A phase I study of the ceramide nanoliposome in patients with advanced solid tumors.

PURPOSE: Ceramide is a sphingolipid metabolite that deactivates multiple oncogenic signaling pathways and promotes cell death. In-vivo data demonstrate single-agent anti-cancer activity and enhanced efficacy with combination strategies. This phase I dose-escalation trial evaluated Ceramide nanoLiposomes (CNL) in patients with advanced solid tumors and no standard treatment option. METHODS: The primary objective was to establish the maximum tolerated dose. Secondary objectives included determining the recommended phase II dose, the safety and tolerability, the pharmacokinetic profile and preliminary anti-tumor efficacy. RESULTS: 15 patients with heavily pretreated metastatic disease enrolled. Safety data were analyzed for all patients, while pharmacokinetic data were available for 14 patients. There were no grade 3 or higher treatment-related adverse events. The maximum tolerated dose was not reached and there were no dose-limiting toxicities. The most common grade 1 or 2 treatment-related adverse events included headache, fatigue, constipation, nausea and transaminitis. The maximum concentration and area under the curve increased with dose. Clearance was consistent between doses and was observed mainly through the liver without significant hepatotoxicity. The half-life ranged from 20 to 30 h and the volume of distribution was consistent with a lipophilic drug. CONCLUSIONS: CNL exhibited an encouraging safety profile and pharmacokinetic parameters, with some signals of efficacy including prolonged stable disease in 1 patient with refractory pancreatic cancer. Pre-clinical data indicate potential synergy between CNL and multiple systemic therapies including chemotherapy, targeted therapy, and immunotherapy. Future studies are planned investigating CNL in combination strategies. TRIAL REGISTRATION: This study is registered under ClinicalTrials.gov ID: NCT02834611.

Siltation negatively affects settlement and gaping behaviour in eastern oysters.

While high levels of siltation are known to be deleterious to eastern oysters (Crassostrea virginica), the collective effect of suspended and bedded sediment is understudied from the perspective of oyster farming and bed restoration. In this study, we used laboratory experiments to explore spat settlement rates on a wild bed proxy substrate (i.e., empty shells on the bottom of experimental tanks) in conditions simulating a siltation event and the presence of suspended spat collectors. Using high-frequency valvometry, we also described the behavioural effects of acute sediment burial on wild adult oysters in situ. The vast majority of larvae settled on bottom substrate as opposed to suspended collectors. Sediment negatively affected overall oyster spat settlement on bottom shell, as spat densities were ≈3 × lower when sediment was present. This negative effect was largely attributed to severely depressed spat densities on the upper side (top) of bottom shells. Settlement on the underside of bottom shell was less affected. Wild adult oyster behaviour was negatively affected by acute burial, which ultimately resulted in death. We suggest that the reduction in settlement in the presence of siltation is likely due to the combined effects of suspended sediment on cue detection and bedded sediment on substrate availability. Given that oysters are ecosystem engineers, the negative effects of siltation on both larval and adult oysters can ultimately result in cascading effects to the surrounding biological community.

Bayesian risk assessment model of human cryptosporidiosis cases following consumption of raw Eastern oysters (Crassostrea virginica) contaminated with Cryptosporidium oocysts in the Hillsborough River system in Prince Edward Island, Canada.

Cryptosporidium spp. has been associated with foodborne infectious disease outbreaks; however, it is unclear to what extent raw oyster consumption poses a risk to public health. Control of Cryptosporidium in shellfish harvest seawater in Canada is not mandatory and, despite relay/depuration processes, the parasite can remain viable in oysters for at least a month (depending on initial loads and seawater characteristics). Risks of human infection and illness from exposure to oysters contaminated with Cryptosporidium oocysts were assessed in a Bayesian framework. Two data sets were used: counts of oocysts in oysters harvested in Approved, Restricted, and Prohibited zones of the Hillsborough River system; and oocyst elimination rate from oysters exposed to oocysts in laboratory experiments. A total of 20 scenarios were assessed according to number of oysters consumed in a single serving (1, 10 and 30) and different relay times. The median probability of infection and developing cryptosporidiosis (e.g. illness) due to the consumption of raw oysters in Prince Edward Island was zero for all scenarios. However, the 95th percentiles ranged from 2% to 81% and from 1% to 59% for probability of infection and illness, respectively. When relay times were extended from 14 to 30 days and 10 oysters were consumed in one serving from the Restricted zones, these probabilities were reduced from 35% to 16% and from 15% to 7%, respectively. The 14-day relay period established by Canadian authorities for harvesting in Restricted zones seems prudent, though insufficient, as this relay period has been shown to be enough to eliminate fecal coliforms but not Cryptosporidium oocysts, which can remain viable in the oyster for over a month. Extending relay periods of 14 and 21 days for oysters harvested in Restricted zones to 30 days is likely insufficient to substantially decrease the probability of infection and illness. The highest risk was found for oysters that originated in Prohibited zones. Our findings suggest that Cryptosporidium oocysts are a potential cause of foodborne infection and illness when consuming raw oysters from Hillsborough River, one of the most important oyster production bays on Prince Edward Island. We discuss data gaps and limitations of this work in order to identify future research that can be used to reduce the uncertainties in predicted risks.

