Search

Optoacoustic imaging has grown in clinical relevance due to inherent advantages in sensitivity, resolution, and imaging depth, but the development of contrast agents is lacking. This study assesses the influence of structural features of squaraine dyes on optoacoustic activity through computational models, in vitro testing, and in vivo experimentation. The squaraine scaffold was decorated with halogens and side-chain extensions. Extension of side chains and heavy halogenation of squaraines both increased optoacoustic signals individually, although they had a more significant effect in tandem. Density functional theory models suggest that the origin of the increased optoacoustic signal is the increase in transition dipole moment and vibrational entropy, which manifested as increased absorbance in near-infrared region (NIR) wavelengths and decreased fluorescence quantum yield. This study provides insight into the structure-function relationships that will lead guiding principles for optimizing optoacoustic contrast agents. Further developments of squaraines and other agents will further increase the relevance of optoacoustic imaging in a clinical setting., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Purpose: G-CSF enhances colon cancer development. This study defines the prevalence and effects of increased G-CSF signaling in human colon cancers and investigates G-CSF inhibition as an immunotherapeutic strategy against metastatic colon cancer., Experimental Design: Patient samples were used to evaluate G-CSF and G-CSF receptor (G-CSFR) levels by IHC with sera used to measure G-CSF levels. Peripheral blood mononuclear cells were used to assess the rate of G-CSFR+ T cells and IFNγ responses to chronic ex vivo G-CSF. An immunocompetent mouse model of peritoneal metastasis (MC38 cells in C57Bl/6J) was used to determine the effects of G-CSF inhibition (αG-CSF) on survival and the tumor microenvironment (TME) with flow and mass cytometry., Results: In human colon cancer samples, the levels of G-CSF and G-CSFR are higher compared to normal colon tissues from the same patient. High patient serum G-CSF is associated with increases in markers of poor prognosis, (e.g., VEGF, IL6). Circulating T cells from patients express G-CSFR at double the rate of T cells from controls. Prolonged G-CSF exposure decreases T cell IFNγ production. Treatment with αG-CSF shifts both the adaptive and innate compartments of the TME and increases survival (HR, 0.46; P = 0.0237) and tumor T-cell infiltration, activity, and IFNγ response with greater effects in female mice. There is a negative correlation between serum G-CSF levels and tumor-infiltrating T cells in patient samples from women., Conclusions: These findings support G-CSF as an immunotherapeutic target against colon cancer with greater potential benefit in women., (©2023 American Association for Cancer Research.)

It is important to develop minimally invasive biomarker platforms to help in the identification and monitoring of patients with Alzheimer's disease (AD). Assisting in the understanding of biochemical mechanisms as well as identifying potential novel biomarkers and therapeutic targets would be an added benefit of such platforms. This study utilizes a simplified and novel serum profiling platform, using mass spectrometry (MS), to help distinguish AD patient groups (mild and moderate) and controls, as well as to aid in understanding of biochemical phenotypes and possible disease development. A comparison of discriminating sera mass peaks between AD patients and control individuals was performed using leave one [serum sample] out cross validation (LOOCV) combined with a novel peak classification valuation (PCV) procedure. LOOCV/PCV was able to distinguish significant sera mass peak differences between a group of mild AD patients and control individuals with a p value of 10 -13 . This value became non-significant ( p = 0.09) when the same sera samples were randomly allocated between the two groups and reanalyzed by LOOCV/PCV. This is indicative of physiological group differences in the original true-pathology binary group comparison. Similarities and differences between AD patients and traumatic brain injury (TBI) patients were also discernable using this novel LOOCV/PCV platform. MS/MS peptide analysis was performed on serum mass peaks comparing mild AD patients with control individuals. Bioinformatics analysis suggested that cell pathways/biochemical phenotypes affected in AD include those involving neuronal cell death, vasculature, neurogenesis, and AD/dementia/amyloidosis. Inflammation, autoimmunity, autophagy, and blood-brain barrier pathways also appear to be relevant to AD. An impaired VWF/ADAMTS13 vasculature axis with connections to F8 (factor VIII) and LRP1 and NOTCH1 was indicated and is proposed to be important in AD development.

Adolescent alcohol use is one of the strongest predictors for the development of an alcohol use disorder (AUD). Notably, this period of risk coincides with the development of affective disorders, which disproportionately impact and drive problematic drinking behavior in women. Stress is a particularly salient factor that drives relapse during periods of abstinence. Previous work in our lab has shown that adolescent intermittent ethanol vapor (AIE) produces sex-dependent changes in glutamatergic activity in the bed nucleus of the stria terminalis (BNST) and behavioral outcomes following acute restraint stress in adulthood. In females, AIE disrupts group 1 metabotropic glutamate (mGlu1/5) receptor activity and enhances anhedonia-like behavior. The current study site-specifically knocked down mGlu5 receptors in the BNST of male and female Grm5 loxp mice, exposed them to AIE, and observed the interaction of AIE and stress on negative affect-like behaviors in adulthood. These negative affect-like behaviors included the novelty-induced hypophagia task following acute restraint stress, open field activity, and contextual fear conditioning. Overall, we replicated our previous findings that AIE enhanced anhedonia-like activity in the novelty-induced hypophagia task in females and fear acquisition in males. The primary effect of BNST-mGlu5 receptor knockdown was that it independently enhanced anhedonia-like activity in females. Correlation analyses revealed that behavior in these paradigms showed poor interdependence. These results indicate that preclinical models of negative affective-like states encompass distinct features that may have independent, clinically relevant mechanisms. Further, modulating mGlu5 receptors is a prospective treatment target for females experiencing anhedonic-like states that make them susceptible to alcohol relapse.

