Some Like It Sweet: Dendritic Cells Add Sugar to Their T(ea).

In this issue of Cell, Liu et al. present FucoID, a glycosyltransferase-mediated tagging platform, to biochemically label and capture antigen-specific T cells. With this technology, the authors isolate and characterize tumor-specific CD8+ and CD4+ T cells in murine tumor models. FucoID shows promise as a tool to enhance the understanding of anti-tumor immune responses.

Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels.

Neuronal (N)-type Ca(2+) channel-selective omega-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new omega-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced (125)I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha1(B-b)/alpha(2)delta1/beta(3)) Ca(2+) channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of omega-conotoxin action was investigated by creating molecular diversity in beta subunits: N-type channels with beta(2a) subunits almost completely recovered from CVIE or CVIF block, whereas those with beta(3) subunits exhibited weak recovery, suggesting that reversibility of the omega-conotoxin block may depend on the type of beta-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca(2+) channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that omega-conotoxin/voltage-gated Ca(2+) channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca(2+) channel-selective omega-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

Synthetic 10FN3-based mono- and bivalent inhibitors of MDM2/X function.

Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay methodology, numerous design iterations addressing insertion site(s) and spacer length were screened for optimal presentation of an MDM2/X dual peptide inhibitor in the 10FN3 scaffold. Lead inhibitors demonstrated robust, on-target cellular inhibition of MDM2/X leading to activation of the p53 tumor suppressor. Significant improvement to target engagement was observed by increasing valency within a single 10FN3 domain, which has not been demonstrated previously. We further established stable reporter cell lines with tunable expression of EGFP-fused 10FN3 domain inhibitors, and showed their intracellular location to be contingent on target engagement. Importantly, competitive inhibition of MDM2/X by small molecules and cell-penetrating peptides led to a readily observable phenotype, indicating significant potential of the developed platform as a robust tool for cell-based drug screening.

Biologic properties of three newly established human esophageal carcinoma cell lines.

Three epithelial cell lines, CE-48T/VGH, CE-69T/-VGH, and CE-81T/VGH, were established from human squamous cell carcinoma of the esophagus. The cells were polygonal with a high nucleus-to-cytoplasm ratio. Many cells were multinucleate. Electron microscopy revealed the presence of tonofilaments and desmosomes. Chromosome analysis showed that these 3 cell lines were heteroploids of human origin. When transplanted into BALB/c (nu/nu) mice, CE-69T/VGH and CE-81T/VGH produced tumors, the histology of which proved to be carcinomas. All 3 cell lines secreted carcinoembryonic antigen. However, the secretion patterns were different. These 3 cell lines may provide useful models for the study of human esophageal cancer.

Ecotin: a serine protease inhibitor with two distinct and interacting binding sites.

The interaction between ecotin and target proteases with trypsin-like specificity has been systematically dissected to understand the structural basis of ecotin's broad inhibitory specificity and the role of the secondary binding site. Site-directed and region-specific mutagenesis were preformed at ecotin's primary site P1 residue (84), the C-terminal dimer interface (133 to 142), and two surface loops of the secondary binding site (67 to 70, 108 to 113). Substitutions at the P1 position resulted in less than fivefold difference in the potency of ecotin binding to rat trypsin, suggesting that the extended binding site is important in binding. A ten amino acid C-terminal truncation variant showed threefold weaker self-association but remained a dimer. The interactions of the secondary binding site of ecotin with bovine trypsin, rat trypsin and human urokinase-type plasminogen activator (uPA) were investigated with alanine substitutions in ecotin at Trp67, Gly68, Tyr69, Asp70, Arg108, Asn110, Lys112 and Leu113, which formed contacts between the inhibitor and protease. By combining these mutations at the secondary binding site with mutations in the primary binding site the molecular recognition between ecotin and its target serine proteases was probed. The contrast in the Ki values of the various ecotin variants towards bovine trypsin, rat trypsin and human uPA established the role of ecotin's secondary binding site in recognizing these homologous serine proteases. Ecotin binds to proteases with a chymotrypsin fold through a combination of primary and secondary site surface loops and is amenable to redesign of its potency and specificity for this class of enzymes.

Phage display of proteases and macromolecular inhibitors.

We describe methods for displaying the protease trypsin and the macromolecular protease inhibitor ecotin on the surface of filamentous phage. Our strategy for selecting variant ecotins against target proteases is also described. We believe that the two proteins that have been displayed serve as ideal models for studying molecular recognition in detail. The ability to search efficiently through a large number of variant proteins for desired properties using phage display technology and the in vitro selection methods described opens a new avenue for studying protein-ligand interactions, as well as creating proteins with novel functions.

Circulating immune complexes in patients with cystic fibrosis.