The Application of a Mathematical Model to Evaluate the Effectiveness of Control Strategies Against Ciona intestinalis in Mussel Production.

The Prince Edward Island (PEI) mussel industry has faced challenges associated with invasive tunicate species over the past two decades. Field experiments to find suitable mitigation strategies require considerable time and are resource intensive. This study demonstrates the application of a mathematical model to assess several control strategies against Ciona intestinalis populations under different temperature conditions in a mussel production area in PEI. A temperature dependent compartmental model was used to model the total abundance of C. intestinalis. Each mitigation strategy was defined in terms of a combination of timing and frequency of treatments. Various strategies were explored to obtain the combination that maximized the difference in predicted abundances between the control (untreated) and the different mitigation strategies. Treatment frequency was allowed to vary between one and four times over a given production year. The model was assessed under baseline conditions, which mimicked water temperatures from Georgetown Harbor, PEI, in 2008; as well as under scenarios that reflected prolonged summer or warm spring temperatures. Furthermore, the sensitivity of the model to variations in presumed treatment efficacy was evaluated. The use of all four available treatments, starting around the first week of July and correctly timed thereafter, provided the most effective strategy, assuming the baseline temperature scenario. However, the effectiveness of this mitigation strategy depended on temperature conditions. The mathematical model developed in this study allows decision makers to explore different strategies to control the abundance of C. intestinalis in mussel production areas under different environmental conditions. In addition, the modeling framework developed could be adapted to simulate comparable ectoparasitic infestation in aquatic environments.

Refining the tethering of American oysters (Crassostrea virginica) to measure the effects of two environmental stressors.

Tethering assays, or the physical restraint of test organisms, has been used in the past to measure selected organisms' response to stressors while removing the observer from the experimental setting. Although informative for monitoring and hypothesis testing, these assays often used microfilaments that have been found to be too invasive or prone to biases given their effects on test organisms' behavior. Here, we describe a new variation of tethering using American oysters (Crassostrea virginica) and illustrate its use in the study of their mortality rates as a result of two stressors: siltation and predation by a non-indigenous species. Our protocol identified a resistant (non-toxic) glue that could be used to attach oysters to stone slabs, thus partially mimicking the natural cementation of the shell to natural substrates. This variation of tethering was harmless and maintained oysters' body position and natural ability to filter feed. Using tethered oysters in separate two-week field cage experiments, we also show how siltation and predation by a non-indigenous species (the European green crab, Carcinus maenas), caused a gradual, easily measurable increase in oyster mortality rates. We argue that this variation of tethering is a cost-effective and advantageous way to monitor or test the effects of these and other stressors on oysters and similar species.

Non-indigenous predators threaten ecosystem engineers: Interactive effects of green crab and oyster size on American oyster mortality.

Non-indigenous green crabs (Carcinus maenas) are emerging as important predators of autogenic engineers like American oysters (Crassostrea virginica) throughout the eastern seaboard of Canada and the United States. To document the spreading distribution of green crabs, we carried out surveys in seven sites of Prince Edward Island during three fall seasons. To assess the potential impact of green crabs on oyster mortality in relation to predator and prey size, we conducted multiple predator-prey manipulations in the field and laboratory. The surveys confirmed an ongoing green crab spread into new productive oyster habitats while rapidly increasing in numbers in areas where crabs had established already. The experiments measured mortality rates on four sizes of oysters exposed to three sizes of crab, and lasted 3-5 days. The outcomes of experiments conducted in Vexar® bags, laboratory tanks and field cages were consistent and were heavily dependent on both crab size and oyster size: while little predation occurred on large oysters, large and medium green crabs preyed heavily on small sizes. Oysters reached a refuge within the 35-55 mm shell length range; below that range, oysters suffered high mortality due to green crab predation and thus require management measures to enhance their survival. These results are most directly applicable to aquaculture operations and restoration initiatives but have implications for oyster sustainability.

Evidence-based tool surpasses expert opinion in predicting probability of eradication of aquatic nonindigenous species.

