Autocrine regulation of wound healing by ATP release and P2Y2 receptor activation.

AIMS: Application of exogenous nucleotides can modulate wound healing via the activation of purinergic receptors. However, evidence for the release of endogenous nucleotides and the subsequent activation of purinergic receptors in this process has not been well defined. Therefore, the current study aimed to investigate wound-mediated nucleotide release and autocrine purinergic signalling during HaCaT keratinocyte wound closure following scratch injury. MAIN METHODS: An in vitro scratch wound apparatus was employed to study wound healing over 24-h in the presence of modulators of ATP release, P2 receptors and pathways downstream of P2 receptor activation. KEY FINDINGS: Adenosine 5'-triphosphate (ATP) was released from scratched cells. The ectonucleotidase apyrase and pharmacological inhibition of the nucleotide release hemichannel, pannexin-1, decreased wound closure over time. The non-selective P2Y receptor antagonist suramin and the selective P2Y2 receptor antagonist AR-C118925XX, but not other P2 antagonists, decreased wound closure. AR-C118925XX decreased wound closure in a concentration-dependent fashion. However, exogenous P2Y2 receptor agonists, ATP or uridine 5'-triphosphate, did not enhance wound closure. PCR and immunoblotting confirmed P2Y2 receptor expression in HaCaT cells. U73122, a phospholipase C antagonist, and 2-aminoethoxydiphenylborate, an inositol 1,4,5-trisphosphate receptor-sensitive Ca2+-release channel antagonist, decreased wound closure consistent with P2Y2 receptor activation. Absence of extracellular or intracellular Ca2+ or inhibition of intracellular Ca2+-release also impaired wound closure. SIGNIFICANCE: These data describe a novel autocrine signalling mechanism in which wound-mediated release of endogenous ATP in response to mechanical scratching of HaCaT cells activates P2Y2 receptors to facilitate wound closure.

The P2X7 receptor antagonist Brilliant Blue G reduces serum human interferon-γ in a humanized mouse model of graft-versus-host disease.

Graft-versus-host disease (GVHD) remains a major problem after allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. Previous studies have demonstrated a role for the adenosine triphosphate (ATP)-gated P2X7 receptor channel in allogeneic mouse models of GVHD. In this study, injection of human peripheral blood mononuclear cells (PBMCs) into immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (NOD-SCID-IL)-2Rγnull (NSG) mice established a humanized mouse model of GVHD. This model was used to study the effect of P2X7 blockade in this disease. From five weeks post-PBMC injection, humanized mice exhibited clinical signs and histopathology characteristic of GVHD. The P2X7 antagonist, Brilliant Blue G (BBG), blocked ATP-induced cation uptake into both murine and human cells in vitro. Injection of BBG (50 mg/kg) into NSG mice did not affect engraftment of human leucocytes (predominantly T cells), or the clinical score and survival of mice. In contrast, BBG injection reduced circulating human interferon (IFN)-γ significantly, which was produced by human CD4+ and CD8+ T cells. BBG also reduced human T cell infiltration and apoptosis in target organs of GVHD. In conclusion, the P2X7 antagonist BBG reduced circulating IFN-γ in a humanized mouse model of GVHD supporting a potential role for P2X7 to alter the pathology of this disease in humans.

Realistic and interactive high-resolution 4D environments for real-time surgeon and patient interaction.

BACKGROUND: Remote consultations that are realistic enough to be useful medically offer considerable clinical, logistical and cost benefits. Despite advances in virtual reality and vision hardware and software, these benefits are currently often unrealised. METHOD: The proposed approach combines high spatial and temporal resolution 3D and 2D machine vision with virtual reality techniques, in order to develop new environments and instruments that will enable realistic remote consultations and the generation of new types of useful clinical data. RESULTS: New types of clinical data have been generated for skin analysis and respiration measurement; and the combination of 3D with 2D data was found to offer potential for the generation of realistic virtual consultations. CONCLUSION: An innovative combination of high resolution machine vision data and virtual reality online methods, promises to provide advanced functionality and significant medical benefits, particularly in regions where populations are dispersed or access to clinicians is limited. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of isoflurane and ethanol administration on c-Fos immunoreactivity in mice.

