Polymeric Micellar Nanoparticles Enable Image-guided Drug Delivery in Solid Tumors.

We report the development of a nanotechnology to co-deliver chemocoxib A with a reactive oxygen species (ROS)-activatable and COX-2 targeted pro-fluorescent probe, fluorocoxib Q (FQ) enabling real time visualization of COX-2 and CA drug delivery into solid cancers, using a di-block PPS 135 - b -POEGA 17 copolymer, selected for its intrinsic responsiveness to elevated reactive oxygen species (ROS), a key trait of the tumor microenvironment. FQ and CA were synthesized independently, then co-encapsulated within micellar PPS 135 - b -POEGA 17 co-polymeric nanoparticles (FQ-CA-NPs), and were assessed for cargo concentration, hydrodynamic diameter, zeta potential, and ROS-dependent cargo release. The uptake of FQ-CA-NPs in mouse mammary cancer cells and cargo release was assessed by fluorescence microscopy. Intravenous delivery of FQ-CA-NPs to mice harboring orthotopic mammary tumors, followed by vital optimal imaging, was used to assess delivery to tumors in vivo . The CA-FQ-NPs exhibited a hydrodynamic diameter of 109.2 ± 4.1 nm and a zeta potential (σ) of -1.59 ± 0.3 mV. Fluorescence microscopy showed ROS-dependent cargo release by FQ-CA-NPs in 4T1 cells, decreasing growth of 4T1 breast cancer cells, but not affecting growth of primary human mammary epithelial cells (HMECs). NP-derived fluorescence was detected in mammary tumors, but not in healthy organs. Tumor LC-MS/MS analysis identified both CA (2.38 nmol/g tumor tissue) and FQ (0.115 nmol/g tumor tissue), confirming the FQ-mediated image guidance of CA delivery in solid tumors. Thus, co-encapsulation of FQ and CA into micellar nanoparticles (FQ-CA-NPs) enabled ROS-sensitive drug release and COX-2-targeted visualization of solid tumors.

Lateralised Behavioural Responses in Livestock to Environmental Stressors: Implications for Using Infrared Thermography to Assess Welfare Conditions.

Lateralised behavioural responses to environmental stressors have become more frequently used as indicators of social welfare in animals. These lateralised behavioural responses are under the control of asymmetrical brain functions as part of the primary functions of most vertebrates and assist in primary social and survival functions. Lateralised behavioural responses originating from the left hemisphere are responsible for processing familiar conditions, while the right hemisphere is responsible for responding to novel stimuli in the environment. The forced lateralisation and side preference tests have been used to determine the visual lateralised behavioural responses in livestock to environmental stressors. Limb preference during movement has also been used to determine motor lateralisation. Although behavioural investigations in livestock have recorded lateralised behavioural responses to environmental stressors, there are still limitations in the implication of lateralisation to other conditions, such as restraint and invasive procedures. Thus, it is important to have a non-invasive measure for these lateralised behavioural responses. Recently, lateralised behavioural responses have been correlated with the use of infrared temperature of external body surfaces, such as the eyes and coronary bands of limbs. This review summarised the different forms of the lateralised behavioural responses in livestock, especially cattle and horses, to environmental stressors, and the association between these responses and the relevant external body surfaces' infrared temperature, with the purpose of improving the use of non-invasive measures in assessing welfare conditions in animals. The combination of the lateralised behavioural responses and infrared temperature of external body surfaces to environmental stressors could improve the assessment strategies of welfare conditions and the related additional husbandry interventions that could be applied to improve the welfare of farm animals.

Infrared thermography as a tool for the measurement of negative emotions in dairy cows.