Background: Women with colorectal cancer (CRC) have a significant survival advantage over men. Sex influences on the tumour microenvironment (TME) are not well characterised, despite the importance of immune response in CRC. We hypothesised that sex-divergent immune responses could contribute to survival., Methods: Using a murine model of metastatic CRC, we examined T cells, macrophages, and cytokines locally and systemically. TME and serum cytokines were measured by multiplex bead-based arrays, while FCA was used to identify cells and phenotypes. IHC provided spatial confirmation of T cell infiltration., Results: Females had increased survival and T cell infiltration. CD8, CD4 and Th2 populations correlated with longer survival. Males had increased serum levels of chemokines and inflammation-associated cytokines. Within the TME, males had lower cytokine levels than females, and a shallower cytokine gradient to the periphery. Female tumours had elevated IL-10+ macrophages, which correlated with survival., Conclusions: These data demonstrate survival-associated differences in the immune response of males and females to metastatic CRC. Females showed changes in cytokine production accompanied by increased immune cell populations, biased toward Th2-axis phenotypes. Key differences in the immune response to CRC correlated with survival in this model. These differences support a multi-faceted shift across the TME.

Adolescent alcohol exposure increases the risk of developing alcohol use disorders (AUDs), yet the mechanisms responsible for this vulnerability remain largely unknown. One potential target for alcohol-induced changes is the circuitry that modulates negative affect and stress, two sexually dependent drivers of alcohol relapse. The bed nucleus of the stria terminalis (BNST) is a sexually dimorphic region that critically regulates negative affective- and stress-induced relapse. Group I metabotropic glutamate receptors (mGluR) are a target of interest due to their regulation of stress, anxiety behaviors, and BNST plasticity. The current studies investigate sex-dependent sensitivity to the effects of adolescent intermittent ethanol vapor exposure (AIE) on negative affect during acute and protracted alcohol withdrawal and following stress in adulthood. This work also assessed whether BNST group I mGluR-mediated long-term depression (LTD) was disrupted at these timepoints. During acute withdrawal, AIE altered LTD induced by the group I mGluR antagonist DHPG in females, but not males. During adulthood, stress unmasked persistent changes in DHPG-induced LTD and behavior that were not present under basal conditions. Females with an AIE history demonstrated enhanced negative affective-like behavior in the novelty-induced hypophagia test following restraint stress-a phenotype that could be blocked with systemic mGluR5 allosteric antagonism via MTEP. Conversely, males with an AIE history demonstrated elevated freezing in a contextual fear conditioning paradigm. These studies demonstrate long-lasting, sex-dependent phenotypes produced by AIE and suggest pharmaceutical interventions for alcohol use and comorbid disorders may be more effective if designed with sex differences in mind.

Persistent alterations in synaptic plasticity and neurotransmission are thought to underlie the heightened risk of adolescent-onset drinkers to develop alcohol use disorders in adulthood. The bed nucleus of the stria terminalis (BNST) is a compelling region to study the consequences of early alcohol, as it is innervated by cortical structures which undergo continued maturation during adolescence and is critically involved in stress and negative affect-associated relapse. In adult mice, chronic ethanol induces long-term changes in GluN2B-containing NMDA receptors (NMDARs) of the BNST. It remains unclear, however, whether the adolescent BNST is susceptible to such persistent alcohol-induced modifications and, if so, whether they are preserved into adulthood. We therefore examined the short- and long-term consequences of adolescent intermittent ethanol exposure (AIE) on NMDAR transmission and plasticity in the BNST of male and female mice. Whole-cell voltage clamp recordings revealed greater glutamatergic tone in the BNST of AIE-treated males and females relative to air-controls. This change, which corresponded to an increase in presynaptic glutamate release, resulted in altered postsynaptic NMDAR metaplasticity and enhanced GluN2B transmission in males but not females. Only AIE-treated males displayed upregulated GluN2B expression (determined by western blot analysis). While these changes did not persist into adulthood under basal conditions, exposing adult males (but not females) to acute restraint stress reinstated AIE-induced alterations in NMDAR metaplasticity and GluN2B function. These data demonstrate that adolescent alcohol exposure specifically modifies NMDARs in the male BNST, that the plastic changes to NMDARs are long-lasting, and that they can be engaged by stress., (Copyright © 2019 Carzoli, Sharfman, Lerner, Miller, Holmgren and Wills.)

Traumatic Brain Injury (TBI) and persistent post-concussion syndrome (PCS) including chronic migraine (CM) are major health issues for civilians and the military. It is important to understand underlying biochemical mechanisms of these conditions, and be able to monitor them in an accurate and minimally invasive manner. This study describes the initial use of a novel serum analytical platform to help distinguish TBI patients, including those with post-traumatic headache (PTH), and to help identify phenotypes at play in these disorders. The hypothesis is that physiological responses to disease states like TBI and PTH and related bodily stresses are reflected in biomolecules in the blood in disease-specific manner. Leave one out (serum sample) cross validations (LOOCV) and sample randomizations were utilized to distinguished serum samples from the following TBI patient groups: TBI +PTSD + CM + severe depression (TBI "most affected" group) vs healthy controls, TBI "most affected" vs TBI, TBI vs controls, TBI + CM vs controls, and TBI + CM vs TBI. Inter-group discriminatory p values were ≤ 10-10, and sample group randomizations resulted in p non-significant values. Peptide/protein identifications of discriminatory mass peaks from the TBI "most affected" vs controls and from the TBI plus vs TBI minus CM groups yielded information of the cellular/molecular effects of these disorders (immune responses, amyloidosis/Alzheimer's disease/dementia, neuronal development). More specific biochemical disease effects appear to involve blood brain barrier, depression, migraine headache, autoimmunity, and autophagy pathways. This study demonstrated the ability for the first time of a novel, accurate, biomarker platform to monitor these conditions in serum, and help identify biochemical relationships leading to better understanding of these disorders and to potential therapeutic approaches., Competing Interests: The authors have declared that no competing interests exist.

