Citations

Abstract 4-1BB, a member of the TNFR superfamily, is a costimulatory receptor primarily expressed on activated T cells. It has been shown that the administration of agonistic anti-4-1BB Abs enhances tumor immunity and allogenic immune responses. Paradoxically, we found that the administration of an agonistic anti-4-1BB mAb (2A) dramatically reduced the incidence and severity of experimental autoimmune encephalomyelitis (EAE). Adoptive transfer of T cells from such treated mice failed to induce EAE, whereas anti-4-1BB treatment following adoptive transfer of encephalitogenic T cells did not prevent EAE pathogenesis. These results suggest that anti-4-1BB treatment during the induction phase inhibits autoreactive T cell immune responses rather than preventing T cell trafficking into the CNS. This was substantiated by the observations that draining lymph node cells from anti-4-1BB-treated mice failed to respond to Ag stimulation in vitro. In addition, we found that such treatment initially promotes the activation and proliferation of Ag-specific CD4+ T cells but subsequently increases their probability of undergoing activation-induced cell death, thereby inhibiting effector T cell responses. More importantly, 2A treatment also inhibits the relapse of EAE in a clinically relevant murine model of multiple sclerosis. This study indicates that the agonistic Ab against 4-1BB can potentially be used as a novel immunotherapeutic agent for treating autoimmune diseases.

Abstract Azadirachta indica, commonly known as neem, has gained worldwide prominence because of its medical properties, namely antitumor, antiviral, anti-inflammatory, antihyperglycemic, antifungal, and antibacterial activities. Despite these promising results, gaps remain in our understanding of the molecular mechanism of action of neem compounds and their potential for use in clinical trials. We investigated supercritical extract of neem leaves (SENL) for the following: molecular targets in vitro, in vivo efficacy to inhibit tumor growth, and bioactive compounds that exert antitumor activity. Treatment of LNCaP-luc2 prostate cancer cells with SENL suppressed dihydrotestosterone-induced androgen receptor and prostate-specific antigen levels. SENL inhibited integrin β1, calreticulin, and focal adhesion kinase activation in LNCaP-luc2 and PC3 prostate cancer cells. Oral administration of SENL significantly reduced LNCaP-luc2 xenograft tumor growth in mice with the formation of hyalinized fibrous tumor tissue, reduction in the prostate-specific antigen, and increase in AKR1C2 levels. To identify the active anticancer compounds, we fractionated SENL by high-pressure liquid chromatography and evaluated 16 peaks for cytotoxic activity. Four of the 16 peaks exhibited significant cytotoxic activity against prostate cancer cells. Mass spectrometry of the isolated peaks suggested the compounds with cytotoxic activity were nimbandiol, nimbolide, 2′,3′-dihydronimbolide, and 28-deoxonimbolide. Analysis of tumor tissue and plasma samples from mice treated with SENL indicated 28-deoxonimbolide and nimbolide as the bioactive compounds. Overall, our data revealed the bioactive compounds in SENL and suggested that the anticancer activity could be mediated through alteration in androgen receptor and calreticulin levels in prostate cancer. Mol Cancer Ther; 13(5); 1067–77. ©2014 AACR.

Improved approximate frequency domain expressions are derived for cylindrical shear wave propagation in viscoelastic media. These expressions extend prior results that describe cylindrical wave propagation in lossless media. Previously, an analytical expression for a cylindrical wave was obtained in terms of a Hankel function and a large argument approximation was applied to the result. A leading frequency-dependent term was then treated as a constant with respect to frequency. In the improved expression, the frequency-dependence of the leading term is retained. For lossless media, the leading term is a fractional integrator, and for viscoelastic media, the leading term is either a fractional integrator or an integer-order integrator, depending on the frequency range. The lossless and the viscoelastic models are evaluated in the frequency domain for simplified source geometries and compared to numerical results. The comparison shows that the agreement between the analytical and the numerical models is excellent. Implications for time-domain calculations in viscous media will also be discussed. [This work was supported in part by NIH Grant Nos. R01 EB012079 and R01 DK092255.]

