circulating lymphocytes, activation status, immune checkpoint expression and damage-associated molecular patterns was assessed by flow cytometry., Results: The frequency of naïve T-cells significantly increased in circulation post-esophagectomy from POD-0 to POD-7 (p<0.01) with a significant decrease in effector memory T-cells by POD7 followed by a subsequent increase by week 6 (p<0.05). A significant increase in activated circulating CD27 + T-cells was observed from POD-0 to POD-7 (p<0.05). The percentage of PD-1 + and CTLA-4 + T-cells peaked on POD-1 and was significantly decreased by week 6 (p<0.01). There was a significant increase in soluble PD-1, PD-L2, TIGIT and LAG-3 from POD-3 to week 6 (p<0.01). Increased checkpoint expression correlated with those who developed metastatic disease early in their postoperative course. Th1 cytokines and co-stimulatory factors decreased significantly in the immediate post-operative setting, with a reduction in IFN-γ, IL-12p40, IL-1RA, CD28, CD40L and TNF-α. A simultaneous increase was observed in Th2 cytokines in the immediate post-operative setting, with a significant increase in IL-4, IL-10, IL-16 and MCP-1 before returning to preoperative levels at week 6., Conclusion: Our study highlights the prevailing Th2-like immunophenotype post-surgery. Therefore, shifting the balance in favour of a Th1-like phenotype would offer a potent therapeutic approach to promote cancer regression and prevent recurrence in the adjuvant setting and could potentially propagate anti-tumour immune responses perioperatively if administered in the immediate neoadjuvant setting. Consequently, this body of work paves the way for further studies and appropriate trial design is needed to further interrogate and validate the use of ICI in the multimodal treatment of locally advanced disease in the neoadjuvant and adjuvant setting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Donlon, Davern, Sheppard, O’Connell, Dunne, Hayes, Mylod, Ramjit, Temperley, Mac Lean, Cotter, Bhardwaj, Butler, Conroy, O’Sullivan, Ravi, Donohoe, Reynolds and Lysaght.)

The last century of research in tumor immunology has culminated in the advent of immunotherapy, most notably immune checkpoint inhibitors. These drugs have shown encouraging results across a multitude of malignancies and have shifted the paradigm of cancer treatment. However, no more than 40% of patients treated with these immune checkpoint blockade inhibitors respond. Thus, resistance is a barrier to therapy that remains poorly understood. All cells require energy and biosynthetic precursors for survival, growth, and functioning, where multiple metabolic pathways allow for flexibility in how nutrients are utilized. A defining hallmark of many cancers is altered cellular metabolism, creating an imbalanced demand for nutrients within the tumor microenvironment. Immunometabolism is increasingly understood to be vital to the functions and phenotypes of a myriad of immune cell subsets. In tumors, the high demand for nutrients by the tumor drives competition between tumor cells and infiltrating immune cells, culminating in dysfunctional immune responses. This chapter discusses the recent successes in cancer immunotherapy and highlights challenges to therapy. We also outline the major metabolic processes involved in the generation of an immune response, how this can become dysregulated in the context of the tumor microenvironment, and how this contributes to resistance to immunotherapy. Finally, we explore the potential for targeting immunometabolic pathways to improve immunotherapy, and examine current trials targeting various aspects of metabolism in an attempt to improve the outcomes from immunotherapy.