Noninvasive functional imaging holds great promise for the future of translational research, due to the ability to directly compare between preclinical and clinical models of psychiatric disorders. Despite this potential, concerns have been raised regarding the necessity to anesthetize rodent and monkey subjects during these procedures, because anesthetics may alter neuronal activity. For example, in studies on drugs of abuse and alcohol, it is not clear to what extent anesthesia can interfere with drug-induced neural activity. Therefore, the current study investigated whole-brain c-Fos activation following isoflurane anesthesia as well as ethanol-induced activation of c-Fos in anesthetized mice. In the first experiment, we examined effects of one or three sessions of gaseous isoflurane on c-Fos activation across the brain in male C57BL/6J mice. Isoflurane administration led to c-Fos activation in several areas, including the piriform cortex and lateral septum. Lower or similar levels of activation in these areas were detected after three sessions of isoflurane, suggesting that multiple exposures may eliminate some of the enhanced neuronal activation caused by acute isoflurane. In the second experiment, we investigated the ability of ethanol injection (1.5 or 2.5g/kgi.p.) to induce c-Fos activation under anesthesia. Following three sessions of isoflurane, 1.5g/kg of ethanol induced c-Fos in the central nucleus of amygdala and the centrally-projecting Edinger-Westphal nucleus (EWcp). This induction was lower after 2.5g/kg of ethanol. These results demonstrate that ethanol-induced neural activation can be detected in the presence of isoflurane anesthesia. They also suggest, that while habituation to isoflurane helps reduce neuronal activation, interaction between effects of anesthesia and alcohol can occur. Studies using fMRI imaging could benefit from using habituated animals and dose-response analyses.

Rapid Resolution of Donor-Derived Glomerular Fibrin Thrombi After Deceased Donor Kidney Transplantation.

The aim of this study was to determine the clinical and histologic outcomes related to transplanting kidneys from deceased donors with glomerular fibrin thrombi (GFT). We included all cases transplanted between October 2003 and October 2014 that had either a preimplantation biopsy or an immediate postreperfusion biopsy showing GFT. The study cohort included 61 recipients (9.9%) with GFT and 557 in the control group without GFT. Delayed graft function occurred in 49% of the GFT group and 39% in the control group (p = 0.14). Serum creatinine at 1, 4, and 12 months and estimated GFR at 12 months were similar in the two groups. Estimated 1-year graft survival was 93.2% in the GFT group and 95.1% in the control group (p = 0.22 by log-rank). Fifty-two of the 61 patients in the GFT group (85%) had a 1-month protocol biopsy, and only two biopsies (4%) showed residual focal glomerular thrombi. At the 1-year protocol biopsy, the prevalence of moderate to severe interstitial fibrosis and tubular atrophy was 24% in the GFT group and 30% in the control group (p = 0.42). We concluded that GFT resolves rapidly after transplantation and that transplanting selected kidneys from deceased donors with GFT is a safe practice.

A 3D machine vision method for non-invasive assessment of respiratory function.

BACKGROUND: Respiratory function testing is important for detecting and monitoring illness, however, it is difficult for some patients, such as the young and severely ill, to perform conventional tests that require cooperation and/or patient contact. METHOD: A new method was developed for non-contact breathing measurement, employing photometric stereo to capture the surface topography of the torso of an unconstrained subject. The surface is integrated to calculate time-dependent volume changes during respiration. RESULTS: The method provides a useful means of continuously measuring volume changes during respiration with high spatial and temporal resolution. The system was tested by comparison with pneumotachometry equipment and a clear periodic signal, of a frequency corresponding to the reference data, was observed. CONCLUSION: The approach is unique in performing breathing monitoring (with potential diagnostic capability) for unconstrained patients in virtually any lighting conditions (including darkness during sleep) and in a non-contact, unobtrusive (i.e. using imperceptible light) fashion. Copyright © 2015 John Wiley & Sons, Ltd.

Atmospheric hydrogen peroxide and Eoarchean iron formations.