Circulating immune complexes (CICs) were measured in the sera of clinically stable and acutely infected patients with cystic fibrosis (CF). Twenty CF patients were studied when clinically stable; and additional 18 patients were studied during an acute exacerbation of pulmonary infection as evidenced by fever, tachypnea, increased white blood cell count, increased sputum production, and acute chest x-ray film changes. Three methods for determining CICs were employed: polyethyelene glycol precipitation, a C1q phase assay, and the Raji cell radioimmune assay. Ten of 20 clinically stable CF patients had one or two positive assays for CICs; two of 20 had two positive assays. In contrast, 16 of 18 acutely infected CF patients had a positive CIC test, and 12 of these were positive with two or three of the assays employed. Serum C3 and C4 concentrations and total hemolytic complement activity did not correlate with the presence of CICs in either patient group. These findings suggest that immune complex formation may mediate some of the tissue damage characteristic of CF, although this usually does not involve intravascular complement activation.

Variable weight training in cystic fibrosis.

A six-month pilot study of variable weight training (VWT) was undertaken to assess its impact on body weight, pulmonary function, muscle size and strength, and social function in 12 adolescent and adult patients with moderately severe cystic fibrosis. Exercise for patients with cystic fibrosis (CF) has often been recommended as an adjunct to physical therapy, although aerobic exercise has not resulted in weight gain in CF. Compared to a three-month control period, six months of VWT resulted in significant increase in weight (2.88 kg, p less than .02), muscle size (1.6 to 1.8 cm upper arm, p less than .01), strength (increase from 16 to 32 muscle groups at normal strength, p less than .005), and decrease in residual volume (1.77 L, p less than .03) and RV/TLC (12.4 percent, p less than .02). There was no significant improvement in other measures of pulmonary or social function. VWT appears to be a form of exercise in which even moderately ill CF patients can engage safely, leading to desired weight gain and increased strength. These results warrant further study of the effects of VWT on pulmonary function and CF morbidity.

Supplemental oxygen and exercise performance in patients with cystic fibrosis with severe pulmonary disease.

Patients with cystic fibrosis (CF) and advanced pulmonary disease have pulmonary limitation of exercise, often associated with arterial oxygen desaturation. Improving oxygenation during exercise by providing supplemental oxygen may improve exercise performance in these patients. To test this, we performed graded exercise stress tests in 22 CF patients with severe pulmonary disease (mean PaO2, 64 +/- 2 mm Hg [+/- SE]; PaCO2 46 +/- 2 mm Hg; RV/TLC, 57 +/- 4 percent; FEV1, 38 +/- 4 percent of predicted; FEF25-75%, 13 +/- 2 percent of predicted; median age, 26 years) and compared them to 21 controls (RV/TLC, 27 +/- 4 percent; FEV1, 112 +/- 2 percent of predicted; FEF25-75%, 80 +/- 4 percent of predicted; median age, 29 years). Each subject performed graded exercise stress tests while breathing FIO2 of 0.21 and FIO2 of 0.30. Subjects were blinded to the composition of the inspired gas, and the order of testing was randomized. We found that CF subjects exercised longer, had a higher maximal VO2, higher O2 pulse, and less arterial oxygen desaturation when receiving supplemental O2. Control subjects exercised longer when breathing supplemental O2 but had no significant change in maximal VO2, O2 pulse, or SaO2. Both CF and control subjects had increased end-tidal PCO2 when exercising while breathing supplemental O2. We conclude that CF patients with advanced pulmonary disease have increased exercise tolerance and aerobic capacity when exercising while breathing supplemental O2.

Technique, indications, and clinical use of 24 hour esophageal pH monitoring.

The technique of 24 hour esophageal pH monitoring (24 hour pH test) is described. Experience with the 24 hour pH test in 393 patients with suspected esophageal disease has shown the clinical usefulness of the test in objectively determining the presence of gastroesophageal reflux. The test was effective in evaluating atypical symptoms of gastroesophageal reflux such as respiratory symptoms and chest pain and, in children, failure to thrive and recurrent pneumonia. The 24 hour pH test was particularly useful in evaluating patients who were referred with other abdominal or thoracic disease and had, in addition, symptoms suggestive of gastroesophageal reflux on history. The test helped to unsnarl the cause of recurrent symptoms after an esophageal myotomy for achalasia or an antireflux procedure. Of 179 patients with typical symptoms of gastroesophageal reflux, 27% had normal 24 hour test results and were subsequently diagnosed as having another cause for their symptoms. Of 146 patients who had normal findings on esophagoscopy, 54% were shown to have abnormal gastroesophageal reflux on 24 hour pH monitoring, indicating lack of sensitivity of endoscopy to detect reflux. In addition, the 24 hour pH test identified patterns of abnormal reflux and indicated those patients most at risk for development of stricture. The test is well tolerated by the patients, simple to use, and dependable when performed and read as described. The clinical use of the 24 hour pH test brings objectivity to the evaluation of exophageal disease that has hitherto not been available.