Mild traumatic brain injury (mTBI) diagnoses have increased due to aggressive sports and blast-related injuries, but the cellular mechanisms and pathology underlying mTBI are not completely understood. Previous reports indicate that Nociceptin Orphanin/FQ (N/OFQ), an endogenous neuropeptide, contributes to post-injury ischemia following mechanical brain injury, yet its specific role in cerebral hypoxia, vestibulomotor function and injury marker expression following blast-induced TBI is not known. This study is the first to identify a direct association of N/OFQ and its N/OFQ peptide (NOP) receptor with TBI-induced changes following a single 80psi head blast exposure in male rats. N/OFQ and NOP receptor expression increased in brain tissue and plasma following TBI, concurrent with vestibular dysfunction but preceding hypoxia and appearance of injury markers compared to sham rats. A single post-blast treatment with the NOP receptor antagonist, SB-612111, transiently improved acute vestibulomotor performance. It also prevented increases in markers of TBI-induced hypoxia, pro-apoptotic proteins and injury seen 8-10days post-blast. This study reveals an apparent role for the N/OFQ-NOP receptor system in blast TBI and suggests potential therapeutic utility of NOP receptor antagonists for mTBI., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Dried plum has unique anabolic effects on bone, but the responsible bioactive components have remained unclear. This study investigated components of dried plum with potential osteoprotective activity utilizing aged, osteopenic Sprague Dawley rats fed diets supplemented with a crude polyphenol extract, potassium, vitamin K or their combination. Whole body and femoral bone mineral density were restored with the polyphenol and combination treatments to a similar extent as the dried fruit. The combination treatment reversed trabecular bone loss in the spine and cortical bone in the femur mid-diaphysis in a similar manner. Biomarkers of bone resorption were reduced by the polyphenol and combination treatments. The polyphenol extract accounted for most of the anabolic effect of dried plum on bone. This study is the first to show the bioactive components in dried plum responsible for restoring bone in vivo.

A stage I non-small cell lung cancer (NSCLC) serum profiling platform is presented which is highly efficient and accurate. Test sensitivity (0.95) for stage I NSCLC is the highest reported so far. Test metrics are reported for discriminating stage I adenocarcinoma vs squamous cell carcinoma subtypes. Blinded analysis identified 23 out of 24 stage I NSCLC and control serum samples. Group-discriminating mass peaks were targeted for tandem mass spectrometry peptide/protein identification, and yielded a lung cancer phenotype. Bioinformatic analysis revealed a novel lymphocyte adhesion pathway involved with early-stage lung cancer.

A gene array was used to profile the expression of 22,875 long non-coding RNAs (lncRNAs) and a large number of protein coding genes in 47 specimens of pancreatic ductal adenocarcinoma (PDAC), adjacent benign pancreas and the pancreas from patients without pancreatic disease. Of the lncRNAs profiled, the expression of 126 were significantly increased and 260 were decreased in the tumors ( p < 0.05, 2-fold). The expression of one lncRNA in particular, heterogeneous nuclear ribonucleoprotein U (HNRNPU) processed transcript (also known as ncRNA00201) was among the most significantly deregulated (increased four-fold) in the tumors compared to normal/adjacent benign tissues. Increased expression of HNRNPU processed transcript was associated with poor prognosis for patients with PDAC. The expression of HNRNPU processed transcript was increased in PDAC cell lines compared to noncancerous pancreatic cell lines. LNA TM gapmer mediated inhibition of HNRNPU processed transcript reduced cell proliferation in Patu-T and PL45 pancreatic cancer cell lines. Reduced invasion and migration was reported upon HNRNPU processed transcript knockdown in Patu-T cells. Small interfering RNA (siRNA) knockdown of the HNRNPU protein coding gene correlated with a 55% reduction in the HNRNPU processed transcript expression and a corresponding reduction in proliferation of Patu-T and PL45 cells. However, gapmer inhibition of HNRNPU processed transcript did not affect HNRNPU mRNA levels. The lncRNA HNRNPU processed transcript expression is increased in both PDAC tissues and cell lines; knockdown of this lncRNA further reduces proliferation and invasion/migration of pancreatic carcinoma cells., Competing Interests: The authors declare no conflict of interest.

