Leptin is capable of modulating the immune response. Proinflammatory cytokines induce leptin production, and we now demonstrate that leptin can directly activate the inflammatory response. RNA expression for the leptin receptor (Ob-R) was detectable in human PBMCs. Ob-R expression was examined at the protein level by whole blood flow cytometry using an anti-human Ob-R mAb 9F8. The percentage of cells expressing leptin receptor was 25 ± 5% for monocytes, 12 ± 4% for neutrophils, and 5 ± 1% for lymphocytes (only B lymphocytes). Incubation of resting PBMCs with leptin induced rapid expression of TNF-α and IL-6 mRNA and a dose-dependent production of TNF-α and IL-6 by monocytes. Incubation of resting PBMCs with high-dose leptin (250 ng/ml, 3–5 days) induced proliferation of resting cultured PBMCs and their secretion of TNF-α (5-fold), IL-6 (19-fold), and IFN-γ (2.5-fold), but had no effect on IL-4 secretion. The effect of leptin was distinct from, and additive to, that seen after exposure to endotoxin or activation by the mixed lymphocyte reaction. In conclusion, Ob-R is expressed on human circulating leukocytes, predominantly on monocytes. At high doses, leptin induces proinflammatory cytokine production by resting human PBMCs and augments the release of these cytokines from activated PBMCs in a pattern compatible with the induction of Th1 cytokines. These results demonstrate that leptin has a direct effect on the generation of an inflammatory response. This is of relevance when considering leptin therapy and may partly explain the relationship among leptin, proinflammatory cytokines, insulin resistance, and obesity.

A study was carried out to evaluate the clinical and haematological effects of dietary supplementation with eicosapentaenoic acid (EPA)-rich fish oil (MaxEPA', 2.8 g EPA daily) compared to placebo (olive oil) in 10 patients with stable angina pectoris. After 3 months, there was a significant increase in red cell deformability (p less than 0.001), reduced whole blood viscosity (p less than 0.02), and prolonged skin bleeding time (p less than 0.001) in the fish oil group compared to the placebo group. Haematocrit, plasma viscosity, fibrinogen concentration, platelet count, and in vitro platelet aggregation were unaltered. No significant symptomatic or objective improvement was noted in angina pectoris in either group despite the significant rheological changes produced in the patients receiving fish oil.

Background Bacterial infection is a common complication in patients with advanced disease, including cancer. Ingestion (phagocytosis) and subsequent killing of bacteria by neutrophils and monocytes are critical in the control of infection. Using a range of different methodologies, previous invitro and in vivo studies have generally reported that some commonly prescribed opioids are immunosuppressive (ie, morphine), whereas others have been shown to be immunoneutral (ie, fentanyl), and even immunostimulatory (ie, tramadol). However, direct, comparative, systematic studies aimed at analysing their relative impact remain lacking. Aims To systematically assess the in vitro effects of clinically-relevant concentrations of commonly used opioids on the phagocytosis of E coli by peripheral blood neutrophils and monocytes. Methods Peripheral blood was collected from healthy volunteers and incubated with clinically-relevant concentrations of morphine, fentanyl, tramadol, buprenorphine, oxycodone, diamorphine, methadone or codeine for 60 min. Samples were then incubated with FITC-conjugated E.coli for 10 min at 37°C, at which time the proportion of neutrophils and monocytes that had phagocytosed E.coli, and the intensity of the phagocytic response was determined using whole blood flow cytometry (PHAGOTEST, Orpegen Pharma GmbH, Germany). Control samples were incubated at 4°C. Results Although morphine, fentanyl, tramadol and buprenorphine inhibited the phagocytic responses of neutrophils and monocytes by up to 75% in some individuals, due to inter-individual variability this did not reach statistical significance. Codeine, oxycodone, diamorphine and methadone had no detectable effect on neutrophil or monocyte phagocytosis. Conclusion Although the true impact of opioid treatment on the susceptibility of patients to bacterial infection in vivo has yet to be defined, these systematically observed in vitro findings indicate that opioid choice might impact on the clinical status of some patients who are at an increased risk of bacterial infection. Future work needs to elucidate the population prevalence to the susceptibility from these potentially immunosuppressive opioids.

