The role of cytokines, coagulation and fibrinolysis in leucocyte and LAK cell cytotoxicity of tumour cells

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Interleukin-2 activates lymphocytes to become highly cytotoxic for a wide range of tumour cell types in vitro (Iymphokine activated killer or LAK cells), and in animal models. However, only limited therapeutic benefit was observed in clinical trials of LAK cell therapy. This project aimed to investigate the molecular and cellular interactions involved in the production and effector functions of LAK cells, to identify factor(s) which might be responsible for the poor clinical responses observed in LAK cell therapy. Tumour cell lines were heterogeneous in their response to killing by cytokines (TNFα, LT, IFNγ and IL-1β), and purified monocytes or lymphocytes, but were consistently highly sensitive to LAK cell cytotoxicity. Autologous monocytes and lymphocytes were not killed by LAK cells, in contrast to human umbilical vein endothelial cells and fibroblasts. Supernatants from LAK cells were considerably less cytotoxic than the effector cells, and physical separation of effector and target cells resulted in inhibition of killing. Lymphocyte and LAK cell cytotoxicity was associated predominantly with the CD8+ (cytotoxic T-cell) lymphocyte sub-population, and was significantly inhibited by anti-TNFα and anti-LT, demonstrating that these cytokines were the primary effector molecules in this system. LAK cells and A375 melanoma cells showed procoagulant activity, predominantly via the tissue factor pathway, and LAK cells also possessed surface factor V. In addition, A375 cells were highly fibrinolytic. Tumour cell killing by LAK cells was inhibited by plasma, and further experiments determined that polymerised fibrin, but not fibrin monomer was responsible. From these results it was suggested that culture of small numbers of cells from tumour biopsies, and the determination of their sensitivity to cytotoxic drugs, cytokines and effector cells may lead to more effective treatment protocols for immunotherapy of individual tumours. In order to enhance the efficacy of immunotherapy, further in vivo research is required to elucidate the interactions between immune effector cells and the coagulation/fibrinolytic systems

Soluble fibrin inhibits monocyte adherence and cytotoxicity against tumor cells: implications for cancer metastasis

BACKGROUND: Soluble fibrin (sFn) is a marker for disseminated intravascular coagulation and may have prognostic significance, especially in metastasis. However, a role for sFn in the etiology of metastatic cancer growth has not been extensively studied. We have reported that sFn cross-linked platelet binding to tumor cells via the major platelet fibrin receptor αIIbβ3, and tumor cell CD54 (ICAM-1), which is the receptor for two of the leukocyte β2 integrins (α(L)β2 and a(M)β2). We hypothesized that sFn may also affect leukocyte adherence, recognition, and killing of tumor cells. Furthermore, in a rat experimental metastasis model sFn pre-treatment of tumor cells enhanced metastasis by over 60% compared to untreated cells. Other studies have shown that fibrin(ogen) binds to the monocyte integrin α(M)β2. This study therefore sought to investigate the effect of sFn on β2 integrin mediated monocyte adherence and killing of tumor cells. METHODS: The role of sFn in monocyte adherence and cytotoxicity against tumor cells was initially studied using static microplate adherence and cytotoxicity assays, and under physiologically relevant flow conditions in a microscope perfusion incubator system. Blocking studies were performed using monoclonal antibodies specific for β2 integrins and CD54, and specific peptides which inhibit sFn binding to these receptors. RESULTS: Enhancement of monocyte/tumor cell adherence was observed when only one cell type was bound to sFn, but profound inhibition was observed when sFn was bound to both monocytes and tumor cells. This effect was also reflected in the pattern of monocyte cytotoxicity. Studies using monoclonal blocking antibodies and specific blocking peptides (which did not affect normal coagulation) showed that the predominant mechanism of fibrin inhibition is via its binding to α(M)β2 on monocytes, and to CD54 on both leukocytes and tumor cells. CONCLUSION: sFn inhibits monocyte adherence and cytotoxicity of tumor cells by blocking α(L)β2 and α(M)β2 binding to tumor cell CD54. These results demonstrate that sFn is immunosuppressive and may be directly involved in the etiology of metastasis. Use of specific peptides also inhibited this effect without affecting coagulation, suggesting their possible use as novel therapeutic agents in cancer metastasis

Zyflamend, a polyherbal mixture, down regulates class I and class II histone deacetylases and increases p21 levels in castrate-resistant prostate cancer cells

