Clinical value of ctDNA in upper-GI cancers: A systematic review and meta-analysis.

BACKGROUND: The recent expanding technical possibilities to detect tumor derived mutations in blood, so-called circulating tumor DNA (ctDNA), has rapidly increased the interest in liquid biopsies. This review and meta-analysis explores the clinical value of ctDNA in malignancies of the upper gastro-intestinal tract. METHODS: PubMed, Cochrane and Embase databases were searched to identify studies reporting the diagnostic, prognostic or predictive value of ctDNA in patients with esophageal, gastric and pancreatic cancer, until January 2017. The diagnostic accuracy and, using random-effect pair-wise meta-analyses, the prognostic value of ctDNA was assessed. RESULTS: A total of 34 studies met the inclusion criteria. For esophageal and gastric cancer, amplification of oncogenes in blood, such as HER2 and MYC, can be relevant for diagnostic purposes, and to predict treatment response in certain patient subpopulations. Given the limited number of studies assessing the role of ctDNA in esophageal and gastric cancer, the meta-analysis estimated the diagnostic accuracy and predictive value of ctDNA in pancreatic cancer only (n=10). The pooled sensitivity and specificity of ctDNA as a diagnostic tool in pancreatic cancer were 28% and 95%, respectively. Patients with pancreatic cancer and detectable ctDNA demonstrated a worse overall survival compared to patients with undetectable ctDNA (HR 1.92, 95% confidence interval (CI) 1.15-3.22, p=0.01). CONCLUSION: The presence of ctDNA is significantly associated with a poor prognosis in patients with pancreatic cancer. The use of ctDNA in clinical practice is promising, although standardization of sequencing techniques and further development of high-sensitive detection methods is needed.

Systematic validation of specific phenotypic markers for in vitro polarized human macrophages.

BACKGROUND: Polarization of macrophages by specific micro-environmental conditions impacts upon their function following subsequent activation. This study aimed to systematically validate robust phenotypic markers for in vitro polarized human macrophages in order to facilitate the study of macrophage subsets in vivo. METHODS: Human peripheral blood monocytes were polarized in vitro with IFN-γ, IL-4, or IL-10. Similar experiments were performed with TNF, IL-13, dexamethasone, M-CSF and GM-CSF as polarizing stimuli. Phenotypic markers were assessed by flow cytometry and qPCR. RESULTS: IFN-γ polarized macrophages (MΦ(IFN-γ)) specifically enhanced membrane expression of CD80 and CD64, IL-4 polarized macrophages (MΦ(IL-4)) mainly upregulated CD200R and CD206, and downregulated CD14 levels, and IL-10 polarized macrophages (MΦ(IL-10)) selectively induced CD163, CD16, and CD32. The expression profiles of the most specific markers were confirmed by qPCR, dose-response experiments, and the use of alternative polarizing factors for each macrophage subset (TNF, IL-13, and dexamethasone, respectively). GM-CSF polarized macrophages (MΦ(GM-CSF)) upregulated CD80 but not CD64 expression, showing a partial phenotypic similarity with MΦ(IFN-γ), and also upregulated the expression of the alternative activation marker CD206. M-CSF polarized macrophages (MΦ(M-CSF)) not only expressed increased levels of CD163 and CD16, resembling MΦ(IL-10,) but also displayed high levels of CD64. The phenotype of MΦ(M-CSF) could be further modulated by additional polarization with IFN-γ, IL-4, or IL-10, whereas MΦ(GM-CSF) showed less phenotypic plasticity. CONCLUSION: This study validated CD80 as the most robust phenotypic marker for human MΦ(IFN-γ), whereas CD200R was upregulated and CD14 was specifically downregulated on MΦ(IL-4). CD163 and CD16 were found to be specific markers for MΦ(IL-10). The GM-CSF/M-CSF differentiation model showed only a partial phenotypic similarity with the IFN-γ/IL-4/IL-10 induced polarization.

[Life-threatening infections with a new strain of Clostridium difficile]. / Levensbedreigende infecties met een nieuwe variant van Clostridium difficile.

