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.     

Influence of Organic-Phosphates on 3-Phosphoglycerate Dependent O2 Evolution in C3 and C4 Mesophyll Chloroplasts

In C₄ plants phosphoenolpyruvate (PEP) of the C₄ cycle may betransported on a chloroplast transporter which also transports3-phosphoglycerate (3-PGA) and triosephosphates. In C₃ plantsPEP is not considered to be effectively transported on the chloroplastphosphate translocator. The influences of certain organic phosphates,having a similar structure to either PEP or triose-phosphates,on 3-PGA dependent O₂ evolution by C₄ (Digitaria sanquinalisL. Scop.) and C₃ (Hordeum vulgare L.) mesophyll chloroplastswere investigated. In the C₄ mesophyll chloroplasts phosphoglycolatewas a competitive inhibitor (K_i = 2.1 mM) of 3-PGA dependentO₂ evolution, and was as effective as previously reported forPEP. 2-Phosphoglycerate was also a competitive inhibitor (K_t= 8.6 mM) of O₂ evolution in the C₄ mesophyll chloroplasts with3-PGA as substrate, while phospholactate was a weak inhibitorand glyphosate had no effect. Neither PEP, phosphoglycolatenor 2-phosphoglycerate were effective inhibitors of 3- PGA dependentO₂ evolution in the C₃ chloroplasts. Phosphohydroxypyruvatewas a competitive inhibitor of 3-PGA dependent O₂2 evolutionin both chloroplast types. The selectivity in inhibition ofO₂ evolution with 3-PGA as substrate suggests that the C₄ mesophyllchloroplasts can recognize certain organic phosphates with thephosphate in the C-2 or C-3 position but that the C₄ mesophyllchloroplasts can only effectively recognize certain organicphosphates with the phosphate in the C-3 position. The resultsalso support the view that 3-PGA and PEP are transported onthe same phosphate translocator in C₄ mesophyll chloroplasts. ¹ Current address: Department of Horticulture, 2001 Fyffe Court,The Ohio State University, Columbus, Ohio 43210-1096. (Received March 24, 1987; Accepted April 16, 1987)     

Experience with Coeliac Axis Compression Syndrome

Seven patients with “coeliac axis compression syndrome” are reported. Five were treated surgically, but only two did well. A survey of 200 healthy adults showed epigastric bruits in 6·5%; only one of these had dyspepsia, though dyspepsia was present in 12·5% overall.Caution is urged in attributing a causal relationship between coeliac axis compression and pain and in proceeding to arteriography when compression is suspected on clinical grounds.     

Recovery of a bacterial sub-population from sewage using immunofluorescent flow cytometry and cell sorting

Abstract The effectiveness of immunofluorescence flow cytometry and cell sorting to detect, quantify and separate indigenous bacterial populations present in low concentrations in sewage outflow was investigated. Preparatory experiments for targeted recovery revealed indigenous, immunoglobulin-G-binding particles present at low levels in sewage outflow samples taken from Coniston Water. Fluorescence-activated cell sorting of this population was employed to enrich for these particles, which were confirmed as bacterial cells. This cell population comprised approximately 23% of the total plate count on MacConkey agar before cell sorting, rising to approximately 95% after sorting. These results corresponded to cell densities of less than 5% of the total plate count on R2A agar. Taxonomic tests suggested the bacterium to be Ochrobactrum anthropi .     

Polymorphism in Two Parasitoids Detected Using Random Amplified Polymorphic DNA Polymerase Chain Reaction

We used Chelex 100 chelating resin to prepare DNA for the polymerase chain reaction (PCR) from two species of Hymenopteran parasitoids, Trioxys pallidus and Diglyphus begini. Chelex 100 produces consistent DNA yields for both species, as measured with Hoescht dye fluorometry. Approximately twice as much DNA was obtained from individual D. begini wasps than from T. pallidus wasps, but there were no differences in yield between sexes. We used this DNA to perform random amplified polymorphic DNA (RAPD) analysis, a PCR technique that amplifies various regions of the genome using arbitrarily chosen 10-base primers. Of the 120 primers tested using T. pallidus, 92 produced a total of 342 scorable bands, 118 of which exhibited presence/absence polymorphism. Of the 25 primers tested using D. begini, 18 produced a total of 53 scorable bands, 30 of which exhibited presence/absence polymorphism. The level of genetic variation detected using this technique was greater than any found in Hymenoptera using allozymes. Scorable bands segregated as dominant Mendelian traits. Potential uses of RAPD-PCR in genetic analyses on parasitic Hymenoptera are discussed.     

A novel method for the evaluation of proximal tubule epithelial cellular necrosis in the intact rat kidney using ethidium homodimer

Background  

Ethidium homodimer is a cell-membrane impermeant nuclear fluorochrome that has been widely used to identify necrotic cells in culture. Here, we describe a novel technique for evaluating necrosis of epithelial cells in the proximal tubule that involves perfusing ethidium homodimer through the intact rat kidney. As a positive control for inducing necrosis, rats were treated with 3.5, 1.75, 0.87 and 0.43 mg/kg mercuric chloride (Hg²⁺, intraperitoneal), treatments which have previously been shown to rapidly cause dose-dependent necrosis of the proximal tubule. Twenty-four h after the administration of Hg²⁺, ethidium homodimer (5 μM) was perfused through the intact left kidney while the animal was anesthetized. The kidney was then removed, placed in embedding medium, frozen and cryosectioned at a thickness of 5 μm. Sections were permeabilized with -20°C methanol and then stained with 4',6-diamidino-2-phenylindole (DAPI) to label total nuclei. Total cell number was determined from the DAPI staining in random microscopic fields and the number of necrotic cells in the same field was determined by ethidium homodimer labeling.     

THE INTRACELLULAR MEMBRANES OF BLASTOMYCES DERMATITIDIS

Edwards , George A., and Mercedes R. Edwards . (Div. Labs, and Research, N.Y.S. Dept. of Health, Albany.) The intracellular membranes of Blastomyces dermatitidis. Amer. Jour. Bot. 47 (8): 622–632. Illus. 1960.—The yeast cells of Blastomyces dermatitidis have been studied in thin sections with the electron microscope. The cell is multinucleate, and the nuclei are frequently interconnected by their outer limiting membranes. The cell is bordered by a cell wall and the plasma membrane, which may be seen in direct continuity with the nuclear envelope. The cytoplasm contains numerous mitochondria, many profiles of the endoplasmic reticulum, and few multivesicular bodies. The membranes of all the constant cellular components are interconnected. Mitochondria appear to be formed from any of several membrane systems. The micromorphology of the cell suggests efficiency of communication and cytoplasmic mobility.     

Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices

Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72–120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.