Colonic polyps and coloduodenal fistula: unusual complications in patient with cystic fibrosis.

Abdominal pain, vomiting, and obstipation often occur in children and young adults with cystic fibrosis (CF). The common causes include meconium ileus equivalent, intussusception, and adhesions from previous surgery. One of our patients with CF who had meconium peritonitis as a neonate presented with duodenal obstruction during childhood. This was caused by colonic polyps arising in the hepatic flexure and eroding through the colonic and duodenal walls into the duodenum. She was treated with total parenteral nutrition, right colectomy, gastric diversion, and a controlled duodenal fistula that healed uneventfully. She has remained well 1 year after discharge from the hospital. To our knowledge, a similar case has not been reported previously.

Intra-abdominal pressure and manometric data of the distal esophageal sphincter. Their relationship to gastroesophageal reflux.

Nineteen patients with abnormal gastroesophageal reflux (13 with and six without hiatal hernia) had esophageal manometry and simultaneous 24-hour monitoring of esophageal pH and intra-abdominal pressure (IAP). Only 8% of all IAP challenges induced a reflux episode. This incidence increased to 13% in patients with a distal esophageal sphincter (DES) pressure of less than 5 mm Hg and an abdominal esophageal length of less than 1 cm, whereas it was only 6% in patients with a greater DES pressure and a longer abdominal esophagus. At the same time, there was an average of 2.7 reflux episodes per hour, of which 38.7% were caused by a challenge of IAP indicating that other mechanisms, besides changes in abdominal pressure, can cause reflux. There was no difference in DES pressure, length of abdominal esophagus, and the effect of IAP challenges in patients with and those without a hiatal hernia.

Efficacy of ivermectin in hookworms as examined in Ancylostoma caninum infections.

Single oral doses of ivermectin were given to dogs with moderate or heavy infections of Ancylostoma caninum (egg counts ranging from 7,100 to 41,700 eggs/g feces) at 100, 50, 30, or 10 micrograms/kg body weight. Each of these dosages was effective in clearing the infection completely, so that numerous worms were passed in the feces on days 1-3, but no worm was recovered from the intestinal tract at necropsy on day 4 after treatment. In contrast, an average of 178 worms per dog was recovered at necropsy from the vehicle-treated control and the untreated animals. Albendazole, a known anti-hookworm agent, even in a dose of 400 mg, eliminated only 21-65% of the worms harbored by the infected animals. No untoward reaction to ivermectin or significant pathological change was noted in the experimental animals. In vitro experiments demonstrated that ivermectin: (1) was highly detrimental to actively motile adult worms in concentrations greater than 5.60 micrograms/ml; (2) was detrimental to eggs inside the uterine tissue of female worms in dosages at or greater than 10 micrograms/kg body weight; and (3) killed infected larvae in concentrations as low as 0.0025 micrograms/ml.

Heat shock treatment decreases the mortality of sepsis in rats.

Sepsis is an increasingly common and lethal diagnosis in hospitalized patients. In spite of the advances in antibiotics and medical equipment, the mortality rate has not been improved in the last decade. Recently, heat shock proteins (Hsps) have been well documented to play a self-protective role in almost all living cells under various pathological and physiological stresses through a mechanism known as thermotolerance or cross tolerance. The present study was designed to investigate the expression of Hsp72 and the protective role of pre-induction of Hsps in the mortality during different phases of sepsis. Adult male Sprague-Dawley rats were employed in the study. Sepsis was induced by cecal ligation and puncture (CLP). Heat shock treatment was performed 16 hrs before sepsis induction by heating the rats whole-bodily with an electric heating pad under general anesthesia. The mortality rates with time in both control and preheated groups were monitored. Hsp72 was detected by SDS-PAGE, Western blotting and immunostaining in the brain, heart, liver, kidney, lung, adrenal gland, muscle and lymphocytes. The results show that both early (9 hrs post-CLP) and late (18 hrs post-CLP) sepsis failed to increase the synthesis of Hsp72. Previous induction of Hsps by heat shock treatment significantly decreased the mortality rate of late sepsis. Applying a sufficient inducer to lymphocytes of late sepsis reversed the synthesis of Hsp72 from inactive state into an over-expressive situation in vitro. These results suggest that Hsps are involved in the pathogenesis of sepsis and the involvement of Hsps during the progression of sepsis could add to a first line of host defense against invasive pathogens. Searching for an acceptable agent or less invasive method that leads to increased Hsps expression may provide a means for better treatment of severe infection.

[Successful axenic cultivation of a local human strain of Giardia lamblia in suckling gerbil].

