The Obesity Epidemic Is Not the Victims' Fault.

The Centers for Disease Control and Prevention recently issued two statements that 1) maintain that obesity causes diabetes and other expressions of the metabolic syndrome and 2) that imply obesity is the victim's fault. Both statements are incorrect and potentially harmful.

PI3K signaling promotes formation of lipid-laden foamy macrophages at the spinal cord injury site.

After spinal cord injury (SCI), infiltrating macrophages undergo excessive phagocytosis of myelin and cellular debris, forming lipid-laden foamy macrophages. To understand their role in the cellular pathology of SCI, investigation of the foamy macrophage phenotype in vitro revealed a pro-inflammatory profile, increased reactive oxygen species (ROS) production, and mitochondrial dysfunction. Bioinformatic analysis identified PI3K as a regulator of inflammation in foamy macrophages, and inhibition of this pathway decreased their lipid content, inflammatory cytokines, and ROS production. Macrophage-specific inhibition of PI3K using liposomes significantly decreased foamy macrophages at the injury site after a mid-thoracic contusive SCI in mice. RNA sequencing and in vitro analysis of foamy macrophages revealed increased autophagy and decreased phagocytosis after PI3K inhibition as potential mechanisms for reduced lipid accumulation. Together, our data suggest that the formation of pro-inflammatory foamy macrophages after SCI is due to the activation of PI3K signaling, which increases phagocytosis and decreases autophagy.

p53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricle Pressure Overload.

BACKGROUND: Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure. METHODS: We used the Tcf21 (transcription factor 21)MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction. RESULTS: Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21-lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation. CONCLUSIONS: This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.

Cytostatic hypothermia and its impact on glioblastoma and survival.

Patients with glioblastoma (GBM) have limited options and require novel approaches to treatment. Here, we studied and deployed nonfreezing "cytostatic" hypothermia to stunt GBM growth. This growth-halting method contrasts with ablative, cryogenic hypothermia that kills both neoplastic and infiltrated healthy tissue. We investigated degrees of hypothermia in vitro and identified a cytostatic window of 20° to 25°C. For some lines, 18 hours/day of cytostatic hypothermia was sufficient to halt division in vitro. Next, we fabricated an experimental tool to test local cytostatic hypothermia in two rodent GBM models. Hypothermia more than doubled median survival, and all rats that successfully received cytostatic hypothermia survived their study period. Unlike targeted therapeutics that are successful in preclinical models but fail in clinical trials, cytostatic hypothermia leverages fundamental physics that influences biology broadly. It is a previously unexplored approach that could provide an additional option to patients with GBM by halting tumor growth.

Fish Oil Contaminated with Persistent Organic Pollutants Induces Colonic Aberrant Crypt Foci Formation and Reduces Antioxidant Enzyme Gene Expression in Rats.

Background: Epidemiologic, clinical, and experimental studies have suggested that fish oil (FO), a rich source of n-3 (ω-3) polyunsaturated fatty acids, protects against colon cancer. However, this message is confounded by the FDA's warning that the consumption of certain types of fish should be restricted because of contamination with persistent organic pollutants (POPs), such as polychlorinated biphenyls (PCBs) and organochlorine pesticides.Objective: We examined FO contaminated with POPs (PCBs, dichlorodiphenyltrichloroethane, and chlordane) compared with unmodified FO on the risk factors of colon cancer development.Methods: Male Sprague-Dawley rats aged 28 d (n = 30) were allocated into 3 groups and fed 15% corn oil (CO), FO, or POP-contaminated FO for 9 wk with a subcutaneous injection of colon carcinogen azoxymethane at weeks 3 and 4. Colonic aberrant crypt foci (ACF) and cell proliferation were enumerated, and the gene expression of inflammation, antioxidant enzymes, and repair enzymes were determined with the use of real-time quantitative polymerase chain reaction analysis.Results: FO-fed rats had a lower number of ACF (mean ± SE: 29 ± 4.0 for FO compared with 53 ± 8.4 for CO and 44 ± 4.6 for POP FO) and higher-multiplicity ACF than the CO and POP FO groups (4.7 ± 0.9 for FO compared with 11 ± 1.5 for CO and 9.6 ± 1.8 for POP FO) (P < 0.05). FO feeding lowered the proliferation index compared with the CO and POP FO feeding groups (18% ± 1.1% for FO compared with 25% ± 1.6% for CO and 23% ± 0.7% for POP FO) (P = 0.009). Superoxide dismutase [2.4 ± 0.6 relative quantification (RQ) for FO compared with 1.2 ± 0.2 RQ for CO and 1.3 ± 0.3 RQ for POP FO] and catalase gene expression (10 ± 2.0 RQ for FO compared with 5.4 ± 1.1 RQ for CO and 6.6 ± 1.5 RQ for POP FO) were higher in the FO group than in the CO and POP FO groups (P < 0.05). There were no differences between CO and POP FO on the variables.Conclusion: These results indicate that POPs in FO reduce the preventive effects of FO on colon carcinogenesis by increasing preneoplastic lesion formation through the downregulation of antioxidant enzyme expression and increasing cell proliferation in rats.