We validate a technique for model-free measurement of shear wave velocity and attenuation. A mechanical shaker was used to excite harmonic plane and cylindrical waves in phantoms and excised tissue. Radiation force was used to excite impulsive cylindrical waves. 2D FFT of the displacement yielded the k-space whose coordinates are frequency and the wave number. The shear wave velocity at each frequency was obtained by finding the maximum at the given frequency in k-space and dividing the frequency coordinate by the wave number coordinate. The attenuation (α) at a given frequency was calculated using α = FWHM × π/√3, where FWHM is the full width at half maximum of the k-space peak along the given frequency. This method was applied to measure shear wave velocity and attenuation of transplanted kidneys and livers, and in the thyroid tumor, and compare it to the healthy tissues. The velocities and attenuations at each frequency for various excitation methods agree within one standard deviation. The k-space estimates of velocity and attenuation agreed with those obtained using the phase gradient (velocity) and amplitude decay (attenuation). The transplanted organs and the thyroid tumor had higher velocity and lower attenuation than healthy tissues.

Background and Purpose— The prognostic significance of subarachnoid extension of intracerebral hemorrhage was determined in the INTEnsive blood pressure Reduction in Acute Cerebral hemorrhage Trial (INTERACT2) study. Methods— INTERACT2 was an open randomized controlled trial of early intensive compared with guideline-recommended blood pressure lowering in patients with elevated systolic blood pressure within 6 hours of intracerebral hemorrhage. Independent predictors of death or major disability (scores 3–6 on the modified Rankin Scale) at 90 days were analyzed in logistic regression models. Results— Of 2582 participants, 192 (7%) had subarachnoid extension, which was associated with larger hematoma volumes ( P <0.0001) and higher National Institute of Health Stroke Scale score ( P <0.0001). Subarachnoid extension predicted death or major disability at 90 days (71% versus 53%; unadjusted odds ratio, 2.25; 95% confidence interval, 1.63–3.10; P <0.0001). The association remained significant after adjusting for age, region, lipid-lowering therapy, systolic blood pressure, glucose, location of hematoma, intraventricular extension, and randomized treatment (odds ratio, 2.17; 95% confidence interval, 1.50–3.14; P <0.0001), but not after further adjustment for baseline hematoma volume ( P =0.62). Conclusions— Subarachnoid extension of intracerebral hemorrhage is associated with poor prognosis, which is determined by a larger volume of the underlying intraparenchymal hematoma. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00716079.

AbstractBackgroundTransforming growth factor (TGF)‐β1 produced in airway epithelia has been suggested as a contributor to the airway remodeling observed in asthma patients. The protein tyrosine phosphatase SHP2 is a demonstrable modulator of TGF‐β1 production and thus a potential regulator of airway remodeling.ObjectivesTo define the signal event by which SHP2 regulates asthmatic responses in airway epithelial cells by using a mouse model of experimental OVA‐induced airway remodeling.MethodsThe airways of Shp2flox/flox mice were infected with recombinant adenovirus vectors expressing a Cre recombinase–green fluorescence protein (GFP) fusion protein as part of allergen provocation studies using mice sensitized with ovalbumin (OVA) and repeatedly challenged with OVA. Several endpoint pathologies were assessed, including airway hyper‐responsiveness (AHR), lung inflammatory score, peribronchial collagen deposition, and α‐smooth muscle actin (SMA) hyperplasia. In vitro studies using airway epithelial cells (BEAS‐2B) were used to investigate the role of SHP2 in the regulation of pulmonary remodeling events, including the expression of collagen, α‐SMA, and TGF‐β1.ResultsChronic OVA challenges in wild‐type mice resulted in airway remodeling and lung dysfunction (e.g., increased inflammatory scores, collagen deposition (fibrosis), smooth muscle hyperplasia, and a significant increase in AHR). These endpoint pathology metrics were each significantly attenuated by conditional shp2 gene knockdown in airway epithelia. In vitro studies using BEAS‐2B cells also demonstrated that the level of TGF‐β1 production by these cells correlated with the extent of shp2 gene expression.ConclusionsSHP2 activities in airway epithelial cells appear to modulate TGF‐β1 production and, in turn, regulate allergic airway remodeling following allergen provocation.Clinical ImplicationsOur findings identify SHP2 as a previously underappreciated contributor to the airway remodeling and lung dysfunction associated with allergen challenge. As such, SHP2 represents a potentially novel therapeutic target for the treatment of asthmatics.Capsule summaryAirway epithelial protein tyrosine phosphatase SHP2 appears to modulate TGF‐β1 activities as part of one or more cellular pathways leading to regulating the airway remodeling and lung dysfunction occurring in mouse models of allergic respiratory inflammation.