The main objective of evidence-based management is to promote use of scientific data in the decision-making process of managers, with data either complementing or replacing expert knowledge. It is expected that this will increase the efficiency of environmental interventions. However, the relative accuracy and precision of evidence-based tools and expert knowledge has seldom been evaluated. It is therefore essential to verify whether such tools provide better decision support before advocating their use. We conducted an elicitation survey in which experts were asked to (1) evaluate the influence of various factors on the success of eradication programs for aquatic nonindigenous species and (2) provide probabilities of success for real case studies for which we knew the outcome. The responses of experts were compared with the results and predictions of a newly developed evidence-based tool: a statistical model calibrated with a meta-analysis of case studies designed to evaluate probability of eradication. Experts and the model generally identified the same factors as influencing the probability of success. However, the model provided much more accurate estimates for the probability of eradication than expert opinion, strongly suggesting that an evidence-based approach is superior to expert knowledge in this case. Uncertainty surrounding the predictions of the evidence-based tool was similar to among-expert variability. Finally, a model based on >30 case studies returned more accurate predictions than expert opinion. We conclude that decision-making processes based on expert judgment would greatly benefit from incorporating evidence-based tools.

Determining brain fitness to fight: Has the time come?

Professional boxing is associated with a risk of chronic neurological injury, with up to 20-50% of former boxers exhibiting symptoms of chronic brain injury. Chronic traumatic brain injury encompasses a spectrum of disorders that are associated with long-term consequences of brain injury and remains the most difficult safety challenge in modern-day boxing. Despite these concerns, traditional guidelines used for return to sport participation after concussion are inconsistently applied in boxing. Furthermore, few athletic commissions require either formal consultation with a neurological specialist (i.e. neurologist, neurosurgeon, or neuropsychologist) or formal neuropsychological testing prior to return to fight. In order to protect the health of boxers and maintain the long-term viability of a sport associated with exposure to repetitive head trauma, we propose a set of specific requirements for brain safety that all state athletic commissions would implement.

Preclinical development of a C6-ceramide NanoLiposome, a novel sphingolipid therapeutic.

Despite the therapeutic potential of sphingolipids, the ability to develop this class of compounds as active pharmaceutical ingredients has been hampered by issues of solubility and delivery. Beyond these technical hurdles, significant challenges in completing the necessary preclinical studies to support regulatory review are necessary for commercialization. This review seeks to identify the obstacles and potential solutions in the translation of a novel liposomal technology from the academic bench to investigational new drug (IND) stage by discussing the preclinical development of the Ceramide NanoLiposome (CNL), which is currently being developed as an anticancer drug for the initial indication of hepatocellular carcinoma (HCC).

Chronic wound repair and healing in older adults: current status and future research.

Older adults are more likely to have chronic wounds than younger people, and the effect of chronic wounds on quality of life is particularly profound in this population. Wound healing slows with age, but the basic biology underlying chronic wounds and the influence of age-associated changes on wound healing are poorly understood. Most studies have used in vitro approaches and various animal models, but observed changes translate poorly to human healing conditions. The effect of age and accompanying multimorbidity on the effectiveness of existing and emerging treatment approaches for chronic wounds is also unknown, and older adults tend to be excluded from randomized clinical trials. Poorly defined outcomes and variables; lack of standardization in data collection; and variations in the definition, measurement, and treatment of wounds also hamper clinical studies. The Association of Specialty Professors, in conjunction with the National Institute on Aging and the Wound Healing Society, held a workshop, summarized in this article, to explore the current state of knowledge and research challenges, engage investigators across disciplines, and identify research questions to guide future study of age-associated changes in chronic wound healing.

Occurrence and antimicrobial resistance of Escherichia coli in oysters and mussels from Atlantic Canada.

Acquiring antimicrobial-resistant (AMR) bacteria through consuming contaminated animal food products is an emerging public health concern, though the sources of contamination are not always clear. This study characterized the occurrence of AMR in Escherichia coli from bivalve molluscs and assessed for the possible sources in the Hillsborough river complex of Prince Edward Island, Canada in areas overlapping with an oyster fishery. Multivariable statistical analysis indicated that the probability of detecting E. coli increased as the estimated dosage of animal effluent contamination decreased. Isolates with AMR were only found from sampling sites closest to untreated human effluent sources. Twenty-seven percent (n=6 of 22) of the isolates were pathogenic, with virulence factors consistent with extraintestinal E. coli of human origin. Though there is more evidence of contamination arising from human effluent, more research is needed to identify driving sources.