Proposed clinical internal carotid artery classification system.

INTRODUCTION: Numerical classification systems for the internal carotid artery (ICA) are available, but modifications have added confusion to the numerical systems. Furthermore, previous classifications may not be applicable uniformly to microsurgical and endoscopic procedures. The purpose of this study was to develop a clinically useful classification system. MATERIALS AND METHODS: We performed cadaver dissections of the ICA in 5 heads (10 sides) and evaluated 648 internal carotid arteries with computed tomography angiography. We identified specific anatomic landmarks to define the beginning and end of each ICA segment. RESULTS: The ICA was classified into eight segments based on the cadaver and imaging findings: (1) Cervical segment; (2) cochlear segment (ascending segment of the ICA in the temporal bone) (relation of the start of this segment to the base of the styloid process: Above, 425 sides [80%]; below, 2 sides [0.4%]; at same level, 107 sides [20%]; P < 0.0001) (relation of cochlea to ICA: Posterior, 501 sides [85%]; posteromedial, 84 sides [14%]; P < 0.0001); (3) petrous segment (horizontal segment of ICA in the temporal bone) starting at the crossing of the eustachian tube superolateral to the ICA turn in all 10 samples; (4) Gasserian-Clival segment (ascending segment of ICA in the cavernous sinus) starting at the petrolingual ligament (PLL) (relation to vidian canal on imaging: At same level, 360 sides [63%]; below, 154 sides [27%]; above, 53 sides [9%]; P < 0.0001); in this segment, the ICA projected medially toward the clivus in 275 sides (52%) or parallel to the clivus with no deviation in 256 sides (48%; P < 0.0001); (5) sellar segment (medial loop of ICA in the cavernous sinus) starting at the takeoff of the meningeal hypophyseal trunk (ICA was medial into the sella in 271 cases [46%], lateral without touching the sella in 127 cases [23%], and abutting the sella in 182 cases [31%]; P < 0.0001); (6) sphenoid segment (lateral loop of ICA within the cavernous sinus) starting at the crossing of the fourth cranial nerve on the lateral aspect of the cavernous ICA and located directly lateral to the sphenoid sinus; (7) ring segment (ICA between the 2 dural rings) starting at the crossing of the third cranial nerve on the lateral aspect of the ICA; (8) cisternal segment starting at the distal dural ring. CONCLUSIONS: The classification may be applied uniformly to all skull base surgical approaches including lateral microsurgical and ventral endoscopic approaches, obviating the need for 2 separate classification systems. The classification allows extrapolation of relevant clinical information because each named segment may indicate potential surgical risk to specific structures.

Intrapleural tissue plasminogen activator and deoxyribonuclease for pleural infection. An effective and safe alternative to surgery.