Members of the Culex pipiens complex differ in physiological traits that facilitate their survival in diverse environments. Assortative mating within the complex occurs in some regions where autogenous (the ability to lay a batch of eggs without a blood meal) and anautogenous populations are sympatric, and differences in mating behaviors may be involved. For example, anautogenous populations mate in flight/swarms, while autogenous populations often mate at rest. Here, we characterized flight activity of males and found that anautogenous strain males were crepuscular, while autogenous strain males were crepuscular and nocturnal, with earlier activity onset times. We conducted quantitative trait locus (QTL) mapping to explore the genetic basis of circadian chronotype (crepuscular vs. crepuscular and nocturnal) and time of activity onset. One major-effect QTL was identified for chronotype, while 3 QTLs were identified for activity onset. The highest logarithm of the odds (LOD) score for the chronotype QTL coincides with a chromosome 3 marker that contains a 15-nucleotide indel within the coding region of the canonical clock gene, cryptochrome 2. Sequencing of this locus in 7 different strains showed that the C-terminus of CRY2 in the autogenous forms contain deletions not found in the anautogenous forms. Consequently, we monitored activity in constant darkness and found males from the anautogenous strain exhibited free running periods of ~24 h while those from the autogenous strain were ~22 h. This study provides novel insights into the genetic basis of flight behaviors that likely reflect adaptation to their distinct ecological niches., (© The American Genetic Association 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Species in the Anopheles farauti complex are major malaria vectors in the Asia Pacific region. Anopheline mosquitoes exhibit circadian and diel rhythms in sugar- and blood-feeding (biting), flight activity, oviposition, and in some species, a short-lived dusk/early night associated swarming behaviour during which mating occurs. A behavioural study of wild-caught mosquitoes from Queensland, Australia was conducted to investigate the differences in diel rhythmic flight activity between two cryptic species in several reproductive states., Results: The 24-hour flight activity of individual adult female mosquitoes under light:dark cycle conditions were monitored with a minute-to-minute time resolution using an infrared beam break method. Mosquitoes were analyzed for reproductive state (insemination and parity) and identified to species [An. farauti (s.s.) Laveran and An. hinesorum Schmidt] by PCR analysis. We compared daily total flight activity, timing of activity onset, the peak in early nocturnal activity, and patterns of activity during the scotophase (night). Species-specific differences between An. farauti and An. hinesorum were observed. Compared to An. farauti, An. hinesorum had an earlier onset of dusk activity, an earlier peak in nocturnal activity, and a higher level of activity at the onset of darkness. Small differences between species were also observed in the pattern of the dusk/early-night bouts of activity. A second nocturnal peak in inseminated nulliparous An. hinesorum was also observed during the middle of the scotophase., Conclusions: The behavioural differences between these two sympatric species of the An. farauti complex might contribute to subtle differences in habitat adaptation, the timing of host-seeking and/or sugar-feeding activity. This study provides baseline data for analysis of populations of mosquitoes from other geographical regions where these species are malaria vectors, such as in the Solomon Islands and Papua New Guinea. This is important as selective pressures due to long-term use of indoor residual spraying of insecticides and insecticide-treated bed nets are shifting the nocturnal profile of biting behaviour of these vectors to earlier in the night.

Background: Host-seeking behaviours in anopheline mosquitoes are time-of-day specific, with a greater propensity for nocturnal biting. We investigated how a short exposure to light presented during the night or late day can inhibit biting activity and modulate flight activity behaviour., Results: Anopheles gambiae (s.s.), maintained on a 12:12 LD cycle, were exposed transiently to white light for 10-min at the onset of night and the proportion taking a blood meal in a human biting assay was recorded every 2 h over an 8-h duration. The pulse significantly reduced biting propensity in mosquitoes 2 h following administration, in some trials for 4 h, and with no differences detected after 6 h. Conversely, biting levels were significantly elevated when mosquitoes were exposed to a dark treatment during the late day, suggesting that light suppresses biting behaviour even during the late daytime. These data reveal a potent effect of a discrete light pulse on biting behaviour that is both immediate and sustained. We expanded this approach to develop a method to reduce biting propensity throughout the night by exposing mosquitoes to a series of 6- or 10-min pulses presented every 2 h. We reveal both an immediate suppressive effect of light during the exposure period and 2 h after the pulse. This response was found to be effective during most times of the night: however, differential responses that were time-of-day specific suggest an underlying circadian property of the mosquito physiology that results in an altered treatment efficacy. Finally, we examined the immediate and sustained effects of light on mosquito flight activity behaviour following exposure to a 30-min pulse, and observed activity suppression during early night, and elevated activity during the late night., Conclusions: As mosquitoes and malaria parasites are becoming increasingly resistant to insecticide and drug treatment respectively, there is a necessity for the development of innovative control strategies beyond insecticide-treated nets (ITNs) and residual spraying. These data reveal the potent inhibitory effects of light exposure and the utility of multiple photic pulses presented at intervals during the night/late daytime, may prove to be an effective tool that complements established control methods.

