Dose-finding based on feasibility and toxicity in T-cell infusion trials

A new modality for treatment of cancer involves the ex vivo growth of cancer-specific T-cells for subsequent infusion into the patient. The therapeutic aim is selective destruction of cancer cells by the activated infused cells. An important problem in the early phase of developing such a treatment is to determine a maximal tolerated dose (MTD) for use in a subsequent phase II clinical trial. Dose may be quantified by the number of cells infused per unit body weight, and determination of an MTD may be based on the probability of infusional toxicity as a function of dose. As in a phase I trial of a new chemotherapeutic agent, this may be done by treating successive cohorts of patients at different dose levels, with each new level chosen adaptively based on the toxicity data of the patients previously treated. Such a dose-finding strategy is inadequate in T-cell infusion trials because the number of cells grown ex vivo for a given patient may be insufficient for infusing the patient at the current targeted dose. To address this problem, we propose an algorithm for trial conduct that determines a feasible MTD based on the probabilities of both infusibility and toxicity as functions of dose. The method is illustrated by application to a dendritic cell activated lymphocyte infusion trial in the treatment of acute leukemia. A simulation study indicates that the proposed methodology is both safe and reliable.     

In Vitro Assessment of Probiotic Potential of an Autochthonous Bacterial Isolate,Pseudomonas mosselii COFCAU_PMP5

Microbiology - Microbiota of the fish gut contains many beneficial bacteria that can be used as probiotics in aquaculture. In this study, several in vitro assays along with an in vivo safety...     

A pump-free tricellular blood–brain barrier on-a-chip model to understand barrier property and evaluate drug response

Disruption of the blood–brain barrier (BBB) leads to various neurovascular diseases. Development of therapeutics required to cross the BBB is difficult due to a lack of relevant in vitro models. We have developed a three-dimensional (3D) microfluidic BBB chip (BBBC) to study cell interactions in the brain microvasculature and to test drug candidates of neurovascular diseases. We isolated primary brain microvascular endothelial cells (ECs), pericytes, and astrocytes from neonatal rats and cocultured them in the BBBC. To mimic the 3D in vivo BBB structure, we used type I collagen hydrogel to pattern the microchannel via viscous finger patterning technique to create a matrix. ECs, astrocytes, and pericytes were cocultured in the collagen matrix. The fluid flow in the BBBC was controlled by a pump-free strategy utilizing gravity as driving force and resistance in a paper-based flow resistor. The primary cells cultured in the BBBC expressed high levels of junction proteins and formed a tight endothelial barrier layer. Addition of tumor necrosis factor alpha to recapitulate neuroinflammatory conditions compromised the BBB functionality. To mitigate the neuroinflammatory stimulus, we treated the BBB model with the glucocorticoid drug dexamethasone, and observed protection of the BBB. This BBBC represents a new simple, cost-effective, and scalable in vitro platform for validating therapeutic drugs targeting neuroinflammatory conditions.     

The chloride channel ClC-4 co-localizes with cystic fibrosis transmembrane conductance regulator and may mediate chloride flux across the apical membrane of intestinal epithelia.

Cystic fibrosis (CF) causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to mislocalization of CFTR protein from the brush border membrane of epithelial tissues and/or its dysfunction as a chloride channel. In initial reports, it was proposed that certain channels from the ClC family of chloride channels may provide compensatory or alternative pathways for epithelial chloride secretion in tissues from cystic fibrosis patients. In the present work, we provide the first evidence that ClC-4 protein is functionally expressed on the surface of the intestinal epithelium and hence, is appropriately localized to act as a therapeutic target in this CF-affected tissue. We show using confocal and electron microscopy that ClC-4 co-localizes with CFTR in the brush border membrane of the epithelium lining intestinal crypts in mouse and human tissues. In Caco-2 cells, a cell line thought to model human enterocytes, ClC-4 protein is expressed on the cell surface and also partially co-localizes with EEA1 and transferrin, marker molecules of early and recycling endosomes, respectively. Hence, like CFTR, ClC-4 may cycle between the plasma membrane and endosomal compartment. Furthermore, we show that ClC-4 functions as a chloride channel on the surface of these epithelial cells as antisense ClC-4 cDNA expression reduced the amplitude of endogenous chloride currents by 50%. These studies provide the first evidence that ClC-4 is endogenously expressed and may be functional in the brush border membrane of enterocytes and hence should be considered as a candidate channel to provide an alternative pathway for chloride secretion in the gastrointestinal tract of CF patients.     