Glioblastoma multiforme (GBM) is the most frequently occurring primary brain tumor; it is a debilitating disease that is associated with poor prognosis, short median patient survival and a limited response to current therapies. As a result, there is a dire need for novel therapeutic interventions that are curative, or at the very least extend patient survival. Immunotherapy is an attractive option for the treatment of GBM due to its high specificity and minimal systemic toxicity. Peripherally activated immune cells have been shown to efficiently penetrate the brain parenchyma and access intracranial tumors, overcoming the blood-brain barrier, which hinders many molecularly targeted therapies. Two antigens, TRP-2 and WT-1, were found to be significantly expressed in GBM tissue sections while being absent from the normal brain. Peptide sequences known to be immunogenic were chosen from both TRP-2 and WT-1 antigens. The DNA sequences corresponding to these peptide sequences were then inserted into the complementarity determining regions of a DNA plasmid encoding an antibody known as ImmunoBody®; this vaccine has been shown to generate a higher avidity response than both peptide and peptide-pulsed dendritic cell vaccinations. As a result of the highly promising preclinical results shown, the ImmunoBody® vaccination is currently being studied in a phase I/II clinical trial for melanoma. As a result the ImmunoBody® DNA vaccine has been selected as our method of vaccination. Syngeneic C57BL/6 mice and humanized C57BL/6 HHDII/DR1 mice were used to assess the TRP-2 and WT-1 directed ImmunoBody® vaccines. Mice were vaccinated biolistically with the ImmmunoBody® plasmid coated onto gold particles using a gene gun. An initial priming dose was given on day 0 followed by a boost on days 7 and 14. Mice were vaccinated with either a TRP-2-ImmunoBody®, a WT-1-ImmunoBody® or a combination of the two. The immune response generated was determined by ex vivo IFN-γ ELISpot using splenocytes derived from the spleen of immunized animals. Results from these dual vaccination experiments reveal that it is possible to use both of these vaccines in tandem without losing the specificity towards each vaccine-containing peptide. High level of peptide-specific IFN-γ-releasing cells were detected directly ex vivo and the ability of these IFN-γ-releasing cells to recognize targeT-cells that naturally express the WT-1 and TRP-2 antigens is being investigated. The efficacy of TRP-2-ImmunoBody® with or without PD-1 at treating syngeneic orthotopic GL-261Luc2 tumors implanted in C57BL/6 mice is also currently being assessed. The combination of the two vaccines will also be assessed in the humanized HHDII/DR1 mice using a humanized B16 cell line that has had murine Beta-2m knocked out and HHDII and HLA-DR1 knocked in. Anti-PD-1 checkpoint blockade will also be incorporated into this vaccination regime with the aim of boosting the antitumor immune response. Citation Format: Joshua R. D. Pearson, Lindy G. Durrant, Victoria A. Brentville, Graham Pockley, Stephanie E.B. McArdle. Development of a new immunotherapy treatment for glioblastoma multiforme [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B122.