Background: Marine and freshwater zooplankton exhibit daily rhythmic patterns of behavior and physiology which may be regulated directly by the light:dark (LD) cycle and/or a molecular circadian clock. One of the best-studied zooplankton taxa, the freshwater crustacean Daphnia, has a 24 h diel vertical migration (DVM) behavior whereby the organism travels up and down through the water column daily. DVM plays a critical role in resource tracking and the behavioral avoidance of predators and damaging ultraviolet radiation. However, there is little information at the transcriptional level linking the expression patterns of genes to the rhythmic physiology/behavior of Daphnia., Results: Here we analyzed genome-wide temporal transcriptional patterns from Daphnia pulex collected over a 44 h time period under a 12:12 LD cycle (diel) conditions using a cosine-fitting algorithm. We used a comprehensive network modeling and analysis approach to identify novel co-regulated rhythmic genes that have similar network topological properties and functional annotations as rhythmic genes identified by the cosine-fitting analyses. Furthermore, we used the network approach to predict with high accuracy novel gene-function associations, thus enhancing current functional annotations available for genes in this ecologically relevant model species. Our results reveal that genes in many functional groupings exhibit 24 h rhythms in their expression patterns under diel conditions. We highlight the rhythmic expression of immunity, oxidative detoxification, and sensory process genes. We discuss differences in the chronobiology of D. pulex from other well-characterized terrestrial arthropods., Conclusions: This research adds to a growing body of literature suggesting the genetic mechanisms governing rhythmicity in crustaceans may be divergent from other arthropod lineages including insects. Lastly, these results highlight the power of using a network analysis approach to identify differential gene expression and provide novel functional annotation.

Chronic alcohol consumption contributes to fatty liver disease. Our studies revealed that the hepatic circadian clock is disturbed in alcohol-induced hepatic steatosis, and effects of chronic alcohol administration upon the clock itself may contribute to steatosis. We extended these findings to explore the effects of chronic alcohol treatment on daily feeding and locomotor activity patterns. Mice were chronically pair-fed ad libitum for 4 weeks using the Lieber-DeCarli liquid diet, with calorie-controlled liquid and standard chow diets as control groups. Locomotor activity, feeding activity, and real-time bioluminescence recording of PERIOD2::LUCIFERASE expression in tissue explants were measured. Mice on liquid control and chow diets exhibited normal profiles of locomotor activity, with a ratio of 22:78% day/night activity and a peak during early night. This pattern was dramatically altered in alcohol-fed mice, marked by a 49:51% ratio and the absence of a distinct peak. While chow-diet fed mice had a normal 24:76% ratio of feeding activity, with a peak in the early night, this pattern was dramatically altered in both liquid-diet groups: mice had a 43:57% ratio, and an absence of a distinct peak. Temporal differences were also observed between the two liquid-diet groups during late day. Cosinor analysis revealed a ∼4-h and ∼6-h shift in the alcohol-fed group feeding and locomotor activity rhythms, respectively. Analysis of hepatic PER2 expression revealed that the molecular clock in alcohol-fed and control liquid-diet mice was shifted by ∼11 h and ∼6 h, respectively. No differences were observed in suprachiasmatic nucleus explants, suggesting that changes in circadian phase in the liver were generated independently from the central clock. These results suggest that chronic alcohol consumption and a liquid diet can differentially modulate the daily rhythmicity of locomotor and feeding behaviors, aspects that might contribute to disturbances in the circadian timing system and development of hepatic steatosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Whole bloods from weaned lambs with severe selenium responsive unthriftiness usually contain <5 ng Se/ml. Mildly or moderately affected lambs have blood levels of 5-10 ng/ml. Selenium responsive infertility in ewes appears to be associated with whole blood selenium levels below 10 ng/ml.