RATIONALE: Intrapleural tissue plasminogen activator (tPA)/deoxyribonuclease (DNase) therapy for pleural infection given at the time of diagnosis has been shown to significantly improve radiological outcomes. Published cases are limited to only a single randomized controlled trial and a few case reports. OBJECTIVES: Multinational observation series to evaluate the pragmatic "real-life" application of tPA/DNase treatment for pleural infection in a large cohort of unselected patients. METHODS: All patients from eight centers who received intrapleural tPA/DNase for pleural infection between January 2010 and September 2013 were included. Measured outcomes included treatment success at 30 days, volume of pleural fluid drained, improvement in radiographic pleural opacity and inflammatory markers, need for surgery, and adverse events. MEASUREMENTS AND MAIN RESULTS: Of 107 patients treated, the majority (92.3%) were successfully managed without the need for surgical intervention. No patients died as a result of pleural infection. Most patients (84%) received tPA/DNase more than 24 hours after failing to respond to initial conservative management with antibiotics and thoracostomy. tPA/DNase increased fluid drained from a median of 250 ml (interquartile range [IQR], 100-654) in the 24 hours preceding commencement of intrapleural therapy to 2,475 ml (IQR 1,800-3,585) in the 72 hours following treatment initiation (P < 0.05). We observed a corresponding clearance of pleural opacity on chest radiographs from a median of 35% (IQR 25-31) to 14% (7-28) of the hemithorax (P < 0.001), as well as significant reduction in C-reactive protein (P < 0.05). Pain necessitating escalation of analgesia occurred in 19.6% patients, and nonfatal bleeding occurred in 1.8%. CONCLUSIONS: This large series of patients who received intrapleural tPA/DNase therapy provides important evidence that the treatment is effective and safe, especially as a "rescue therapy" in patients who do not initially respond to antibiotics and thoracostomy drainage.

Bacteria-induced static batch fungal fermentation of the diterpenoid cyathin A(3), a small-molecule inducer of nerve growth factor.

Cyathin A(3), produced by the fungus Cyathus helenae, is a member of the cyathane family of diterpene natural products. While many of the cyathanes display antibacterial/antimicrobial activity or have cytotoxic activity against human cancer cell lines, their most exciting therapeutic potential is derived from their ability to induce nerve growth factor (NGF) release from glial cells, making the cyathanes attractive lead molecules for the development of neuroprotective therapeutics to prevent/treat Alzheimer's disease. To investigate if cyathin A(3) has NGF-inducing activity, we set out to obtain it using published C. helenae bench-scale fungal fermentations. However, to overcome nonproducing fermentations, we developed an alternative, bacteria-induced static batch fermentation approach to the production of cyathin A(3), as described in this report. HPLC, UV absorption spectra, and mass spectrometry identify cyathin A(3) in fungal fermentations induced by the timely addition of Escherichia coli K12 or Bacillus megabacterium. Pre-filtration of the bacterial culture abolishes cyathin A(3) induction, suggesting that bacteria-associated media changes or physical interaction between the fungus and bacteria underlie the induction mechanism. Through alteration of incubation conditions, including agitation, the timing of induction, and media composition, we optimized the fermentation to yield nearly 1 mg cyathin A(3)/ml media, a sixfold increase over previously described yields. Additionally, by comparison of fermentation profiles, we reveal that cyathin A(3) biosynthesis is regulated by carbon catabolite repression. We have used an enzyme-linked immunosorbent assay to illustrate that cyathin A(3) induces NGF release from cultured glial cells, and therefore cyathin A(3) warrants further examination in the development of neuroprotective therapeutics.

BRCA1 modulates sensitivity to 5F-203 by regulating xenobiotic stress-inducible protein levels and EROD activity.

PURPOSE: We have investigated the effects of BRCA1 over-expression and knockdown on 5F-203-induced gene expression and cytotoxicity in human breast cancer cells. 5F-203 is a chemotherapeutic prodrug that both induces a p450 enzyme, CYP1A1, and is metabolically activated by CYP1A1. METHODS: We used several molecular biological techniques to confirm our findings. BRCA1 regulates sensitivity to 5F-203 by regulating the expression of CYP1A1 mRNA and its EROD activity. XRE-Luc reporter assays, semi-quantitative RT-PCR, Western blot analysis, EROD activity measurements, gene knockdown and MTT cell survival assays were used for this study. RESULTS: Our results show that the ability of 5F-203 treatments to increase CYP1A1 mRNA level and CYP1A1 enzymatic activity (EROD activity) are affected by BRCA1 protein levels. In addition, the ability of 5F-203 treatments to induce proteins, P53 and P53 target genes such as P21, is significantly decreased in BRCA1 knockdown cells, suggesting that BRCA1-related effects could at least partially explain why BRCA1 knockdown increases resistance to 5F-203-mediated cytotoxicity. We also observed altered expression of the two major transcription factors (AhR and ARNT) that affect CYP1A1 expression when BRCA1 protein levels are altered. CONCLUSION: BRCA1 is an important protein, which affects 5F-203-mediated cytotoxicity. Our findings are potentially clinically significant; they suggest that those patients most likely to respond to this new prodrug will have tumors containing normal amounts of BRCA1.

