Antibody responses to galectin-8, TARP and TRAP1 in prostate cancer patients treated with a GM-CSF-secreting cellular immunotherapy

A critical factor in clinical development of cancer immunotherapies is the identification of tumor-associated antigens that may be related to immunotherapy potency. In this study, protein microarrays containing >8,000 human proteins were screened with serum from prostate cancer patients (N = 13) before and after treatment with a granulocyte–macrophage colony-stimulating factor (GM-CSF)-secreting whole cell immunotherapy. Thirty-three proteins were identified that displayed significantly elevated (P ≤ 0.05) signals in post-treatment samples, including three proteins that have previously been associated with prostate carcinogenesis, galectin-8, T-cell alternative reading frame protein (TARP) and TNF-receptor-associated protein 1 (TRAP1). Expanded analysis of antibody induction in metastatic, castration-resistant prostate cancer (mCRPC) patients (N = 92) from two phase 1/2 trials of prostate cancer immunotherapy, G-9803 and G-0010, indicated a significant (P = 0.03) association of TARP antibody induction and median survival time (MST). Antibody induction to TARP was also significantly correlated (P = 0.036) with an increase in prostate-specific antigen doubling time (PSADT) in patients with a biochemical (PSA) recurrence following prostatectomy or radiation therapy (N = 19) from in a previous phase 1/2 trial of prostate cancer immunotherapy, G-9802. RNA and protein encoding TARP and TRAP1 was up-regulated in prostate cancer tissue compared to matched normal controls. These preliminary findings suggest that antibody induction to TARP may represent a possible biomarker for treatment response to GM-CSF secreting cellular immunotherapy in prostate cancer patients and demonstrates the utility of using protein microarrays for the high-throughput screening of patient-derived antibody responses.     

Association between FABP2 Ala54Thr polymorphisms and type 2 diabetes mellitus risk: a HuGE Review and Meta‐Analysis

Many studies have examined the association between the FABP2 (rs1799883) Ala54Thr gene polymorphism and type 2 diabetes mellitus risk (T2DM) in various populations, but their results have been inconsistent. To assess this relationship more precisely, A HuGE review and meta‐analysis were performed. The PubMed and CNKI database was searched for case‐control studies published up to April 2014. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. Ultimately, 13 studies, comprising 2020 T2DM cases and 2910 controls were included. Overall, for the Thr carriers (Ala/Thr and Thr/Thr) versus the wild‐type homozygotes (Ala/Ala), the pooled OR was 1.18 (95% CI = 1.04–1.34, P = 0.062 for heterogeneity), for Thr/Thr versus Ala/Ala the pooled OR was 1.17 (95% CI = 1.05–1.41 P = 0.087 for heterogeneity). In the stratified analysis by ethnicity, the significantly risks were found among Asians but not Caucasians. This meta‐analysis suggests that the FABP2 (rs1799883) Ala54Thr polymorphisms are associated with increased susceptibility to T2DM risk among Asians but not Caucasians.     

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Probing the U-Shaped Conformation of Caveolin-1 in a Bilayer

Caveolin induces membrane curvature and drives the formation of caveolae that participate in many crucial cell functions such as endocytosis. The central portion of caveolin-1 contains two helices (H1 and H2) connected by a three-residue break with both N- and C-termini exposed to the cytoplasm. Although a U-shaped configuration is assumed based on its inaccessibility by extracellular matrix probes, caveolin structure in a bilayer remains elusive. This work aims to characterize the structure and dynamics of caveolin-1 (D82–S136; Cav1_82–136) in a DMPC bilayer using NMR, fluorescence emission measurements, and molecular dynamics simulations. The secondary structure of Cav1_82–136 from NMR chemical shift indexing analysis serves as a guideline for generating initial structural models. Fifty independent molecular dynamics simulations (100 ns each) are performed to identify its favorable conformation and orientation in the bilayer. A representative configuration was chosen from these multiple simulations and simulated for 1 μs to further explore its stability and dynamics. The results of these simulations mirror those from the tryptophan fluorescence measurements (i.e., Cav1_82–136 insertion depth in the bilayer), corroborate that Cav1_82–136 inserts in the membrane with U-shaped conformations, and show that the angle between H1 and H2 ranges from 35 to 69°, and the tilt angle of Cav1_82–136 is 27 ± 6°. The simulations also reveal that specific faces of H1 and H2 prefer to interact with each other and with lipid molecules, and these interactions stabilize the U-shaped conformation.     