Transcribed ultraconserved regions (T-UCRs) are a class of non-coding RNAs with 100% sequence conservation among human, rat and mouse genomes. T-UCRs are differentially expressed in several cancers, however their expression in pancreatic adenocarcinoma (PDAC) has not been studied. We used a qPCR array to profile all 481 T-UCRs in pancreatic cancer specimens, pancreatic cancer cell lines, during experimental pancreatic desmoplasia and in the pancreases of P48Cre/wt; KrasLSL-G12D/wt mice. Fourteen, 57 and 29% of the detectable T-UCRs were differentially expressed in the cell lines, human tumors and transgenic mouse pancreases, respectively. The vast majority of the differentially expressed T-UCRs had increased expression in the cancer. T-UCRs were monitored using an in vitro model of the desmoplastic reaction. Twenty-five % of the expressed T-UCRs were increased in the HPDE cells cultured on PANC-1 cellular matrix. UC.190, UC.233 and UC.270 were increased in all three human data sets. siRNA knockdown of each of these three T-UCRs reduced the proliferation of MIA PaCa-2 cells up to 60%. The expression pattern among many T-UCRs in the human and mouse pancreases closely correlated with one another, suggesting that groups of T-UCRs are co-activated in PDAC. Successful knockout of the transcription factor EGR1 in PANC-1 cells caused a reduction in the expression of a subset of T-UCRs suggesting that EGR1 may control T-UCR expression in PDAC. We report a global increase in expression of T-UCRs in both human and mouse PDAC. Commonalties in their expression pattern suggest a similar mechanism of transcriptional upregulation for T-UCRs in PDAC., Competing Interests: The authors have no conflicts of interest to disclose.

Blood tests are needed to aid in the early detection of pancreatic ductal adenocarcinoma (PDAC), and monitoring pancreatitis development into malignancy especially in high risk patients. This study exhibits efforts and progress toward developing such blood tests, using electrospray-mass spectrometry (MS) serum profiling to distinguish patients with early-stage PDAC or pancreatitis from each other and from controls. Identification of significant serum mass peak differences between these individuals was performed using t tests and "leave one out" cross validation. Serum mass peak distributions of control individuals were distinguished from those of patients with chronic pancreatitis or early-stage PDAC with P values <10(-15), and patients with chronic pancreatitis were distinguished from those of patients with early-stage PDAC with a P value <10(-12). Sera from 12 out of 12 patients with PDAC stages I, IIA and IIB were blindly validated from controls. Tandem MS/MS identified a cancer phenotype with elements of PDAC involved in early-stage PDAC/control discrimination. These studies indicate electrospray-MS mass profiling can detect serum changes in patients with pancreatitis or early-stage pancreatic cancer. Such technology has the potential to aid in early detection of pancreatic cancer, biomarker development, and in monitoring development of pancreatitis into PDAC., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Keratinocyte growth factor (KGF)/KGF receptor (KGFR) signaling produces a rapid increase in the progression of breast cancer. Molecular modeling was used to create a group of KGFR-selective kinase inhibitors (TKI). Compound L-27 is a potent and selective KGFR TKI. The present study examined the oncolytic potential of L-27 using a breast cancer xenograft model., Materials and Methods: An orthotopic xenograft model was developed with KGF-transfected MCF-7 cells to examine the influence of L-27 upon KGFR-mediated tumor progression., Results: L-27 was found to produce a dose-related reduction in the growth and metastasis of mouse xenograft tumors. Furthermore, L-27 treatment did not produce any signs of gross toxicity., Conclusion: L-27 was found to reduce the growth and metastasis of MCF-7 tumor xenografts with elevated expression of KGF. Thus, KGFR TKI may provide a new therapeutic approach for the treatment of breast and other types of cancer., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Purpose: The neutrophil-derived granular protein, CAP37, an innate immune system molecule, has antibiotic and immunomodulatory effects on host cells, including corneal epithelial cells. We previously showed that CAP37 modulates corneal epithelial cell migration, adhesion, and proliferation, and that protein kinase C delta (PKCδ) mediates CAP37-induced chemotaxis of these cells. The objective of this study was to investigate the hypothesis that CAP37 facilitates corneal wound healing through the PKC signaling pathway., Methods: The standard "scratch" assay performed on monolayers of corneal epithelial cells was used to measure the in vitro effect of CAP37 on wound closure. In vivo wound healing in response to CAP37 was measured using a mouse corneal epithelium abrasion model. In vitro and in vivo wound closure were monitored over 48 hours. The PKCδ was visualized during wound closure in cell monolayers and corneal epithelium by immunohistochemistry. The importance of PKCδ in CAP37-induced corneal wound healing was assessed by siRNA., Results: We found that CAP37 accelerated wound closure in vitro and in vivo. Maximal closure occurred with concentrations of CAP37 between 250 and 500 ng/mL. Topical applications on mouse cornea led to re-epithelialization of the cornea by 24 hours. Immunohistochemistry of in vitro and in vivo wounds revealed a local increase of PKCδ along the wound edge in CAP37-treated cell monolayers and corneas, compared to untreated controls. CAP37-induced corneal wound healing was significantly reduced in vivo upon treatment with PKCδ siRNA., Conclusions: These findings support the hypothesis that CAP37 facilitates corneal wound healing through the activation of PKCδ., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Serum mass profiling can discern physiological changes associated with specific disease states and their progression. Sera (86 total) from control individuals and patients with stage I nonsmall cell lung cancer or benign small pulmonary nodules were discriminated retrospectively by serum changes discerned by mass profiling. Control individuals were distinguished from patients with Stage I lung cancer or benign nodules with test sensitivities of 89% and 83%. Lung cancer patients versus those with benign nodules were distinguished with 80% sensitivity. This study exhibits progress toward a minimally-invasive aid in early detection of lung cancer and monitoring small pulmonary nodules for malignancy.