The efficacy and retention of prototype and commercial (Cosecure) soluble-glass boluses containing selenium, cobalt and copper and having a similar size, density and composition were evaluated in separate 12-month trials with sheep grazing low selenium pastures but with adequate cobalt and copper levels. In both trials, sheep confirmed by X-ray as containing a bolus grew at a significantly greater rate than control sheep. This was attributed to the correction of a selenium deficiency. Although liver copper, and liver and serum vitamin B12 levels were significantly greater in treated sheep than controls on some occasions, the differences were not as great or consistent as with blood selenium levels. The rate of bolus loss was high and in both trials, approximately 40% of the sheep had lost the bolus after 6 months. No prototype boluses were present after 12 months. Intact Cosecure boluses were recovered from 3 sheep out of 30 after 12 months whilst one animal contained a part bolus. The recovered boluses were approximately 45% lighter than when originally administered. Bolus loss did not appear to be due to complete dissolution. In sheep which had lost the bolus, blood selenium levels fell with a half-life of 43 +/- 10 days.

Two groups of adult Merino sheep, initially grazed on pasture, were dosed daily with zinc sulphate (1 mg Zn/kg LW) or zinc oxide (15 mg Zn/kg LW) for six and seven weeks, respectively. On the 18th day, both groups were transferred to indoor pens together with unmedicated control sheep, and five days later the feet of all animals were infected artificially with a virulent strain of Bacteroides nodosus. By the fourth week after challenge, 85% or more of the feet challenged had developed advanced footrot, and no significant differences in the incidence and severity of lesions between dosed and un-dosed groups were recorded. Plasma zinc concentrations, monitored throughout the experiment, remained at similar levels in sheep receiving the lower dose rate and in the controls. At the higher dose rate, plasma zinc levels increased till Day 23, but thereafter declined to values comparable to those of undosed animals. At post mortem, no evidence of zinc toxicosis was found, and only the kidneys of sheep receiving the higher dosage showed a significant accumulation of zinc compared with the controls.

Blood was collected from a group of ten sheep immediately after removal from pasture and thereafter at regular intervals for 50 hours whilst maintaining them without food but with adequate water. The samples were analysed for parameters normally examined as an aid to the diagnosis of zinc, copper, selenium, cobalt and iodine status. Serum vitamin B12 levels increased with time. Mean plasma zinc concentrations and mean serum concentrations of both T4 and fT4 followed significant parabolic trends. Maximum mean zinc levels (50 +/- 10% higher than initial values) were recorded after 30 hours. T4 and fT4 levels peaked at approximately 26 hours. Zinc levels at two hours were 20 +/- 5% lower than the initial values. Whole blood copper, selenium and glutathione peroxidase levels did not change significantly with time the largest differences were 11 +/- 6%, 5 +/- 4% and 10 +/- 8% respectively.

In grasses and clovers the highest concentrations of selenium were found in the leaves. The selenium content of different species in spring pastures on the same soil type did not vary widely although the slower growing and more deeply rooting species contained higher levels. The concentration of selenium in a pasture varied with time of the year, the lower levels being usually associated with conditions that stimulated rapid growth of the herbage, although factors other than growth rate were also involved. Lucerne appeared to be unusual in that in some areas it accumulated much higher levels of selenium than was found in other associated species. A very wide variation in levels of selenium in pastures growing on different soils has been observed 3-1500 ng/g). Selenium-responsive unthriftiness in grazing lambs occurred where the selenium content of spring pastures was <20 ng/g. Pastures containing >30 ng/g were apparently adequate while intermediate levels were probably marginally deficient.

Two trials were undertaken with lambs grazing selenium deficient pasture to determine if copper would enhance liveweight and fleece-weight responses to selenium supplementation. In the first trial, lambs given selenium or selenium plus copper gained significantly more weight and had higher fleece-weights after 260 days than did control lambs or lambs given copper alone. Copper given alone or together with selenium had no significant effect on liveweight or fleece-weight when compared with control lambs and lambs that were given selenium alone respectively. This finding was confirmed in a second trial when growth and fleece-weights of selenium and selenium plus copper treated lambs were compared and no significant differences found. In both trials copper significantly raised liver copper levels. In selenium supplemented but not in selenium deficient sheep, copper significantly increased blood selenium levels.