Differential distribution of pigment-protein complexes in the Thylakoid membranes of Synechocystis 6803

Thylakoids in Synechocystis 6803, though apparently uniform in appearance in ultrastructure, were found to consist of segments which were functionally dissimilar and had distinct proteomes. These thylakoid segments can be isolated from Synechocystis 6803 by successive ultracentrifugation of cell free extracts at 40,000×g (40?k segments), 90,000×g (90?k segments) and 150,000×g (150?k segments). Electron microscopy showed differences in their appearance. 40?k segments looked feathery and fluffy, whereas the 90?k and 150?k thylakoid membrane segments appeared tiny and less fluffy. The absorption spectra showed heterogeneous distribution of pigment-protein complexes in the three types of segments. The photochemical activities of Photosystem I (PSI) and Photosystem II (PSII) showed unequal distributions in 40?k, 90?k and 150?k segments which were substantiated with low temperature fluorescence measurements. The ratio of PSII/PSI fluorescence emission at 77?K (λ(ex)?=?435?nm) was highest in 150?k segments indicating higher PSII per unit PSI in these segments. The chlorophyll fluorescence lifetimes in the membranes, determined with a time-correlated single-photon counting technique, could be resolved in three components: τ(1) (=)?<40?ps, τ(2) (=)?425-900?ps and τ(3) (=)?2.4-3.2?ns. The percentage contribution of the fastest component (τ(1)) decreased in the order 40?k?>?90?k?>?150?k segments whereas that of the other two components showed a reversed trend. These studies indicated differential distribution of pigment-protein complexes in the three membrane segments suggesting heterogeneity in the thylakoids of Synechocystis 6803.     

Luminescence properties of a nanoporous freshwater diatom

Freshwater diatom frustules show special optical properties. In this paper we observed luminescence properties of the freshwater diatom Cyclotella meneghiniana. To confirm the morphological properties we present scanning electron microscopy (SEM) images. X‐ray diffraction (XRD) studies were carried out to visualize the structural properties of the frustules, confirming that silica present in diatom frustules crystallizes in an α‐quartz structure. Study of the optical properties of the silica frustules of diatoms using ultra‐violet‐visible (UV‐vis) spectroscopy and photoluminescence spectroscopy confirmed that the diatom C. meneghiniana shows luminescence in the blue region of the electromagnetic spectrum when irradiated with UV light. This property of diatoms can be exploited to obtain many applications in day‐to‐day life. Also, using time‐resolved photoluminescence spectroscopy (TRPL) it was confirmed that this species of diatom shows bi‐exponential decay. Copyright © 2011 John Wiley & Sons, Ltd.     

Structural analysis of a peptide fragment of transmembrane transporter protein bilitranslocase

Using a combination of genomic and post-genomic approaches is rapidly altering the number of identified human influx carriers. A transmembrane protein bilitranslocase (TCDB 2.A.65) has long attracted attention because of its function as an organic anion carrier. It has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its structure. However, at present, only the primary structure of bilitranslocase is known. In our work, transmembrane subunits of bilitranslocase were predicted by a previously developed chemometrics model and the stability of these polypeptide chains were studied by molecular dynamics (MD) simulation. Furthermore, sodium dodecyl sulfate (SDS) micelles were used as a model of cell membrane and herein we present a high-resolution 3D structure of an 18 amino acid residues long peptide corresponding to the third transmembrane part of bilitranslocase obtained by use of multidimensional NMR spectroscopy. It has been experimentally confirmed that one of the transmembrane segments of bilitranslocase has alpha helical structure with hydrophilic amino acid residues oriented towards one side, thus capable of forming a channel in the membrane.     