Objective: The incidence of cardiovascular disease dramatically increases during menopause, and postmenopausal women seek natural alternatives to hormone therapy. Flaxseed can slow the progression of atherosclerotic lesion formation; however, it is not known whether it can reverse formation that has already occurred., Methods: Seventy-two female Golden Syrian hamsters were randomly divided into six groups (n = 12), sham-operated (sham) or ovariectomized (ovx), and kept on the same diet for 120 days to allow for atherosclerotic lesion development. After this 120-day period, whole flaxseed was introduced to the diets of hamsters in three of the groups: group 1 (sham + casein); group 2 (ovx + casein); group 3 (ovx + 7.5% flaxseed); group 4 (ovx + 15% flaxseed); group 5 (ovx + 22.5% flaxseed); and group 6 (ovx + 17β-estradiol). This diet was maintained for an additional 120 days. Lesion regression was examined histologically, and serum was analyzed for total cholesterol, triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, Apo A, Apo B, and lipoprotein(a)., Results: Results showed that 15% and 22.5% flaxseed, compared with ovx animals, significantly reduced lipoprotein(a) (4.4 mg/dL [ovx] vs 2.15 mg/dL [15% flaxseed] and 0.3 mg/dL [22.5% flaxseed]; P < 0.05) and Apo B (2.8 mg/dL [ovx] vs 2.4 mg/dL [15% flaxseed] and 2.5 mg/dL [22.5% flaxseed]). Flax reduced by 67% the number of animals with aortic arch lesions., Conclusions: All three doses of flax reduce the severity of lesion formation compared with ovx controls. These results support the efficacy of flaxseed in reducing cardiovascular disease risk.

A novel scaffold composed of loosely branched hollow silica microfibers that has been proven to be highly biocompatible is proposed for the 3D culture of cancer cells. The MCF-7 cancer cells can grow and proliferate freely inside the scaffold in the form of multicellular spheroids. MCF-7 cancer cells cultured on the current 3D silica scaffold retained significantly more oncological characters than those cultured on the conventional 2D substrate and can serve as in vitro tumor model for studying cancer treatment., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Patch arteriotomies are performed during many vascular procedures. Whereas synthetic materials are generally felt to be inappropriate for infected environments, the suitability of glutaraldehyde-treated bovine pericardium (GBP), a biologic material, in such instances is unknown. Our main objectives were to develop an animal model to study vascular prostheses while comparing the infectability of polyester (Dacron) and GBP in a topically infected environment., Methods: Twenty-three pigs underwent transabdominal patch arteriotomy of the infrarenal aorta with either Dacron or GBP. The patches were inoculated with sterile saline (1 per group), Staphylococcus aureus 10(4) colony-forming units (CFUs) (4 per group), or S. aureus 10(5) CFUs (6 per group). At 3 wk, the animals were euthanized, and the patches were removed via a left retroperitoneal approach. Specimens were collected for microbiologic and histologic analysis., Results: One animal from each group inoculated with 10(5) CFUs died during the study period, and another died immediately postoperatively of an airway complication. All aortas were patent and without evidence of pseudoaneurysm formation. Gross abscesses were found in 4/6 Dacron and 5/6 GBP animals receiving 10(5) CFUs. Similarly, 4/6 animals implanted with Dacron and 5/6 animals implanted with GBP had positive tissue cultures. A histologic grading system of inflammation substantiated the culture results., Conclusions: No significant difference exists between Dacron and GBP to resist bacterial infection at 3 wk. We have established a reproducible in vivo model to study arterial patch materials in a topically infected environment., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Goals of this study were to analyze the ability of mass spectrometry serum profiling to distinguish non-small cell lung adenocarcinoma from squamous cell carcinoma patients and healthy controls. Sera were obtained from 19 adenocarcinoma patients, 24 squamous cell carcinoma patients, and 21 controls. Identifications of significant mass-to-charge ratio (m/z) peak differences between these groups were performed using t-tests. A "leave one out" cross-validation procedure yielded discriminatory lung adenocarcinoma versus squamous cell carcinoma p and ROC curve values of <.0001 and 0.92, respectively. Test sensitivity and specificity were 84% and 79%, respectively. This approach could aid in lung cancer diagnosis and sub-typing.

The goal of this study was to evaluate the usefulness of electrospray ionization-mass spectrometry (ESI-MS) technology to distinguish sera of early-stage lung cancer patients from control individuals. ESI-MS m/z (mass divided by charge) data were generated from sera of 43 non-small cell lung cancer patients (pathological stages I and II) and 21 control individuals. Identifications of m/z peak area significances between cancer and control ESI-MS sera spectra were performed using t-tests. A "leave one out" cross validation procedure, which mimics blinded sera analysis and corrects for "over-fitting" of data, yielded discriminatory cancer versus control distribution p value and ROC curve area value of <0.001 and 0.87, respectively. Analysis without the "leave one out" cross validation procedure yielded a ROC curve area of 0.99 for discrimination of sera from lung cancer patients versus control individuals. Predictive value measurements revealed overall test efficiency and sensitivity for distinguishing sera from lung cancer patients from controls (using "leave one out" cross validation) of 80% and 84%, respectively. ESI-MS serum analysis between control individuals and lung cancer patients who smoked or did not smoke had p values in ranges indicating that smoking effects are not pronounced in our analysis. These studies indicate that ESI-MS analyses of sera from early stage non-small cell lung cancer patients were helpful in distinguishing these patients from control individuals., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Numerous microRNAs (miRNAs) are reported as differentially expressed in cancer, however the consequence of miRNA deregulation in cancer is unknown for many miRNAs. We report that two miRNAs located on chromosome 17p13, miR-132 and miR-212, are over-expressed in pancreatic adenocarcinoma (PDAC) tissues. Both miRNAs are predicted to target the retinoblastoma tumor suppressor, Rb1. Validation of this interaction was confirmed by luciferase reporter assay and western blot in a pancreatic cancer cell line transfected with pre-miR-212 and pre-miR-132 oligos. Cell proliferation was enhanced in Panc-1 cells transfected with pre-miR-132/-212 oligos. Conversely, antisense oligos to miR-132/-212 reduced cell proliferation and caused a G(2)/M cell cycle arrest. The mRNA of a number of E2F transcriptional targets were increased in cells over expressing miR-132/-212. Exposing Panc-1 cells to the β2 adrenergic receptor agonist, terbutaline, increased the miR-132 and miR-212 expression by 2- to 4-fold. We report that over-expression of miR-132 and miR-212 result in reduced pRb protein in pancreatic cancer cells and that the increase in cell proliferation from over-expression of these miRNAs is likely due to increased expression of several E2F target genes. The β2 adrenergic pathway may play an important role in this novel mechanism., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study evaluated the usefulness of electrospray mass spectrometry to distinguish sera of early-stage pancreatic cancer patients from disease-free individuals. Sera peak data were generated from 33 pancreatic cancer patients and 30 disease-free individuals. A "leave one out" cross-validation procedure discriminated stage I/II pancreatic cancer versus disease-free sera with a p value <.001 and a receiver-operator characteristic curve area value of 0.85. Predictive values for cancer stage I/II test efficiency, specificity, and sensitivity were 78%, 77%, and 79%, respectively. These studies indicate that electrospray mass spectrometry is useful for distinguishing sera of early-stage pancreatic cancer patients from disease-free individuals.