PurposeAutoantibodies are implicated in the pathogenesis of cardiovascular diseases and cardiac arrhythmias. In this pilot study, we tested the hypothesis that autoantibodies are present in patients with postural orthostatic tachycardia syndrome (POTS).Experimental designSeven control subjects (6 F:1 M, average age 36.1 years) and ten patients with the diagnosis of POTS (7 F: 3 M, average age 35.1 years) provided informed consent and 30 mL of venous blood. Human heart membrane proteins were resolved by 2DE and immunoblotted against purified IgGs from controls and patients.ResultsEighteen protein spots immunoreactive specifically against patient IgGs were detected and they were excised from gels, trypsin‐digested, and analyzed by nanoLC‐electrospray MS/MS. Forty unique proteins were identified and these include proteins that are associated with cardiac hypertrophy (mimecan, myozenin), cardiac remodeling (periostin), cardiomyopathy (desmin, desmoplakin), cell survival (laminin), structural integrity (filamin), chaperone proteins (crystalline, HSP70), mitochondrial enzymes, and channel proteins. Ingenuity Pathway Analysis showed multiple pathways were involved including those that regulate energy metabolism, redox, fibrosis, cardiac hypertrophy, and degeneration.Conclusions and clinical relevanceAutoantibodies are present in patients with POTS. These autoantibodies cross‐react with a wide range of cardiac proteins and may induce alterations in cardiac function. Autoimmune pathogenetic mechanisms should be further explored in these patients.

Activity of the vascular large conductance Ca2+-activated K+ (BK) channel is tightly regulated by its accessory β1 subunit (BK-β1). Downregulation of BK-β1 expression in diabetic vessels is associated with upregulation of the forkhead box O subfamily transcription factor-3a (FOXO-3a)–dependent F-box–only protein (FBXO) expression. However, the upstream signaling regulating this process is unclear. Overproduction of reactive oxygen species (ROS) is a common finding in diabetic vasculopathy. We hypothesized that ROS signaling cascade facilitates the FOXO-3a/FBXO-mediated BK-β1 degradation and leads to diabetic BK channel dysfunction. Using cellular biology, patch clamp, and videomicroscopy techniques, we found that reduced BK-β1 expression in streptozotocin (STZ)-induced diabetic mouse arteries and in human coronary smooth muscle cells (SMCs) cultured with high glucose was attributable to an increase in protein kinase C (PKC)-β and NADPH oxidase expressions and accompanied by attenuation of Akt phosphorylation and augmentation of atrogin-1 expression. Treatment with ruboxistaurin (a PKCβ inhibitor) or with GW501516 (a peroxisome proliferator–activated receptor δ activator) reduced atrogin-1 expression and restored BK channel-mediated coronary vasodilation in diabetic mice. Our results suggested that oxidative stress inhibited Akt signaling and facilitated the FOXO-3a/FBXO-dependent BK-β1 degradation in diabetic vessels. Suppression of the FOXO-3a/FBXO pathway prevented vascular BK-β1 degradation and protected coronary function in diabetes.

In the field of tissue engineering, monitoring the elasticity of developing tissue cultures is important for evaluating their growth and maturation. In this work, we used wave propagation methods to measure the elasticity of thin tissue-mimicking layers to test the feasibility of measuring the elasticity of developing tissue cultures. We developed finite element and analytical models for investigating this problem and found good agreement between the results from both models. We used ultrasound radiation force to induce propagating waves, and the waves are measured using high frame rate ultrasound imaging at 10 kHz. The wave modes were identified and compared with an analytic model for Rayleigh wave propagation in a thin elastic layer bound to a solid substrate. The same gelatin mixture was used to make phantoms 1, 4, and 50 mm thick, where the phantom with 50 mm thickness was used as control and evaluated with shear wave imaging methods. The analytic model was used to fit data from experiments in the large block and thin layers of 1 and 4 mm thick, and the measured shear moduli were 22.7, 21.8, and 22.5 kPa, respectively. These results provide a validation for measurement of elasticity in thin elastic layers.