Background Zyflamend, a mixture containing extracts of ten herbs, has shown promise in a variety of preclinical cancer models, including prostate cancer. The current experiments were designed to investigate the effects of Zyflamend on the expression of class I and II histone deacetylases, a family of enzymes known to be over expressed in a variety of cancers. Methods CWR22Rv1 cells, a castrate-resistant prostate cancer cell line, were treated with Zyflamend and the expression of class I and II histone deacetylases, along with their downstream target the tumor suppressor gene p21, was investigated. Involvement of p21 was confirmed with siRNA knockdown and over expression experiments. Results Zyflamend down-regulated the expression of all class I and II histone deacetylases where Chinese goldthread and baikal skullcap (two of its components) appear to be primarily responsible for these results. In addition, Zyflamend up regulated the histone acetyl transferase complex CBP/p300, potentially contributing to the increase in histone 3 acetylation. Expression of the tumor suppressor gene p21, a known downstream target of histone deacetylases and CBP/p300, was increased by Zyflamend treatment and the effect on p21 was, in part, mediated through Erk1/2. Knockdown of p21 with siRNA technology attenuated Zyflamend-induced growth inhibition. Over expression of p21 inhibited cell growth and concomitant treatment with Zyflamend enhanced this effect. Conclusions Our results suggest that the extracts of this polyherbal combination increase histone 3 acetylation, inhibit the expression of class I and class II histone deacetylases, increase the activation of CBP/p300 and inhibit cell proliferation, in part, by up regulating p21 expression

West Nile Virus–associated Flaccid Paralysis Outcome

We report 1-year follow-up data from a longitudinal prospective cohort study of patients with West Nile virus–associated paralysis. As in the 4-month follow-up, a variety of recovery patterns were observed, but persistent weakness was frequent. Respiratory involvement was associated with considerable illness and death

Results from the centers for disease control and prevention's predict the 2013-2014 Influenza Season Challenge

Background: Early insights into the timing of the start, peak, and intensity of the influenza season could be useful in planning influenza prevention and control activities. To encourage development and innovation in influenza forecasting, the Centers for Disease Control and Prevention (CDC) organized a challenge to predict the 2013-14 Unites States influenza season. Methods: Challenge contestants were asked to forecast the start, peak, and intensity of the 2013-2014 influenza season at the national level and at any or all Health and Human Services (HHS) region level(s). The challenge ran from December 1, 2013-March 27, 2014; contestants were required to submit 9 biweekly forecasts at the national level to be eligible. The selection of the winner was based on expert evaluation of the methodology used to make the prediction and the accuracy of the prediction as judged against the U.S. Outpatient Influenza-like Illness Surveillance Network (ILINet). Results: Nine teams submitted 13 forecasts for all required milestones. The first forecast was due on December 2, 2013; 3/13 forecasts received correctly predicted the start of the influenza season within one week, 1/13 predicted the peak within 1 week, 3/13 predicted the peak ILINet percentage within 1 %, and 4/13 predicted the season duration within 1 week. For the prediction due on December 19, 2013, the number of forecasts that correctly forecasted the peak week increased to 2/13, the peak percentage to 6/13, and the duration of the season to 6/13. As the season progressed, the forecasts became more stable and were closer to the season milestones. Conclusion: Forecasting has become technically feasible, but further efforts are needed to improve forecast accuracy so that policy makers can reliably use these predictions. CDC and challenge contestants plan to build upon the methods developed during this contest to improve the accuracy of influenza forecasts. © 2016 The Author(s)

West Nile Virus–associated Flaccid Paralysis

The causes and frequency of acute paralysis and respiratory failure with West Nile virus (WNV) infection are incompletely understood. During the summer and fall of 2003, we conducted a prospective, population-based study among residents of a 3-county area in Colorado, United States, with developing WNV-associated paralysis. Thirty-two patients with developing paralysis and acute WNV infection were identified. Causes included a poliomyelitislike syndrome in 27 (84%) patients and a Guillain-Barré–like syndrome in 4 (13%); 1 had brachial plexus involvement alone. The incidence of poliomyelitislike syndrome was 3.7/100,000. Twelve patients (38%), including 1 with Guillain-Barré–like syndrome, had acute respiratory failure that required endotracheal intubation. At 4 months, 3 patients with respiratory failure died, 2 remained intubated, 25 showed various degrees of improvement, and 2 were lost to followup. A poliomyelitislike syndrome likely involving spinal anterior horn cells is the most common mechanism of WNV-associated paralysis and is associated with significant short- and long-term illness and death