It is widely accepted that photosynthetic bacteria played a crucial role in Fe(II) oxidation and the precipitation of iron formations (IF) during the Late Archean-Early Paleoproterozoic (2.7-2.4 Ga). It is less clear whether microbes similarly caused the deposition of the oldest IF at ca. 3.8 Ga, which would imply photosynthesis having already evolved by that time. Abiological alternatives, such as the direct oxidation of dissolved Fe(II) by ultraviolet radiation may have occurred, but its importance has been discounted in environments where the injection of high concentrations of dissolved iron directly into the photic zone led to chemical precipitation reactions that overwhelmed photooxidation rates. However, an outstanding possibility remains with respect to photochemical reactions occurring in the atmosphere that might generate hydrogen peroxide (H2 O2 ), a recognized strong oxidant for ferrous iron. Here, we modeled the amount of H2 O2 that could be produced in an Eoarchean atmosphere using updated solar fluxes and plausible CO2 , O2 , and CH4 mixing ratios. Irrespective of the atmospheric simulations, the upper limit of H2 O2 rainout was calculated to be <10(6) molecules cm(-2) s(-1) . Using conservative Fe(III) sedimentation rates predicted for submarine hydrothermal settings in the Eoarchean, we demonstrate that the flux of H2 O2 was insufficient by several orders of magnitude to account for IF deposition (requiring ~10(11) H2 O2 molecules cm(-2) s(-1) ). This finding further constrains the plausible Fe(II) oxidation mechanisms in Eoarchean seawater, leaving, in our opinion, anoxygenic phototrophic Fe(II)-oxidizing micro-organisms the most likely mechanism responsible for Earth's oldest IF.

Genetic variant predicts bevacizumab-induced hypertension in ECOG-5103 and ECOG-2100.

BACKGROUND: Bevacizumab has broad anti-tumour activity, but substantial risk of hypertension. No reliable markers are available for predicting bevacizumab-induced hypertension. METHODS: A genome-wide association study (GWAS) was performed in the phase III bevacizumab-based adjuvant breast cancer trial, ECOG-5103, to evaluate for an association between genotypes and hypertension. GWAS was conducted in those who had experienced systolic blood pressure (SBP) >160 mm Hg during therapy using binary analysis and a cumulative dose model for the total exposure of bevacizumab. Common toxicity criteria (CTC) grade 3-5 hypertension was also assessed. Candidate SNP validation was performed in the randomised phase III trial, ECOG-2100. RESULTS: When using the phenotype of SBP>160 mm Hg, the most significant association in SV2C (rs6453204) approached and met genome-wide significance in the binary model (P=6.0 × 10(-8); OR=3.3) and in the cumulative dose model (P=4.7 × 10(-8); HR=2.2), respectively. Similar associations with rs6453204 were seen for CTC grade 3-5 hypertension but did not meet genome-wide significance. Validation study from ECOG-2100 demonstrated a statistically significant association between this SNP and grade 3/4 hypertension using the binary model (P-value=0.037; OR=2.4). CONCLUSIONS: A genetic variant in SV2C predicted clinically relevant bevacizumab-induced hypertension in two independent, randomised phase III trials.

Medullary thyroid cancer and pseudocirrhosis: case report and literature review.

Pseudocirrhosis is a rare form of liver disease that can cause clinical symptoms and radiographic signs of cirrhosis; however, its histologic features suggest a distinct pathologic process. In the setting of cancer, hepatic metastases and systemic chemotherapy are suspected causes of pseudocirrhosis. Here, we present a patient with medullary thyroid carcinoma metastatic to the liver who developed pseudocirrhosis while on maintenance sunitinib after receiving 5-fluorouracil, leucovorin, and oxaliplatin (folfox) in combination with sunitinib. Cirrhotic change in liver morphology was accompanied by diffusely infiltrative carcinomatous disease resembling the primary tumor. We discuss the diagnosis of pseudocirrhosis in this case and review the literature regarding pseudocirrhosis in cancer.

Tissue/fluid correlation study for the depletion of sulfadimethoxine in bovine kidney, liver, plasma, urine, and oral fluid.

Sulfonamides are among the oldest, but still effective, antimicrobial veterinary medicines. In steers and dairy cows, the sulfonamides are effective in the treatment of respiratory disease and general infections. Sulfadimethoxine (SDM) has been approved by US Food and Drug Administration (FDA) for use in steers and dairy cows with a tolerance of 100 ng/g (ppb) in edible tissues and 10 ppb in milk. The detection of SDM residue above tolerance in the animal slaughtered for food process will result in the whole carcass being discarded. This report describes a comprehensive depletion study of SDM (and its main metabolite) in plasma, urine, oral fluid, kidney, and liver. In this study, nine steers were injected intravenously with the approved dose of SDM; the loading dose was 55 mg/kg, followed by 27.5 mg/kg dose at 24 h and again at 48 h. Fluids (blood, urine, and saliva) and tissue (liver and kidney) samples were collected at intervals after the last dose of SMD. The combination of laparoscopic serial sampling technique with the liquid chromatography/mass spectrometry method provided the data to establish the tissue/fluid correlation in the depletion of SMD. A strong correlation and linearity of the log-scale concentration over time in the depletion stage has been confirmed for kidney, liver, and plasma.