Limited information is available regarding systemic changes in mammals associated with exposures to petroleum/hydrocarbon fuels. In this study, systemic toxicity of JP-8 jet fuel was observed in a rat inhalation model at different JP-8 fuel vapor concentrations (250, 500, or 1000 mg/m(3), for 91 days). Gel electrophoresis and mass spectrometry sequencing identified the alpha-2 microglobulin protein to be elevated in rat kidney in a JP-8 dose-dependent manner. Western blot analysis of kidney and lung tissue extracts revealed JP-8 dependent elevation of inducible heat shock protein 70 (HSP70). Tissue changes were observed histologically (hematoxylin and eosin staining) in liver, kidney, lung, bone marrow, and heart, and more prevalently at medium or high JP-8 vapor phase exposures (500-1000 mg/m(3)) than at low vapor phase exposure (250 mg/m(3)) or non-JP-8 controls. JP-8 fuel-induced liver alterations included dilated sinusoids, cytoplasmic clumping, and fat cell deposition. Changes to the kidneys included reduced numbers of nuclei, and cytoplasmic dumping in the lumen of proximal convoluted tubules. JP-8 dependent lung alterations were edema and dilated alveolar capillaries, which allowed clumping of red blood cells (RBCs). Changes in the bone marrow in response to JP-8 included reduction of fat cells and fat globules, and cellular proliferation (RBCs, white blood cells-WBCs, and megakaryocytes). Heart tissue from JP-8 exposed animals contained increased numbers of inflammatory and fibroblast cells, as well as myofibril scarring. cDNA array analysis of heart tissue revealed a JP-8 dependent increase in atrial natriuretic peptide precursor mRNA and a decrease in voltage-gated potassium (K+) ion channel mRNA.

Autophagy is the regulated process cells use to recycle nonessential, redundant, or inefficient components and is an adaptive response during times of stress. In addition to its function in enabling the cell to gain vital nutrients in times of stress, autophagy can also be involved in elimination of intracellular microorganisms, tumor suppression, and antigen presentation. Because of difficulty in diagnosing autophagy, few clinical studies have been performed. This study examined whether autophagy occurs in hepatocytes during sepsis. Electron microscopy (EM) was performed on liver samples obtained from both an observational clinical cohort of six septic patients and four control patients as well as liver specimens from mice with surgical sepsis (by cecal ligation and puncture) or sham operation. EM demonstrated increased autophagic vacuoles in septic vs nonseptic patients. Randomly selected fields (3000 microm(2)) from control and septic patients contained 1.2+/-1.5 vs 5.3+/-3.3 (mean+/-s.d.) complex lysosomal/autophagolysosomal structures per image respectively (P<0.001). In rare instances, hepatocytes with autophagic vacuoles appeared to be unequivocally committed to death. Membrane alterations (membrane vacuoles, invagination into adjacent organelles, and myelin figure-like changes) occur in a subpopulation of mitochondria in sepsis, but other hepatocyte organelles showed no consistent ultrastructural injury. Findings in murine sepsis paralleled those of patients, with 7.2+/-1.9 vs 38.7+/-3.9 lysosomal/autophagolysosomal structures in sham and septic mice, respectively (P=0.002). Quantitative RT-PCR demonstrated that sepsis induced the upregulation of select apoptosis and cytokine gene expression with minimal changes in the core autophagy genes in liver. In conclusion, hepatocyte autophagic vacuolization increases during sepsis and is associated with mitochondrial injury. However, it is not possible to determine whether the increase in autophagic vacuolization is an adaptive response or a harbinger of cell death.