Secretion of Soluble Vascular Endothelial Growth Factor Receptor 1 (sVEGFR1/sFlt1) Requires Arf1, Arf6, and Rab11 GTPases

The soluble form of vascular endothelial growth factor receptor 1 (sVEGFR-1/sFlt1) is generated by alternative splicing of the FLT1 gene. Secretion of sFlt1 from endothelial cells plays an important role in blood vessel sprouting and morphogenesis. However, excess sFlt1 secretion is associated with diseases such as preeclampsia and chronic kidney disease. To date, the secretory transport process involved in the secretion of sFlt1 is poorly understood. In the present study, we investigated the itinerary of sFlt1 trafficking along the secretory pathway. To understand the timecourse of sFlt1 secretion, endothelial cells stably expressing sFlt1 were metabolically radiolabeled with [(35)S]-methionine and cysteine. Our results indicate that after initial synthesis the levels of secreted [(35)S]-sFlt1 in the extracellular medium peaks at 8 hours. Treatment with brefeldin A (BFA), a drug which blocks trafficking between the endoplasmic reticulum (ER) and the Golgi complex, inhibited extracellular release of sFlt1 suggesting that ER to Golgi and intra-Golgi trafficking of sFlt1 are essential for its secretion. Furthermore, we show that ectopic expression of dominant-negative mutant forms of Arf1, Arf6, and Rab11 as well as siRNA-mediated knockdown of these GTPases block secretion of sFlt1 during normoxic and hypoxic conditions suggesting role for these small GTPases. This work is the first to report role of regulatory proteins involved in sFlt1 trafficking along the secretory pathway and may provide insights and new molecular targets for the modulation of sFlt-1 release during physiological and pathological conditions.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INCORPORATION OF N-GLYCOLYLHEXOSAMINES INTO MAMMALIAN GLYCANS BY FEEDING N-GLYCOLYLGALACTOSAMINE

The outermost positions of mammalian cell-surface glycans are predominantly occupied by the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). To date, hydroxylation of CMP-Neu5Ac resulting in the conversion into CMP-Neu5Gc is the only known enzymatic reaction in mammals to synthesize a monosaccharide carrying an N-glycolyl group. In our accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, jbc.M112.363549), we report a metabolic pathway for degradation of Neu5Gc, demonstrating that N-acetylhexosamine pathways are tolerant toward the N-glycolyl substituent of Neu5Gc breakdown products. In this study, we show that exogenously added N-glycolylgalactosamine (GalNGc) serves as a precursor for Neu5Gc de novo biosynthesis, potentially involving seven distinct mammalian enzymes. Following the GalNAc salvage pathway, UDP-GalNGc is epimerized to UDP-GlcNGc, which might compete with the endogenous UDP-GlcNAc for the sialic acid biosynthetic pathway. Using UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase-deficient cells, we confirm that conversion of GalNGc into Neu5Gc depends on this key enzyme of sialic acid biosynthesis. Furthermore, we demonstrate by mass spectrometry that the metabolic intermediates UDP-GalNGc and UDP-GlcNGc serve as substrates for assembly of most major classes of cellular glycans. We show for the first time incorporation of GalNGc and GlcNGc into chondroitin/dermatan sulfates and heparan sulfates, respectively. As demonstrated by structural analysis, N-glycolylated hexosamines were found in cellular gangliosides and incorporated into Chinese hamster ovary cell O-glycans. Remarkably, GalNAc derivatives altered the overall O-glycosylation pattern as indicated by the occurrence of novel O-glycan structures. This study demonstrates that mammalian N-acetylhexosamine pathways and glycan assembly are surprisingly tolerant toward the N-glycolyl substituent.