The anorectic effect of neuropeptide AF is associated with satiety-related hypothalamic nuclei.

Neuropeptide AF (NPAF), a member of the RFamide family, is encoded by the same gene as neuropeptide FF (NPFF), which causes short-term anorexia. However, reports on the role of NPAF on appetite-related process are lacking. Thus, i.c.v. injections of 4.0, 8.0 and 16.0 nmol NPAF were administered to chicks to observe its effect on food and water intake. Chicks treated with 8.0 and 16.0 nmol i.c.v. NPAF decreased both their food and water intake. Additionally, all doses of NPAF injected caused a similar reduction in whole blood glucose concentration 180 min after injection. In a second experiment, chicks that received i.c.v. NPAF had an increased number of c-Fos immunoreactive cells in the dorsomedial, paraventricular (magnocellular and parvicellular parts) and ventromedial nuclei. The arcuate nucleus and lateral hypothalamic area were not affected. In a third experiment, NPAF-treated chicks exhibited fewer feeding pecks and spent less time perching, whereas they spent an increased time in deep rest. Other behaviours, including exploratory pecking, escape attempts, defecations, distance moved, and time spent standing, sitting and preening, were not affected by NPAF injection. We conclude that NPAF causes anorectic effects that are associated with the hypothalamus.

Display of peptides on the surface of tobacco mosaic virus particles.

In this review, we focus on the potential that tobacco mosaic virus (TMV) has as a carrier for immunogenic epitopes, and the factors that must be considered in order to bring products based on this platform to the market. Large Scale Biology Corporation developed facile and scaleable methods for manufacture of candidate peptide display vaccines based on TMV. We describe how rational design of peptide vaccines can improve the manufacturability of particular TMV products. We also discuss downstream processing and purification of the vaccine products, with particular attention to the metrics that a product must attain in order to meet criteria for regulatory approval as injectable biologics.

Differential appetite-related responses to central neuropeptide S in lines of chickens divergently selected for low or high body weight.

The anorexigenic 20 amino acid neuropeptide S (NPS) has not been studied in an animal model of hypo- or hyperphagia. The present study aimed to elucidate whether central NPS appetite-related effects are different in lines of chickens that had undergone long-term divergent selection for low (LWS) or high (HWS) body weight and that were hypo- and hyperphagic, respectively. It took a longer time for food intake to be reduced in LWS than HWS chicks administered the lowest dose of NPS tested (0.14 nmol) and, at the highest dose tested (0.56 nmol), they had a greater reduction in food intake than did HWS chicks. HWS chicks responded with a similar magnitude of food intake reduction that was independent of NPS dose. Although water intake was reduced concurrently with food intake after central NPS in both lines, blood glucose concentrations were not affected. Hypothalamic signalling was different between the lines. Although both lines respond to central NPS with decreased c-Fos immunoreactivity in the lateral hypothalamus, the periventricular nucleus had increased c-Fos immunoreactivity in LWS but not HWS chicks. After central NPS treatment, there was increased c-Fos immunoreactivity in the paraventricular nucleus in HWS but not LWS chicks. These data support the notion of differences in the central NPS system between the LWS and HWS lines and infer that central NPS may differentially affect appetite-related processes in other species that contain hypo- and hyperphagic individuals.

Base excision DNA repair defect in Gadd45a-deficient cells.