Immunocytochemical study of anti-Müllerian hormone in sheep ovarian follicles during fetal and post-natal development

Anti-Müllerian hormone (AMH) was detected in perinatal and postnatal sheep ovaries, using avidin-biotin immunohistochemistry with a monoclonal antibody specific for ruminant AMH. Immunoreactivity was limited to granulosa cells, and was influenced both by the degree of follicular development, and by the age of the animal. In the fetus, only the most advanced follicles exhibited a faint immunoreactivity at 120 days gestation, and no reaction was observed in younger animals. Immediately before and after birth, primordial follicles were still negative, but a faint reaction was elicited in young growing follicles, increasing with follicle size. Strong immunoreactivity was visible in antral follicles, especially in the innermost granulosa cell layers, close to the oocyte and lining the antral cavity.     

Tumor necrosis factor induces acute phase proteins in rats

Inoculation of WAG rats with recombinant mouse tumor necrosis factor results in a rapid and marked increase in several acute phase proteins in the serum (haptoglobin, alpha 1 acid glycoprotein, alpha 2 macroglobulin) and in the plasma (fibrinogen). We conclude that TNF may play an important role in the inflammatory response in vivo and possibly in the pathogenesis of inflammatory disorders.     

Purified bovine AMH induces a characteristic freemartin effect in fetal rat prospective ovaries exposed to it in vitro

To determine whether anti-Müllerian hormone (AMH) is responsible for the gonadal lesions observed in bovine genetic females united by placental anastomoses to male twins (freemartins), prospective ovaries of fetal rats were exposed to purified bovine AMH in vitro. In cultures initiated at 14 days p.c. and maintained 3 to 10 days, AMH consistently induced a characteristic 'freemartin effect', namely reduction of gonadal volume, germ cell depletion and differentiation, in the gonadal blastema, of epithelial cells with large clear cytoplasm linked by interdigitations, resembling rat fetal Sertoli cells. These cells tend to become polarized and form cords, delineated by a continuous basal membrane containing laminin and fibronectin. Such structures, resembling developing seminiferous cords, were not detected in control ovarian cultures. These data strongly suggest that AMH is the testicular factor responsible for triggering the morphological abnormalities of freemartin gonads.     

Neuronal gene expression in aluminum myelopathy

1. Aluminum administration to susceptible animal species results in neurofilament accumulation in neuronal perikarya and proximal axons. Pathogenetic studies in vivo have shown that aluminum rapidly associates with neuronal chromatin. Whether the effect of aluminum on DNA components plays a role in the production of the neurofibrillary lesion remains unclear. 2. In this study we used Northern analysis and in situ hybridization to evaluate mRNA levels of specific neuronal and glial components in the rabbit spinal cord at various times following aluminum administration. 3. Our results show that (a) all neuronal mRNAs evaluated (neurofilament triplet components, neuronal-specific enolase, and amyloid precursor protein) are markedly decreased, with no decrease in glial fibrillary acidic protein; (b) the effect on neuronal gene expression occurs early and concurrently with the development of the neurofibrillary lesion and reverses rapidly after a single dose of aluminum; and (c) there is a direct correlation between the severity of the neurofibrillary lesion and the decrease in neuronal mRNA levels. 4. We interpret our results to mean that the accumulation of neurofilaments in this model is not due to a selective effect on neurofilament gene expression but may be due to an inhibition of genes coding for components involved in processing of neurofilament proteins.