In addition to being less toxic and having a lower impact on a patient’s quality of life, evidence suggests that low-dose or metronomic chemotherapy modulates adaptive and innate antitumor immune responses. In this study, we examined whether the treatment of breast cancer cells and animals bearing breast cancer cell-derived tumors with low, nontoxic doses of chemotherapies promotes sensitivity to natural killer (NK) cells, based on target cell killing in vitro and the control of tumor growth and metastasis in vivo. We have developed a low-dose doxorubicin treatment protocol that sensitizes triple-negative breast cancer (TNBC) cells (MDA-MB-231, MDA-MB-468) to killing by NK cells. Specifically, low-dose doxorubicin treatment induced a “senescent-like” state in breast cancer cells concomitant with an impaired proliferative capacity, as shown by a decreased expression of Ki-67. Treatment of breast cancer cells also upregulated their expression of ligands for activatory NK cell receptors (e.g., MICA/B, ULBP1, ULBP2, ULBP3) and markedly increased their sensitivity to lysis by donor-derived, primary NK cells and the NK-92 cell line, as assessed using an in vitro flow cytometry-based assay. The cytotoxicity of donor-derived NK cells was markedly increased by prior stimulation with IL-2. The therapeutic potential of low-dose chemotherapy alone and in combination with adoptively transferred resting and activated NK cells was evaluated using immunodeficient NSG mice bearing MDA-MB-231/RFP/LUC-derived human TNBC xenografts. Although low-dose doxorubicin treatment alone reduced the growth of the primary tumors, as assessed using caliper measurements and in vivo imaging, the growth of the primary tumor was more markedly reduced following the adoptive transfer of healthy-donor derived NK cells (mean tumor volume: 70.56mm3 versus 228.8mm3 in control groups at day 49). Importantly, although treatment with low-dose doxorubicin alone also delayed the onset of metastasis by 7 days (day 49 in control animals, day 56 in treated animals), no signs of metastasis were observed in healthy-donor derived NK cell-treated animals at the time of culling on day 50. The expression of MICA/B was 8.5 times greater on tumor cells derived from mice that received the combination treatment (p=0.0001) and these cells were more sensitive to killing by NK-92 cells in vitro (3-fold increase at E:T 1:5). Furthermore, in vitro studies demonstrating that the treatment of MDA-MB-231 cells with low-dose doxorubicin reduces the number of CD44High/CD24−/low/EpCAM+ Cancer Initiating Cells (CICs) were confirmed by there being a significantly lower number of CICs in tumors derived from treated mice. These novel findings indicate that this approach has the capacity of preventing the primary tumor “fueling” the dissemination of aggressive, metastatic disease. Taken together, these studies demonstrate that the treatment of breast cancer with low, nontoxic doses of chemotherapies promotes their sensitivity to killing by NK cells in vitro and in vivo. We have shown that administering human NK cells to mice bearing human breast tumors that have been treated with low-dose doxorubicin reduces the growth and metastasis of tumors and eliminates the aggressive cells that “feed” this process. Ongoing studies are further interrogating the influence of this approach on breast cancer metastasis. In summary, this strategy offers a promising opportunity for more effectively treating patients with aggressive, triple-negative breast cancer. Citation Format: Sarra Idri, Graham Pawelec, Yvonne Barnett, Graham Pockley. Combining low-dose chemotherapy with an NK cell-based immunotherapy as a treatment for triple-negative breast cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A135.

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Background Opioids are commonly used in the management of pain, diarrhoea, cough and dyspnoea. However, their ability to modulate the ubiquitous endogenous opioid system can lead to many side effects. A potentially covert side effect which has been attracting increasing interest relates to opioid-induced immune suppression. This may be of particular importance in patients with cancer or infection. The immunomodulatory effects of opioids require an increased awareness within palliative care given that patients are potentially living longer with a palliative diagnosis. Aims To systematically evaluate the in vitro effects on opioids on human immune system. Methods Peripheral blood was obtained from healthy volunteers and the influence of clinically relevant concentrations of morphine, tramadol, fentanyl, buprenorphine, methadone oxycodone codeine and diamorphine on innate and adaptive immunity were evaluated. These tests included investigation of neutrophil and monocyte bacterial defence mechanisms, the capacity of natural killer (NK) cells to kill tumour cells and T cell responsiveness to stimulation. Results None of the opioids had a consistent effect on any of the immune parameters tested when analysed on a group basis. However, there was marked variability between individuals, both in their baseline and the effect of opioids. This was particularly evident with respect to the influence of morphine, tramadol, fentanyl and buprenorphine on neutrophil and monocyte bacterial defence mechanisms. Conclusion These in vitro findings suggest that although morphine, tramadol, fentanyl and buprenorphine have little direct effect on anticancer or antiviral protection they could influence the susceptibility to bacterial infection. Clinical studies aimed at assessing the in vivo effects of opioid administration on innate and adaptive immune capacity in relevant patient groups are required in order to assess the potential clinical significance of opioids used for palliation.

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