Glucocorticoid-induced TNFR family-related protein (GITR) activation exacerbates murine asthma and collagen-induced arthritis.

Glucocorticoid-induced TNFR family-related protein (GITR) is expressed at low levels on resting T cells, B cells and macrophages but at high levels on regulatory T cells (Treg). Although GITR expression is up-regulated on CD4+ effector cells upon activation, the role of GITR in Th1 and Th2 cell development is unclear. We report here that activation of GITR signalling by anti-GITR antibody markedly enhanced the induction of both Th1 and Th2 cytokine production by naive CD4+CD25- T cells. Consistent with this observation, anti-GITR antibody significantly enhanced the expression of the key Th1 (T-bet) and Th2 (GATA3) transcription factors in vitro. Administration of anti-GITR mAb in a murine model of arthritis significantly exacerbated the severity and onset of joint inflammation with elevated production of TNF-alpha, IFN-gamma, IL-5, and collagen-specific IgG1. Administration of anti-GITR mAb also significantly exacerbated murine allergic airways inflammation with elevated production of OVA-specific IFN-gamma, IL-2, IL-4, IL-5, and IgE. Finally, we demonstrated that adoptive transfer of CD4+GITR+ T cells effectively abolished airway inflammation induced in SCID mice reconstituted with CD4+GITR- T cells. Our results therefore provide direct evidence that GITR can modulate both Th1- and Th2-mediated inflammatory diseases, and may be a potential target for therapeutic intervention.

TNF-blocking therapies: an alternative mode of action?

Despite expanding use of drugs blocking tumour necrosis factor (TNF), their precise mechanisms of action remain unclear. Early assumptions that they act by direct neutralization of the toxic inflammatory effects of TNF might be too simplistic because they explain neither the range of effects observed nor the varying properties of different TNF-blocking agents. Recent studies have demonstrated a key role for mast cell-derived TNF in the increase in lymph node size and the organizational complexity that accompanies a developing immune response. Regulation of this phenomenon might comprise a novel mode of action for TNF-directed therapy: by preventing this lymph node hyperplasia, TNF blockade could modulate immune responses, ameliorating pathology in autoimmune diseases, such as rheumatoid arthritis.

TLR2 agonist ameliorates established allergic airway inflammation by promoting Th1 response and not via regulatory T cells.

TLRs are primary sensors of both innate and adaptive immune systems, where they play a pivotal role in the response directed against structurally conserved components of pathogens. Synthetic bacterial lipopeptide Pam3CSK4 is a TLR2 agonist capable of modulating Th1 and Th2 responses. This study examines the therapeutic effect of Pam3CSK4 in established airway inflammation in a murine model of asthma. In mice previously sensitized and challenged with OVA, Pam3CSK4 given i.p. markedly reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid. Pam3CSK4 therapy was associated with a reduction in OVA-induced IL-4 and IL-5 secretion from thoracic lymph node culture, airways inflammation, bronchial hyperresponsiveness, and serum levels of IgE. Pam3CSK4 therapy was also associated with an increase in OVA-induced IFN-gamma, IL-12, and IL-10 production. However, the anti-inflammatory effect of Pam3CSK4 was independent of IL-10 or TGF-beta, but was critically dependent on IL-12, the production of which by dendritic cells was enhanced by Pam3CSK4 in vitro. Our results provide direct evidence that Pam3CSK4 could represent a novel therapeutic agent in allergic airways disease.