Three men, aged 39, 73, and 66 years, respectively, developed an infection with a new strain ofClostridium difficile, ribotype 027.C.difficile-associated diarrhoea (CDAD) occurred in two patients after multiple abdominal surgery and in the third patient one week after autologous haematopoietic cell transplantation. Within a few days, despite antibiotic therapy, all three patients developed severe (pseudomembranous) colitis with sepsis for which admission to the Intensive Care Unit was required. Two patients underwent (sub)total colectomy and received an intensive course of oral and/or rectal vancomycin. In all patients who develop diarrhoea in hospital, especially during or after treatment with antibiotics or chemotherapeutic agents, an infection with C. difficile ribotype 027 should be suspected. Recent outbreaks of this hypervirulent strain of C. difficile have been reported in Canada, the United States, United Kingdom, and The Netherlands. Demonstration of C. difficile toxin in faeces confirms the clinical suspicion of CDAD and ribotyping of the strain may reveal whether the 027 strain is present. For treatment of these 027 infections, vancomycin is preferred to metronidazole. After a severe course of colitis or in case of recurrence a 'tapering and pulse' course ofvancomycin can be prescribed; alternatively, treatment with bovine antibody-enriched whey may be considered. The introduction of this hypervirulent strain has led to reinforcement of the hygienic measures in accordance with the recommendations of the Dutch Working Party on Infection Prevention and a policy to deter the use of fluoroquinolones.

Helical perturbations of the flagellar filament: rhizobium lupini H13-3 at 13 A resolution

Flagellar filaments are highly conserved structures in terms of the underlying symmetry of the polymer, subunit domain organization of the flagellin monomer, amino acid composition and primary sequence at the N and C termini. Traditionally, filaments are classified as "plain" or "complex." In complex filaments, the helical lattice is perturbed in a pairwise manner such that the symmetry is reduced along the 6-start helical lines. Both plain (unperturbed) and complex (helically perturbed) components are helically symmetric and share a common lattice. The perturbation in Rhizobium lupini H13-3 results in a subunit composed of a dimer of flagellin. We have generated a approximately 13 A resolution three-dimensional density map of the complex filament of R. lupini H13-3 from low-dose images of negatively stained filaments. Compared to a previous map, which extended to only approximately 25 A resolution and which was generated from only five filaments containing six layer-lines each, the current map is a product of merging 139 data sets containing 66 layer-lines each. The higher resolution and improved signal-to-noise yield a detailed and interpretable density map. The density map is divided into four concentric rings. These amount to two dense cylinders interconnected by low density radial spokes and wrapped by a three-start external winding. The unperturbed component of the map is strikingly similar to the known plain filament maps and, in particular, to that of Caulobacter crescentus. The helically perturbed component contributes mainly to the filaments's exterior (domain D3) where it comprises the tips of the outer domains interconnecting, pairwise, along the 11-start protofilaments and, again, laterally along the 6-start lines forming vertical and horizontal loops. Strong intersubunit connectivity occurs in the D2 shell and in the inner shell which is dominated by 3-start densities. The contribution of the complex component to the radial spokes seems negligible. Copyright 1998 Academic Press.

Detection of local structures in reduced unfolded bovine pancreatic trypsin inhibitor.

The structure of BPTI and reduced BPTI in concentrated guanidinium HCl (GUHCl) in the presence of glycerol has been probed by measurements of dynamic nonradiative excitation energy transfer between probes attached to its amino groups. Interprobe distance distributions were obtained from analysis of donor fluorescence decay curves and used to characterize local structures in unordered states of the protein. Site specifically fluorescently labeled BPTI derivatives (1-n)BPTI (n = 15, 20, 41, 46) were used, each carrying a 2-methoxy-naphthyl-1-methylenyl group (MNA) at the N-terminal amino group of arg1 and 7-(dimethylamino)-coumarin-4-yl-acetyl residue (DA-coum) at one of its epsilon-NH2 groups of the lysine side chains. Analysis of donor fluorescence decay kinetics gave the interprobe distance distributions in the native and denatured states. The N-terminal-segment, residues 1-15, is in an extended conformation (with an average interprobe distance of 34 +/- 2 A) in the native state. Upon unfolding by reduction with DTT or beta-mercapto ethanol in 6 M GUHCl/glycerol mixture, the conformation of this segment relaxed to a state characterized by a reduced average interprobe distance and a larger width of the distances distribution. The average distance between residues 1 and 26, i.e., between the N-terminus and the turn of the twisted beta sheet element (residues 18-35), increased upon unfolding. At -30 degrees C in the above solvent, the distribution between these two sites was probably composed of two conformational subpopulations. About 45 +/- 20% of the molecules were characterized by a short interprobe distance (like the native state) representing a compact conformation, and 55 +/- 20% of the molecules showed large interprobe distances representing an expanded (unfolded) conformation. Thus local structures seem to exist in reduced denatured BPTI even under denaturing conditions in 6 M GUHCl/glycerol mixtures. Some of those structures are unstable in guanidinium isothiocyanate (GUSCN). The method introduced here is suitable for probing local structures and very long range interactions in unfolded proteins and for search for folding initiation sites (FISs) and early folding intermediates.