In commercial dairy cows, the conditions in which they are kept may lead to negative emotional states associated with the development of chronic physiological and behavioural abnormalities that may compromise their health, welfare and productivity. Such states include fear, stress or anxiety. Behavioural rather than physiological tests are more likely to be used to indicate these states but can be limited by their subjectivity, need for specialised infrastructure and training (of the operator and sometimes the animal) and the time-consuming nature of data collection. Popularly used physiological measures such as blood cortisol may be more appropriate for acute rather than chronic assessments but are easily confounded, for example by a response to the act of measurement per se. More sophisticated physiological measures such as functional magnetic resonance imaging (fMRI) and electroencephalography (EEG) may be impractical due to cost and time and, like blood cortisol, have the confounding associated with the act of measurement. By contrast, infrared thermography of external body surfaces is remote, non-invasive, easily repeated and follows an objective methodology, allowing longitudinal data acquisition for the inference of changes in chronic emotional state over time. The objective of this review was to investigate the potential of infrared thermography to measure cow emotions. In lactating dairy cows, maximum IRT of the eyes and coronary band of the limbs seem to be most representative of thermoregulatory changes, which are repeatable and correlate with behavioural and physiological indicators of emotional state. IRT methodologies have the potential to become a fundamental tool for the objective assessment of welfare state in dairy cows.

Effect of heat stress mitigations on physiological, behavioural, and hormonal responses of Buffalo calves.

This study assessed the effect of heat stress mitigations on the physiological, behavioural, and hormonal responses of buffalo during the hot summer season. Twenty Murrah buffalo male calves were distributed randomly into controlled (C, n = 10) and treatment groups (T, n = 10). The buffalo calves in the C group were housed in the existing shed (10-12-ft height and 10-ft width). Buffalo calves of the T group were allocated in the modified shed: 15-ft height and 20-ft width along with time-controlled pressure mist with fans and rubber mats on the floor. Fans were running all days. The cool water was misted on calves at the rate of 1 min in 5 min, from 11:00 to 18:00 h. The water misting system was installed below the roof, but at 3.5 m above the floor. The calves' body weight, rectal temperature, infrared temperature of the eye, blood samples, respiration rate, and pulse rate were recorded fortnightly for two consecutive months. In one-way ANOVA, rectal temperature, eye temperature, cortisol level, and afternoon's respiration and pulse rate were higher in the calves of C group than that of T group (P < 0.05). Conversely, eating and resting time (min/day) and triiodothyronine were lower in the calves of C group than that of T group (P < 0.05). Therefore, an increase in shed's height and width, using rubber mats on the floor, and cool water misting to buffaloes during the hot summer seasons positively influence their physiological, hormonal, and behavioural responses.

Optimizing an Antioxidant TEMPO Copolymer for Reactive Oxygen Species Scavenging and Anti-Inflammatory Effects in Vivo.

Oxidative stress is broadly implicated in chronic, inflammatory diseases because it causes protein and lipid damage, cell death, and stimulation of inflammatory signaling. Supplementation of innate antioxidant mechanisms with drugs such as the superoxide dismutase (SOD) mimetic compound 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) is a promising strategy for reducing oxidative stress-driven pathologies. TEMPO is inexpensive to produce and has strong antioxidant activity, but it is limited as a drug due to rapid clearance from the body. It is also challenging to encapsulate into micellar nanoparticles or polymer microparticles, because it is a small, water soluble molecule that does not efficiently load into hydrophobic carrier systems. In this work, we pursued a polymeric form of TEMPO [poly(TEMPO)] to increase its molecular weight with the goal of improving in vivo bioavailability. High density of TEMPO on the poly(TEMPO) backbone limited water solubility and bioactivity of the product, a challenge that was overcome by tuning the density of TEMPO in the polymer by copolymerization with the hydrophilic monomer dimethylacrylamide (DMA). Using this strategy, we formed a series of poly(DMA-co-TEMPO) random copolymers. An optimal composition of 40 mol % TEMPO/60 mol % DMA was identified for water solubility and O2•- scavenging in vitro. In an air pouch model of acute local inflammation, the optimized copolymer outperformed both the free drug and a 100% poly(TEMPO) formulation in O2•- scavenging, retention, and reduction of TNFα levels. Additionally, the optimized copolymer reduced ROS levels after systemic injection in a footpad model of inflammation. These results demonstrate the benefit of polymerizing TEMPO for in vivo efficacy and could lead to a useful antioxidant polymer formulation for next-generation anti-inflammatory treatments.

Non-Invasive Physiological Indicators of Heat Stress in Cattle.