The 2015 Plains Elevated Convection at Night Field Project

The central Great Plains region in North America has a nocturnal maximum in warm-season precipitation. Much of this precipitation comes from organized mesoscale convective systems (MCSs). This nocturnal maximum is counterintuitive in the sense that convective activity over the Great Plains is out of phase with the local generation of CAPE by solar heating of the surface. The lower troposphere in this nocturnal environment is typically characterized by a low-level jet (LLJ) just above a stable boundary layer (SBL), and convective available potential energy (CAPE) values that peak above the SBL, resulting in convection that may be elevated, with source air decoupled from the surface. Nocturnal MCS-induced cold pools often trigger undular bores and solitary waves within the SBL. A full understanding of the nocturnal precipitation maximum remains elusive, although it appears that bore-induced lifting and the LLJ may be instrumental to convection initiation and the maintenance of MCSs at night. To gain insight into nocturnal MCSs, their essential ingredients, and paths toward improving the relatively poor predictive skill of nocturnal convection in weather and climate models, a large, multiagency field campaign called Plains Elevated Convection At Night (PECAN) was conducted in 2015. PECAN employed three research aircraft, an unprecedented coordinated array of nine mobile scanning radars, a fixed S-band radar, a unique mesoscale network of lower-tropospheric profiling systems called the PECAN Integrated Sounding Array (PISA), and numerous mobile-mesonet surface weather stations. The rich PECAN dataset is expected to improve our understanding and prediction of continental nocturnal warm-season precipitation. This article provides a summary of the PECAN field experiment and preliminary findings

Effects of temperature on the transmission of Yersinia Pestis by the flea, Xenopsylla Cheopis, in the late phase period

<p>Abstract</p> <p>Background</p> <p>Traditionally, efficient flea-borne transmission of <it>Yersinia pestis</it>, the causative agent of plague, was thought to be dependent on a process referred to as blockage in which biofilm-mediated growth of the bacteria physically blocks the flea gut, leading to the regurgitation of contaminated blood into the host. This process was previously shown to be temperature-regulated, with blockage failing at temperatures approaching 30°C; however, the abilities of fleas to transmit infections at different temperatures had not been adequately assessed. We infected colony-reared fleas of <it>Xenopsylla cheopis </it>with a wild type strain of <it>Y. pestis </it>and maintained them at 10, 23, 27, or 30°C. Naïve mice were exposed to groups of infected fleas beginning on day 7 post-infection (p.i.), and every 3-4 days thereafter until day 14 p.i. for fleas held at 10°C, or 28 days p.i. for fleas held at 23-30°C. Transmission was confirmed using <it>Y. pestis</it>-specific antigen or antibody detection assays on mouse tissues.</p> <p>Results</p> <p>Although no statistically significant differences in per flea transmission efficiencies were detected between 23 and 30°C, efficiencies were highest for fleas maintained at 23°C and they began to decline at 27 and 30°C by day 21 p.i. These declines coincided with declining median bacterial loads in fleas at 27 and 30°C. Survival and feeding rates of fleas also varied by temperature to suggest fleas at 27 and 30°C would be less likely to sustain transmission than fleas maintained at 23°C. Fleas held at 10°C transmitted <it>Y. pestis </it>infections, although flea survival was significantly reduced compared to that of uninfected fleas at this temperature. Median bacterial loads were significantly higher at 10°C than at the other temperatures.</p> <p>Conclusions</p> <p>Our results suggest that temperature does not significantly effect the per flea efficiency of <it>Y. pestis </it>transmission by <it>X. cheopis</it>, but that temperature is likely to influence the dynamics of <it>Y. pestis </it>flea-borne transmission, perhaps by affecting persistence of the bacteria in the flea gut or by influencing flea survival. Whether <it>Y. pestis </it>biofilm production is important for transmission at different temperatures remains unresolved, although our results support the hypothesis that blockage is not necessary for efficient transmission.</p

COVID-19 reopening strategies at the county level in the face of uncertainty: Multiple Models for Outbreak Decision Support

Policymakers make decisions about COVID-19 management in the face of considerable uncertainty. We convened multiple modeling teams to evaluate reopening strategies for a mid- sized county in the United States, in a novel process designed to fully express scientific uncertainty while reducing linguistic uncertainty and cognitive biases. For the scenarios considered, the consensus from 17 distinct models was that a second outbreak will occur within 6 months of reopening, unless schools and non-essential workplaces remain closed. Up to half the population could be infected with full workplace reopening; non-essential business closures reduced median cumulative infections by 82%. Intermediate reopening interventions identified no win-win situations; there was a trade-off between public health outcomes and duration of workplace closures. Aggregate results captured twice the uncertainty of individual models, providing a more complete expression of risk for decision-making purposes.Integrative Biolog