Static tank depuration and chronic short-term experimental contamination of Eastern oysters (Crassostrea virginica) with Giardia duodenalis cysts.

Cysts of the protozoan parasite Giardia have been detected in many bivalve shellfish species worldwide. The detection of zoonotic Giardia duodenalis assemblages A and B is of public health concern, yet there is limited data available demonstrating the bioaccumulation and elimination of Giardia cysts in bivalve shellfish. This study quantified G. duodenalis cysts that were filtered and retained by oysters (Crassostrea virginica) over a one week chronic exposure period, or 24 hour exposure followed by a 6 day depuration period, using static tank systems containing 10 L of 29 ppt water inoculated with 1000 or 10,000 cysts. Under chronic exposure, each oyster retained a mean of 13.4 and 87.4 cysts during the first 24h of exposure at low and high doses, respectively, and the cysts bioaccumulated at a rate of 1.2 and 6.8 cysts/oyster/day, respectively, for the remaining duration of the trials. In acute exposure trials, oysters retained 13.8 cysts or 78.9 cysts at low and high doses, respectively, during the initial 24 hour exposure and naturally depurated cysts at a rate of -0.92 cysts/oyster/day and -2.2 cysts/oyster/day, respectively, after transfer. Although most G. duodenalis cysts were eliminated within the first 24h via pseudofeces and feces, detection of some cysts in the fecal material on day 7 of acute exposure trials was indicative of cysts which passed through the digestive tract and released in feces. Only 48-53% of the initial tank inocula were recovered and may indicate that some cysts were selectively filtered by oysters but degraded through digestion.

Chronic wound repair and healing in older adults: current status and future research.

The incidence of chronic wounds is increased among older adults, and the impact of chronic wounds on quality of life is particularly profound in this population. It is well established that wound healing slows with age. However, the basic biology underlying chronic wounds and the influence of age-associated changes on wound healing are poorly understood. Most studies have used in vitro approaches and various animal models, but observed changes translate poorly to human healing conditions. The impact of age and accompanying multi-morbidity on the effectiveness of existing and emerging treatment approaches for chronic wounds is also unknown, and older adults tend to be excluded from randomized clinical trials. Poorly defined outcomes and variables, lack of standardization in data collection, and variations in the definition, measurement, and treatment of wounds also hamper clinical studies. The Association of Specialty Professors, in conjunction with the National Institute on Aging and the Wound Healing Society, held a workshop, summarized in this paper, to explore the current state of knowledge and research challenges, engage investigators across disciplines, and identify key research questions to guide future study of age-associated changes in chronic wound healing.

Bioaccumulation and elimination of Cryptosporidium parvum oocysts in experimentally exposed Eastern oysters (Crassostrea virginica) held in static tank aquaria.

A variety of human enteropathogens, including viruses, bacteria, and parasites, have been shown to bioaccumulate in suspension-feeding bivalve shellfish. Cryptosporidium parvum is a zoonotic protozoan parasite that has been detected in many shellfish species within both fecally contaminated and clean oyster growing areas across the globe. For this study, C. parvum oocysts (1000 and 10,000) were spiked into 10 L of water in static tank systems housing Crassostrea virginica. Oysters were either held in the contaminated aquaria for 7 days of exposure or were exposed for 24h and subsequently placed in a clean static tank system for the remainder of the trial. Individual oysters, fecal material, and tank water were analyzed for oocysts up to 7 days post-exposure via direct immunofluorescence. Oysters held under chronic exposure conditions gradually accumulated oocysts (1.5 or 34.4 oocysts/oyster/day for low or high dose exposure groups, respectively) between days 1 and 7, with an exponential uptake in oocysts observed within the first 24h post-exposure (mean uptake of 29.6 or 241.9 oocysts/oyster, respectively). Oysters that were transferred to clean water after 24h were capable of slowly depurating oocysts, following a linear trend. During chronic exposure trials 48-49% of the total spiked inoculum was recovered from oyster tissue, whereas 4.8-5.9% and 38-40% was recovered from tank water and from fecal material at day 7, respectively. In acute exposure trials, 30-31% of the total tank inoculum was found in oysters, suggesting that chronically exposed oysters were likely re-filtering some oocysts. Examinations of oyster fecal material from acute exposures revealed that 72-82% of oocysts recovered were already excreted at the time of oyster transfer (day 1), with only 18-28% being excreted during the static depuration phase. These data support that although most C. parvum oocysts are removed by C. virginica oysters within 24h, elimination after this point occurs slowly. Additionally, chronic exposures demonstrate that wild or cultured oysters in saline environments that are frequently exposed to sources of Cryptosporidium may be unable to eliminate the parasites at a rate that balances initial uptake.