Cattle are susceptible to heat stress, especially those kept on high levels of nutrition for the purpose of maximising growth rates, which leads to a significant heat increment in their bodies. Consequences include compromised health and productivity and mortalities during extreme events, as well as serious economic loss. Some measures of heat stress, such as plasma cortisol and temperature in the rectum, vagina, or rumen, are invasive and therefore unlikely to be used on farms. These may cause additional stress to the animal due to handling, and that stress in itself can confound the measure. Consequently, it is desirable to find non-invasive alternatives. Panting score (PS), cortisol metabolites in faeces, milk, or hair, and the infrared temperature of external body surfaces are all potentially useful. Respiratory indicators are difficult and time consuming to record accurately, and cortisol metabolites are expensive and technically difficult to analyse. Infrared temperature appears to offer the best solution but requires further research to determine the thresholds that define when corrective actions are required to ensure optimal health and productivity. Research in this area has the potential to ultimately improve the welfare and profitability of cattle farming.

The Clinical Presentation of Culture-positive and Culture-negative, Quantitative Polymerase Chain Reaction (qPCR)-Attributable Shigellosis in the Global Enteric Multicenter Study and Derivation of a Shigella Severity Score: Implications for Pediatric Shigella Vaccine Trials.

BACKGROUND: Shigella is a leading cause of childhood diarrhea and target for vaccine development. Microbiologic and clinical case definitions are needed for pediatric field vaccine efficacy trials. METHODS: We compared characteristics of moderate to severe diarrhea (MSD) cases in the Global Enteric Multicenter Study (GEMS) between children with culture positive Shigella to those with culture-negative, quantitative polymerase chain reaction (qPCR)-attributable Shigella (defined by an ipaH gene cycle threshold <27.9). Among Shigella MSD cases, we determined risk factors for death and derived a clinical severity score. RESULTS: Compared to culture-positive Shigella MSD cases (n = 745), culture-negative/qPCR-attributable Shigella cases (n = 852) were more likely to be under 12 months, stunted, have a longer duration of diarrhea, and less likely to have high stool frequency or a fever. There was no difference in dehydration, hospitalization, or severe classification from a modified Vesikari score. Twenty-two (1.8%) Shigella MSD cases died within the 14-days after presentation to health facilities, and 59.1% of these deaths were in culture-negative cases. Age <12 months, diarrhea duration prior to presentation, vomiting, stunting, wasting, and hospitalization were associated with mortality. A model-derived score assigned points for dehydration, hospital admission, and longer diarrhea duration but was not significantly better at predicting 14-day mortality than a modified Vesikari score. CONCLUSIONS: A composite severity score consistent with severe disease or dysentery may be a pragmatic clinical endpoint for severe shigellosis in vaccine trials. Reliance on culture for microbiologic confirmation may miss a substantial number of Shigella cases but is currently required to measure serotype specific immunity.

A sampling strategy for the determination of infrared temperature of relevant external body surfaces of dairy cows.

Infrared thermography is a tool to investigate the welfare of cattle. This study aimed to identify a sampling strategy for recording infrared thermograms in dairy cows, in order to most efficiently determine biologically relevant changes in the maximum infrared temperature (IRT) of the eyes and coronary band of forelimbs. Thirty-one dairy cows were used for the study. They were assessed with four replicates of thermograms for each of the head and lower forelimb per cow for 6 mostly consecutive days (sessions). The data obtained were subjected to random effects Analysis of Variance which was used to estimate the variance components for this sampling model, using maximum IRT of both eyes; (left + right eye)/2 and both limbs; (left + right coronary band of forelimb)/2 as dependant variables. The variance components were used to calculate least significant differences (LSD) between two theoretical treatment groups under different sampling scenarios. Analysis showed that there was minimal improvement in precision beyond 2 thermograms within a session but there was improvement with increasing the number of sessions from 2 to 3. The LSD of both eyes and both limbs reached a biologically relevant difference (0.4 and 0.9 °C, respectively) at a minimum number of 14 - 16 cows monitored for 2 consecutive thermography sessions, or 10 - 12 cows for 3 sessions. We conclude that no more than 2 replicate IRT measures are required per session but that measuring on 3 consecutive days should be considered, depending on whether time or number of cows used is the primary limitation.

Gut microbiome communication with bone marrow regulates susceptibility to amebiasis.

The microbiome provides resistance to infection. However, the underlying mechanisms are poorly understood. We demonstrate that colonization with the intestinal bacterium Clostridium scindens protects from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone marrow from C. scindens-colonized mice into naive mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Lachnoclostridia C. scindens can metabolize the bile salt cholate, so we measured deoxycholate and discovered that it was increased in the sera of C. scindens-colonized specific pathogen-free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone increased GMPs and provided protection from amebiasis. We elucidated a mechanism by which C. scindens and the microbially metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with E. histolytica.