G. lamblia cysts isolated from the fresh feces of a Giardia-infected boy in Beijing rural area were inoculated into suckling gerbils (Meriones unguiculatus). Trophozoites of G. lamblia obtained from the intestines of infected gerbils were cultivated in modified TYI-S-33 medium enriched with dehydrated bovine bile. The parasites grew luxuriantly and formed an intensive monolayer on the surface of the culture tube on day 14 after initial cultivation. The culture has been maintained for more than 12 months and more than 120 subcultures have been made. The growth curve of the organism showed that the peak growth of the trophozoites was attained at the 120th hour after seeding. The generation time was 15 +/- 2.0 hours. Periodic examinations of Giardia cultures for bacteria contamination, with Petri dishes of blood agar and beef broth, proved negative. After being cryopreserved in liquid nitrogen for 1 week or longer, the average viable rate of the organism was 65.7% and the resuscitated parasites grew luxuriantly in subcultures.

Hydrophobic residues at position 10 of α-conotoxin PnIA influence subtype selectivity between α7 and α3ß2 neuronal nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are a diverse class of ligand-gated ion channels involved in neurological conditions such as neuropathic pain and Alzheimer's disease. α-Conotoxin [A10L]PnIA is a potent and selective antagonist of the mammalian α7 nAChR with a key binding interaction at position 10. We now describe a molecular analysis of the receptor-ligand interactions that determine the role of position 10 in determining potency and selectivity for the α7 and α3ß2 nAChR subtypes. Using electrophysiological and radioligand binding methods on a suite of [A10L]PnIA analogs we observed that hydrophobic residues in position 10 maintained potency at both subtypes whereas charged or polar residues abolished α7 binding. Molecular docking revealed dominant hydrophobic interactions with several α7 and α3ß2 receptor residues via a hydrophobic funnel. Incorporation of norleucine (Nle) caused the largest (8-fold) increase in affinity for the α7 subtype (Ki=44nM) though selectivity reverted to α3ß2 (IC50=0.7nM). It appears that the placement of a single methyl group determines selectivity between α7 and α3ß2 nAChRs via different molecular determinants.

Site-specific effect of thymine dimer formation on dAn.dTn tract bending and its biological implications.

dAn.dTn sequences, otherwise known as A tracts, are hotspots for cis-syn thymine dimer formation and deletion mutations induced by UV light. Such A tracts are also known to bend DNA, suggesting that some biological effects of UV light might be related to the distinctive structure and properties of cis-syn dimer-containing A tracts. To investigate the effect of thymine dimer formation on A-tract bending multimers of all possible dimer monoadducts of a dA6.dT6-containing decamer known to bend DNA were prepared along with multimers of a dimer-containing 21-mer of heterogeneous sequence. The characteristic anomalous electrophoretic behavior of the phased A-tract multimers was essentially abolished by dimer formation at the center of the A tract and was only slightly reduced by dimer formation at the ends. These effects are attributed to disruption of the A-tract structure at the site of the dimer, resulting in intact A tracts of reduced length and, hence, reduced bending. This model was suggested by the ability to formulate the estimated bend angles of the dimer-containing A tracts as approximately equal to the sum of the bend angles induced by the dimer and the remaining intact portion of the A tract. Contrary to a previous experimental study that concluded that the thymine dimer bends DNA by approximately 30 degrees, the dimer was determined to bend DNA by only approximately 7 degrees. Reduction of the bending of a DNA sequence by dimer formation may have a number of unpredicted and important biological consequences.

The trans-syn-I thymine dimer bends DNA by approximately 22 degrees and unwinds DNA by approximately 15 degrees.

Irradiation of DNA with ultraviolet light leads to the formation of two classes of cyclobutane dimers at adjacent thymines sites, of which the cis-syn is the major class and the trans-syn is the minor class. While the structure and properties of DNA containing cis-syn thymine dimers have been extensively studied, virtually nothing is known about DNA containing trans-syn thymine dimers. To investigate the bending and unwinding of DNA induced by the trans-syn-I thymine dimer, the electrophoretic properties of oligomers of trans-syn-I dimer-containing DNA duplexes were studied. Oligonucleotides 10, 11, and 12 bp in length containing a centrally located trans-syn-I thymine dimer were synthesized, polymerized, and analyzed by polyacrylamide gel electrophoresis. In contrast to the small bending angle (approximately 7 degrees) induced by the cis-syn thymine dimer, we found that trans-syn-I thymine dimer bends DNA significantly (approximately 22 degrees). Both dimers, however, are found to unwind DNA by the same amount (approximately 15 degrees). On the basis of previous NMR studies, it appears that the bend of the trans-syn-I dimer is localized at the 5'-side of the dimer. Gel electrophoretic analysis of multimers of two 11-mers containing a cis-syn thymine dimer at the 5'-end and at the center of a dT6.dA6 tract confirmed our previous estimates for the bending angles of thymine dimer-containing T6 tracts. The substrates reported may be useful in determining how general repair enzymes recognize DNA damage.