Objective: Epidemiological and clinical evidence indicate that inflammatory processes play a pivotal role in a number of conditions associated with aging, including osteoporosis and cardiovascular diseases. The purpose of this study was to evaluate cardiovascular pathology and select inflammatory mediators of interest in a model of low-grade inflammation-induced osteopenia., Methods: Slow-release pellets were subcutaneously implanted in male rats to deliver 0, 3.3, or 33.3 microg of lipopolysaccharide (LPS)/day for 90 days. Tail blood was collected at 1, 2, and 3 months for differential white cell counts, and at the end of the study, hearts were harvested for histological and immunohistochemical evaluation., Results: The low-grade inflammatory response was characterized by elevated peripheral blood neutrophils and monocytes. Histological examination of heart cross sections revealed increased fibrous tissue, infiltration of lymphocytes, accumulation of mast cells, and roughened intimal borders within the arteries and arterioles, consistent with vascular disease. Inflammatory mediators (cyclooxygenase-2, tumor necrosis factor-alpha, and interleukin-1 beta) were up-regulated, and increased expression of platelet endothelial cell adhesion molecule-1 and receptor activator for NF-kappaB ligand was localized to the microvasculature endothelium., Conclusions: These findings suggest that inflammation induced by chronic exposure to LPS produces cardiovascular pathology in the smaller intramural arteries and arterioles and support the utility of this model for further mechanistic and therapeutic studies focused on the role of chronic inflammation in cardiovascular disease.

Enterococcus faecalis is an intestinal commensal that cannot synthesize porphyrins and only expresses a functional respiratory chain when provided with exogenous haematin. In the absence of haematin, E. faecalis reverts to fermentative metabolism and produces extracellular superoxide that can damage epithelial-cell DNA. The acute response of the colonic mucosa to haematin-starved E. faecalis was identified by gene array. E. faecalis was inoculated into murine colons using a surgical ligation model that preserved tissue architecture and homeostasis. The mucosa was exposed to haematin-starved E. faecalis and compared with a control consisting of the same strain grown with haematin. At 1 h post-inoculation, 6 mucosal genes were differentially regulated and this increased to 42 genes at 6 h. At 6 h, a highly significant biological interaction network was identified with functions that included nuclear factor-kappaB (NF-kappaB) signalling, apoptosis and cell-cycle regulation. Colon biopsies showed no histological abnormalities by haematoxylin and eosin staining. Immunohistochemical staining, however, detected NF-kappaB activation in tissue macrophages using antibodies to the nuclear localization sequence for p65 and the F4/80 marker for murine macrophages. Similarly, haematin-starved E. faecalis strongly activated NF-kappaB in murine macrophages in vitro. Furthermore, primary and transformed colonic epithelial cells activated the G2/M checkpoint in vitro following exposure to haematin-starved E. faecalis. Modulation of this cell-cycle checkpoint was due to extracellular superoxide produced as a result of the respiratory block in haematin-starved E. faecalis. These results demonstrate that the uniquely dichotomous metabolism of E. faecalis can significantly modulate gene expression in the colonic mucosa for pathways associated with inflammation, apoptosis and cell-cycle regulation.

Purpose: Visually guided ocular growth is facilitated by scleral extracellular matrix remodeling at the posterior pole of the eye. Coincident with scleral remodeling, significant changes in choroidal morphology, blood flow, and protein synthesis have been shown to occur in eyes undergoing ocular growth changes. The current study is designed to identify gene expression changes that may occur in the choroid/retinal pigment epithelium (RPE) of marmoset eyes during their compensation for hyperopic defocus as compared to eyes compensating for myopic defocus., Methods: Total RNA was isolated from choroid/RPE from four common marmosets (Callithrix jacchus) undergoing binocular lens treatment using extended wear soft contact lenses of equal magnitude but opposite sign (+/-5 diopter [D]). After reverse transcription, cDNA was labeled and hybridized to a human oligonucleotide microarray and gene transcript expression profiles were determined. Real-time polymerase chain reaction (PCR) and western blot analysis were used to confirm genes and proteins of interest, respectively., Results: Microarray analyses in choroid/RPE indicated 204 genes were significantly changed in minus lens-treated as compared with plus lens-treated eyes (p<0.05, Student's t-test). Differential choroid/RPE expression of protein tyrosine phosphatase, receptor type, B (PTPRB), transforming growth factor beta-induced (TGFBI), and basic fibroblast growth factor 2 (FGF-2) were confirmed by real-time PCR. TGFBIp was confirmed at the protein level by western blot analysis in marmoset and human cornea, choroid/RPE, and sclera., Conclusions: The present study demonstrated that significant gene expression changes occur in the marmoset choroid/RPE during visually guided ocular growth. The identification of novel candidate genes in choroid/RPE of marmoset eyes actively accelerating or decelerating their rates of ocular elongation may elucidate the choroidal response during the regulation of postnatal ocular growth and may lead to the identification of choroid/RPE signaling molecules that participate in scleral remodeling.

Blood borne Listeria monocytogenes enter the CNS via migration of parasitized Ly-6Chigh monocytes, but the signals that trigger this migration are not known. To understand more completely events leading to monocyte recruitment, experiments presented here combined microarray analysis of gene expression in the brains of experimentally infected mice with measurements of bacterial CFU and serum cytokines following i.v. infection with L. monocytogenes. At 24 and 48 h postinfection, the brain was sterile but there were significant changes in transcriptional activity related to serum proinflammatory cytokines. Real-time PCR confirmed mRNA up-regulation of genes related to IFN-gamma, IL-1, and TNF-alpha, although IFN-gamma itself was not up-regulated in the brain. Infection with Deltaacta, but not Deltahly mutants, increased serum concentrations of IFN-gamma, IL-6, and to a lesser extent TNF-alpha. The brain was not infected but there was widespread mRNA up-regulation in it and an influx of Ly-6Chigh monocytes in Deltaacta-infected mice. Moreover, DeltaactA-infected IFN-gamma-/- mice had no brain influx of Ly-6Chigh monocytes despite normal monocyte trafficking from bone marrow to blood and spleen. Additionally, IFN-gamma-/- mice showed diminished mRNA expression for monocyte-attracting chemokines, and significantly less CXCL9 and CXCL10 protein in the brain compared with normal mice. These data demonstrate that monocyte recruitment to the brain is independent of bacterial invasion of the CNS and is triggered by proinflammatory cytokines, in particular IFN-gamma, produced by the innate immune response to intracellular infection in peripheral organs.