Aquaculture fouling: Efficacy of potassium monopersulphonate triple salt based disinfectant (Virkon® Aquatic) against Ciona intestinalis.

With the increasing spread of invasive marine species and their detrimental effects on aquaculture operations globally, mitigation strategies need to be optimized to mitigate economic impacts. The efficacy of a potassium monopersulphonate triple salt based disinfectant used in the aquaculture industry (Virkon® Aquatic at 0.5-5%) was evaluated against the solitary tunicate Ciona intestinalis, as well as the susceptibility of three different age groups of C. intestinalis to the treatment and the effect of the disinfectant on mussel mortality. Younger C. intestinalis were most affected by all treatments, and almost all immersion applications significantly decreased the biomass of C. intestinalis compared to untreated plates. Disinfectant solutions of ≥ 1% reduced biomass below pre-treatment levels. Mussel mortality was low, especially for solutions <3%. C. intestinalis should be treated 4 weeks post-settlement to maximize antifouling treatment effects. Immersion in 3% disinfectant for 30 s reduced the biomass of C. intestinalis by up to 89% and would be feasible in field applications using existing treatment equipment.

Diagnosing PNH with FLAER and multiparameter flow cytometry.

BACKGROUND: PNH is an acquired hematopoietic stem cell disorder leading to a partial or absolute deficiency of all glycophosphatidyl-inositol (GPI)-linked proteins. The classical approach to diagnosis of PNH by cytometry involves the loss of at least two GPI-linked antigens on RBCs and neutrophils. While flow assays are more sensitive and specific than complement-mediated lysis or the Hams test, they suffer from several drawbacks. Bacterial aerolysin binds to the GPI moiety of cell surface GPI-linked molecules and causes lysis of normal but not GPI-deficient PNH cells. FLAER is an Alexa488-labeled inactive variant of aerolysin that does not cause lysis of cells. Our goals were to develop a FLAER-based assay to diagnose and monitor patients with PNH and to improve detection of minor populations of PNH clones in other hematologic disorders. METHODS: In a single tube assay, we combined FLAER with CD45, CD33, and CD14 allowing the simultaneous analysis of FLAER and the GPI-linked CD14 structure on neutrophil and monocyte lineages. RESULTS: Comparison to standard CD55 and CD59 analysis showed excellent agreement. Because of the higher signal to noise ratio, the method shows increased sensitivity in our hands over single (CD55 or CD59) parameter analysis. Using this assay, we were able to detect as few as 1% PNH monocytes and neutrophils in aplastic anemia, that were otherwise undetectable using CD55 and CD59 on RBC's. We also observed abnormal FLAER staining of blast populations in acute leukemia. In these cases, the neutrophils stained normally with FLAER, while the gated CD33bright cells failed to express normal levels of CD14 and additionally showed aberrant CD45 staining and bound lower levels of FLAER. CONCLUSION: FLAER combined with multiparameter flow cytometry offers an improved assay for diagnosis and monitoring of PNH clones and may have utility in detection of unsuspected myeloproliferative disorders.

Physiologic properties of small intestine submucosa.

BACKGROUND: Porcine small intestine submucosa (SiS) has been introduced as a bioprosthesis in herniorrhaphy. This study evaluates in vivo properties of SiS that would affect clinical use. MATERIALS AND METHODS: Twelve pigs underwent implantation of SiS (perforated and nonperforated) on the peritoneal surface. Gross characteristics were evaluated and random samples harvested for histological study at 2 (n = 6) and 8 (n = 6) weeks. Collagen deposition was determined by polarized microscopy. Neovascularity (percent area blood vessels, %A(bv)) was determined by immunohistochemical staining with a polyclonal CD-31 antibody. RESULTS: Perforated SiS had a higher density of capillary ingrowth compared with nonperforated at both 2 (5.6%A(bv) versus 1.4%A(bv), P < 0.05) and 8 weeks (6.0%A(bv) versus 1.6%A(bv), P < 0.05). Compared with 2 weeks, 8-week SiS had a larger proportion of incorporation (25% versus 83%, P < 0.05) and new collagen deposition (50% versus 94%, P < 0.05). Significant contraction was observed in SiS 8 weeks after implantation (preimplant area 98 cm2 versus post-implant area 50 cm2, P < 0.05). CONCLUSION: SiS incorporated well 8 weeks after implantation, with deposition of new collagen. Perforated SiS demonstrated a more rapid and greater amount of neovascularity. The degree of contraction suggests that larger areas of SiS should be selected for herniorrhaphy than would be necessary if synthetic materials were used.