Detection of tyrosine kinase inhibitors-induced COX-2 expression in bladder cancer by fluorocoxib A.

Among challenges of targeted therapies is the activation of alternative pro-survival signaling pathways in cancer cells, resulting in an acquired drug resistance. Cyclooxygenase-2 (COX-2) is overexpressed in bladder cancer cells, making it an attractive molecular target for the detection and treatment of cancer. Fluorocoxib A is an optical imaging agent that selectively targets COX-2. In this study, we evaluated the ability of fluorocoxib A to monitor the responses of bladder cancer to targeted therapies in vivo. The effects of several tyrosine kinase inhibitors (TKIs: axitinib, AB1010, toceranib, imatinib, erlotinib, gefitinib, imatinib, sorafenib, vandetanib, SP600125, UO126, and AZD 5438) on COX-2 expression were validated in ten human and canine bladder cancer cell lines (J82, RT4, T24, UM-UC-3, 5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#5Lilly) in vitro. The effects of TKIs on bladder cancer in vivo were evaluated using the COX-2-expressing K9TCC#5Lilly xenograft mouse model and detected by fluorocoxib A. The increased COX-2 expression was detected by all tested TKIs in at least one of the tested COX-2-expressing bladder cancer cell lines (5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, and K9TCC#5Lilly) in vitro. In addition, fluorocoxib A uptake correlated with the AB1010- and imatinib-induced COX-2 expression in the K9TCC#5Lilly xenografts in vivo. In conclusion, these results indicate that fluorocoxib A could be used for the monitoring the early responses to targeted therapies in COX-2-expressing bladder cancer.

Inhibition of Diacylglycerol Lipase Impairs Fear Extinction in Mice.

Elucidating the underlying molecular mechanisms regulating fear and extinction learning may offer insights that can lead to novel treatments for debilitating anxiety and trauma-related disorders including posttraumatic stress disorder. The endocannabinoid (eCB) system is a retrograde inhibitory signaling pathway involved in regulating central responses to stress. The eCB 2-arachidonoylglycerol (2-AG) has recently been proposed to serve as a homeostatic signal mitigating adverse effects of stress exposure, however, less well understood is 2-AG's role in fear learning and fear extinction. In this study, we have sought to explore 2-AG's role in fear conditioning and fear extinction by disrupting 2-AG synthesis utilizing the DAGL inhibitor (DO34) and DAGLα knock-out mice (DAGLα-/-). We found that DAGLα-/- mice, and male and female C57B6/J mice treated with DO34, exhibited impairment in extinction learning in an auditory cue fear-conditioning paradigm. DO34 did not increase unconditioned freezing. Interestingly, inhibition of fatty-acid amide hydrolase was not able to restore normal fear extinction in DO34-treated mice suggesting increased Anandamide cannot compensate for deficient 2-AG signaling in the regulation of fear extinction. Moreover, augmentation of CB1R signaling with tetrahydrocannabinol also failed to restore normal fear extinction in DO34-treated mice. Overall, these data support the hypothesis that DAGLα plays an important role in fear extinction learning. Although genetic and pharmacological disruption of DAGL activity causes widespread lipidomic remodeling, these data combined with previous studies putatively suggest that deficient 2-AG signaling could be a susceptibility endophenotype for the development of trauma-related psychiatric disorders.

Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study.