Objective: The aim of this study is to identify biomarkers in sera of pancreatic cancer patients using mass spectrometry (MS) approaches., Methods: Sera from patients diagnosed with pancreatic adenocarcinoma and sera from normal volunteers were subjected to gel electrophoresis to resolve and quantify differences in protein levels. Protein bands that differed quantitatively were digested with trypsin, and peptides were identified by electrospray ionization (ESI) ion-trap tandem MS. Mass spectra were also collected directly from pancreatic cancer sera as well as healthy control sera using ESI-MS., Results: Three large-mass proteins were found to be elevated in pancreatic cancer sera versus normal sera, alpha-2 macroglobulin, ceruloplasmin, and complement 3C. Complement 3C is a major regulator of inflammatory responses. The ESI-MS of human pancreatic cancer sera versus normal sera revealed greater heterogeneity in cancer sera than control sera, especially in the low-mass region. Bootstrapping statistical analysis identified 20 low-mass serum peaks that correlated with control sera and 20 different peaks that correlated with pancreatic cancer sera., Conclusions: The fact that inflammation-sensitive proteins were identified as increased in pancreatic cancer sera supports the hypothesis that inflammatory-driven processes are involved in pancreatic carcinogenesis. Liquid ESI-MS analyses of sera hold promise for future pancreatic cancer blood tests as well as for understanding mechanisms of pancreatic carcinogenesis. The variability observed between the low-mass regions of normal versus pancreatic cancer spectra may aid in diagnosis and therapy.

Background: Evidence from epidemiological, clinical and animal studies suggests a link may exist between low bone density and cardiovascular disease, with inflammatory mediators implicated in the pathophysiology of both conditions. This project examined whether supplementation with soy isoflavones (IF), shown to have anti-inflammatory properties, could prevent tissue expression of TNF-alpha and the development of skeletal pathology in an animal model of chronic inflammation., Methods: Eight-week old, intact, female C57BL/6J mice were used. In Phase 1, a lipopolysaccharide (LPS)-dose response study (0, 0.133, 1.33 and 13.3 mug/d) was conducted to determine the LPS dose to use in Phase 2. The results indicated the 1.33 mug LPS/d dose produced the greatest decrease in lymphocytes and increase in neutrophils. Subsequently, in Phase 2, mice were randomly assigned to one of six groups (n = 12-13 per group): 0 or 1.33 mug LPS/d (placebo or LPS) in combination with 0, 126 or 504 mg aglycone equivalents of soy IF/kg diet (Control, Low or High dose IF). Mice were fed IF beginning 2 wks prior to the 30-d LPS study period., Results: At the end of the study, no differences were detected in final body weights or uterine weights. In terms of trabecular bone microarchitecture, muCT analyses of the distal femur metaphysis indicated that LPS significantly decreased trabecular bone volume (BV/TV) and number (TbN), and increased separation (TbSp). Trabecular bone strength (i.e. total force) and stiffness were also compromised in response to LPS. The High IF dose provided protection against these detrimental effects on microarchitecture, but not biomechanical properties. No alterations in trabecular thickness (TbTh), or cortical bone parameters were observed in response to the LPS or IF. Immunohistomchemical staining showed that tumor necrosis factor (TNF)-alpha was up-regulated by LPS in the endothelium of small myocardial arteries and arterioles as well as the tibial metaphysis and down-regulated by IF., Conclusion: These results suggest IF may attenuate the negative effects of chronic inflammation on bone and cardiovascular health. Additional research is warranted to examine the anti-inflammatory properties of the soy isoflavones and the mechanisms underlying their prevention of chronic inflammation-induced bone loss.

microRNAs are functional, 22 nt, noncoding RNAs that negatively regulate gene expression. Disturbance of microRNA expression may play a role in the initiation and progression of certain diseases. A microRNA expression signature has been identified that is associated with pancreatic cancer. This has been accomplished with the application of real-time PCR profiling of over 200 microRNA precursors on specimens of human pancreatic adenocarcinoma, paired benign tissue, normal pancreas, chronic pancreatitis and nine pancreatic cancer cell lines. Hierarchical clustering was able to distinguish tumor from normal pancreas, pancreatitis and cell lines. The PAM algorithm correctly classified 28 of 28 tumors, 6 of 6 normal pancreas and 11 of 15 adjacent benign tissues. One hundred microRNA precursors were aberrantly expressed in pancreatic cancer or desmoplasia (p < 0.01), including microRNAs previously reported as differentially expressed in other human cancers (miR-155, miR-21, miR-221 and miR-222) as well as those not previously reported in cancer (miR-376a and miR-301). Most of the top aberrantly expressed miRNAs displayed increased expression in the tumor. Expression of the active, mature microRNA was validated using a real-time PCR assay to quantify the mature microRNA and Northern blotting. Reverse transcription in situ PCR showed that three of the top differentially expressed miRNAs (miR-221, -376a and -301) were localized to tumor cells and not to stroma or normal acini or ducts. Aberrant microRNA expression may offer new clues to pancreatic tumorigenesis and may provide diagnostic biomarkers for pancreatic adenocarcinoma., (Copyright 2006 Wiley-Liss, Inc.)