As one of a number of p53-regulated genes, Gadd45a (growth arrest and DNA damage inducible gene) has been shown to delay carcinogenesis and decrease mutation frequency. Gadd45a is known to regulate nucleotide excision DNA repair (NER) in response to UV radiation. Here, we report an emerging role for Gadd45a in base excision repair (BER). Gadd45a-null mouse embryo fibroblasts MEF and gadd45a-deficient human colon cancer cells exhibited slow BER after treatment with methyl methanesulfonate (MMS) a pure base-damaging agent. In addition, removal of AP sites by apurinic/apyrimidinic endonuclease 1/redox factor 1 (APE1/Ref1) was significantly delayed in gadd45a-null cells. Moreover, the localization of APE1/Ref1 within the nucleus was observed in gadd45a wild-type cells, whereas APE1 become mainly distributed in the cytoplasm, and there is a reduced interaction with proliferating cell nuclear antigen (PCNA) in Gadd45a-deficient cells. Inasmuch as p53 has been shown to regulate BER in addition to the NER pathway, our data suggest that p53-regulated gene Gadd45a contributes to the BER response by affecting the interaction of cellular APE1/Ref1 with PCNA. Gadd45a might be a key component gene of the p53 pathway involved in protection from carcinogenic base damage and maintenance of genomic stability, although the downstream mechanism including APE1/Ref1 will need further study.

Bcl-2 family proteins are essential for platelet survival.

Platelets are relatively short-lived, anucleated cells that are essential for proper hemostasis. The regulation of platelet survival in the circulation remains poorly understood. The process of platelet activation and senescence in vivo is associated with processes similar to those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, and cell shrinkage. ABT-737, a potent antagonist of Bcl-2, Bcl-X(L), and Bcl-w, induces apoptosis in nucleated cells dependent on these proteins for survival. In vivo, ABT-737 induces a reduction of circulating platelets that is maintained during drug therapy, followed by recovery to normal levels within several days after treatment cessation. Whole body scintography utilizing ([111])Indium-labeled platelets in dogs shows that ABT-737-induced platelet clearance is primarily mediated by the liver. In vitro, ABT-737 treatment leads to activation of key apoptotic processes including cytochrome c release, caspase-3 activation, and PS externalization in isolated platelets. Despite these changes, ABT-737 is ineffective in promoting platelet activation as measured by granule release markers and platelet aggregation. Taken together, these data suggest that ABT-737 induces an apoptosis-like response in platelets that is distinct from platelet activation and results in enhanced clearance in vivo by the reticuloendothelial system.

A rare ganglioneuroblastoma secreting dopamine and the value of its measurement in diagnosis and prognosis.

A case is described of a patient with a ganglioneuroblastoma, initially located in the right adrenal, which produced an excess of dopamine (7646 and 7959 nmol/24 h), approximately two and a half times the upper limit of the normal daily urine output. The urinary excretion of noradrenaline, adrenaline and methylated derivatives was always within the normal reference ranges. The patient was generally well, with normal blood pressure and only mild flushes. Two years after surgical resection, recurrence was indicated by an increase in urinary dopamine (8507 nmol/24 h); it was located in the tumour bed and left side of the neck by CT and (123)I MIBG scans. The patient was treated with a high dose of (131)I MIBG, with subsequent reduction in dopamine production. This was repeated on four other occasions, the latest being in January 2005. The output of dopamine was thus used as a marker of tumour diagnosis and progression and it is recommended that the assay of dopamine be included in the screening of catecholamine-secreting tumours to avoid possible misdiagnosis.

Gene deletion of cystatin C aggravates brain damage following focal ischemia but mitigates the neuronal injury after global ischemia in the mouse.

Cystatin C is distributed in all human tissues and fluids with a particular abundance in the cerebrospinal fluid. Cystatin C is a strong endogenous inhibitor of lysosomal cysteine proteases, such as cathepsin B, L, H and S, that are involved in various biological processes such as degradation of cellular proteins and regulation of enzymes, as well as in pathological processes. Pharmacological inhibition of cathepsins has been shown to reduce neuronal damage after brain ischemia, suggesting that cystatin C is an endogenous neuroprotectant. Cystatin C has also amyloidogenic properties and is co-localized with beta-amyloid in degenerated neurons in Alzheimer's disease, suggesting a role in neuronal degeneration. To test the hypothesis that endogenous cystatin C is neuroprotective during brain ischemia, global and focal brain ischemia was induced in mice with the cystatin C gene knocked out. Following focal ischemia, larger brain infarcts were found in cystatin C knockout mice, probably due to a reduced inhibition of the cathepsins during ischemia. In contrast, brain damage after global ischemia was diminished in cystatin C knockout mice, suggesting that cystatin C has an aggravating effect on selective neuronal damage after global ischemia.