BACKGROUND: Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS). METHODS: GEMS was a study of moderate to severe diarrhoea in children younger than 5 years in Africa and Asia. We used quantitative real-time PCR (qPCR) to test for 32 enteropathogens in stool samples from cases and matched asymptomatic controls from GEMS, and compared pathogen-specific attributable incidences with those found with the original GEMS microbiological methods, including culture, EIA, and reverse-transcriptase PCR. We calculated revised pathogen-specific burdens of disease and assessed causes in individual children. FINDINGS: We analysed 5304 sample pairs. For most pathogens, incidence was greater with qPCR than with the original methods, particularly for adenovirus 40/41 (around five times), Shigella spp or enteroinvasive Escherichia coli (EIEC) and Campylobactor jejuni o C coli (around two times), and heat-stable enterotoxin-producing E coli ([ST-ETEC] around 1·5 times). The six most attributable pathogens became, in descending order, Shigella spp, rotavirus, adenovirus 40/41, ST-ETEC, Cryptosporidium spp, and Campylobacter spp. Pathogen-attributable diarrhoeal burden was 89·3% (95% CI 83·2-96·0) at the population level, compared with 51·5% (48·0-55·0) in the original GEMS analysis. The top six pathogens accounted for 77·8% (74·6-80·9) of all attributable diarrhoea. With use of model-derived quantitative cutoffs to assess individual diarrhoeal cases, 2254 (42·5%) of 5304 cases had one diarrhoea-associated pathogen detected and 2063 (38·9%) had two or more, with Shigella spp and rotavirus being the pathogens most strongly associated with diarrhoea in children with mixed infections. INTERPRETATION: A quantitative molecular diagnostic approach improved population-level and case-level characterisation of the causes of diarrhoea and indicated a high burden of disease associated with six pathogens, for which targeted treatment should be prioritised. FUNDING: Bill & Melinda Gates Foundation.

PET radiotracer [¹8F]-P6 selectively targeting COX-1 as a novel biomarker in ovarian cancer: preliminary investigation.

Cyclooxygenase-1 (COX-1), but not COX-2, is expressed at high levels in the early stages of human epithelial ovarian cancer where it seems to play a key role in cancer onset and progression. As a consequence, COX-1 is an ideal biomarker for early ovarian cancer detection. A series of novel fluorinated COX-1-targeted imaging agents derived from P6 was developed by using a highly selective COX-1 inhibitor as a lead compound. Among these new compounds, designed by structural modification of P6, 3-(5-chlorofuran-2-yl)-5-(fluoromethyl)-4-phenylisoxazole ([(18/19)F]-P6) is the most promising derivative [IC50 = 2.0 µM (purified oCOX-1) and 1.37 µM (hOVCAR-3 cell COX-1)]. Its tosylate precursor was also prepared and, a method for radio[(18)F]chemistry was developed and optimized. The radiochemistry was carried out using a carrier-free K(18)F/Kryptofix 2.2.2 complex, that afforded [(18)F]-P6 in good radiochemical yield (18%) and high purity (>95%). In vivo PET/CT imaging data showed that the radiotracer [(18)F]-P6 was selectively taken up by COX-1-expressing ovarian carcinoma (OVCAR 3) tumor xenografts as compared with the normal leg muscle. Our results suggest that [(18)F]-P6 might be an useful radiotracer in preclinical and clinical settings for in vivo PET-CT imaging of tissues that express elevated levels of COX-1.

Seeing it through: translational validation of new medical imaging modalities.

Medical imaging is an invaluable tool for diagnosis, surgical guidance, and assessment of treatment efficacy. The Network for Translational Research (NTR) for Optical Imaging consists of four research groups working to "bridge the gap" between lab discovery and clinical use of fluorescence- and photoacoustic-based imaging devices used with imaging biomarkers. While the groups are using different modalities, all the groups face similar challenges when attempting to validate these systems for FDA approval and, ultimately, clinical use. Validation steps taken, as well as future needs, are described here. The group hopes to provide translational validation guidance for itself, as well as other researchers.

Nitric oxide deficiency promotes vascular side effects of cyclooxygenase inhibitors.

The cardiovascular safety of COX-2 selective and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) has recently been called into question. The factors that predispose to adverse events by NSAIDs are unknown. Because patients with arthritis have decreased nitric oxide (NO) bioavailability, the in vivo effects of NSAIDs on murine vascular tone and platelet activity in the presence or absence of NO were examined. Here, we show that acute hypertensive and prothrombotic activities of the COX-2-selective inhibitor celecoxib are revealed only after in vivo inhibition of NO generation. The nonselective NSAID indomethacin was hypertensive but antithrombotic when NO was absent. In vitro myography of aortic rings confirmed that vasoconstriction required inhibition of both NOS and COX-2 and was abolished by supplementation with exogenous NO. These data indicate that NO suppresses vascular side effects of NSAIDs, suggesting that risk will be greatest in patients with impaired vascular function associated with decreased NO bioavailability.