Conformational studies of a peptide corresponding to a region of the C-terminus of ribonuclease A: implications as a potential chain-folding initiation site.

Conformational properties of the OT-16 peptide, the C-terminal 20 amino acids of RNase A, were examined by nonradiative energy transfer. A modified OT-16 peptide was prepared by solid-phase synthesis with the inclusion of diaminobutyric acid (DABA) at the C-terminus. The OT-16-DABA peptide was labeled with a fluorescent 1,5-dimethylaminonaphthalene sulfonyl (dansyl, DNS) acceptor at the N-terminal amine and a fluorescent naphthoxyacetic acid (NAA) donor at the gamma-amine of the DABA located at the C-terminus of the peptide by using an orthogonal protection scheme. Energy transfer was monitored in DNS-OT-16-DABA-NAA by using both fluorescence intensity (sensitized emission) and lifetime (donor quenching) experiments. The lifetime data indicate that the peptide system is a dynamic, flexible one. A detailed analysis, based on a dynamic model that includes a skewed Gaussian function to model the equilibrium distribution of interprobe distances and a mutual diffusion coefficient between the two probes to model conformational dynamics in the peptide [Beechem & Haas (1989) Biophys. J. 55, 1225.], identified the existence of a partially ordered structure (relatively narrow distribution of interprobe distances) at temperatures greater than or equal to 20 degrees C in the absence of denaturant. The width and the position of the average of the distributions decrease with increasing temperature, in this range; this suggests that the structure is stabilized by hydrophobic interactions. In addition, the peptide undergoes cold denaturation at around 1.5 degrees C as indicated by broadening of the distance distribution. The addition of 6 M guanidine hydrochloride (Gdn-HCl) also broadens the distance distribution significantly, presumably by eliminating the hydrophobic interactions and unfolding the peptide. The results of the analysis of the distance distribution demonstrate that (1) nonradiative energy transfer can be used to study the conformational dynamics of peptides on the nanosecond time scale, (2) a partially ordered structure of OT-16-DABA exists in solution under typical refolding conditions, and (3) structural constraints (presumably hydrophobic interactions) necessary for the formation of a chain-folding initiation site in RNase A are also present in the OT-16-DABA peptide in the absence of denaturant and are disrupted by Gdn-HCl.

Cardiorespiratory effects of hypothermia and bicarbonate alkalosis.

The cardiorespiratory effects of reducing body temperature to 30 degrees C (by packing in ice) and subsequent metabolic alkalosis (by infusion of NaHCO3) were studied in six anesthetized, paralyzed, and artificially ventilated (FIO2 = 0.4) dogs. Heart rate decreased from 135 +/- 6 beats/min (mean +/- S.E.) at 37 degrees C to 84 +/- 4 at 30 degrees C; it increased to 96 +/- 4 after 2 h alkalosis. Cardiac output decreased from 1.84 +/- 0.14 to 0.66 +/- 0.08 l/min and then increased to 0.83 +/- 0.07. pHa increased, as expected on cooling, from 7.41 +/- 0.07 to 7.49 +/- 0.03; with bicarbonate it increased to 7.79 +/- 0.03. PaCO2 decreased on cooling from 32.9 +/- 1.4 to 21.7 +/- 1.2 torr, increasing with bicarbonate to 27.9 +/- 1.4 torr. VO2 decreased from 104.9 +/- 5.1 ml . min-1 . m-2 at 37 degrees C to 51.3 +/- 2.0 at 30 degrees C; with alkalosis it increased by 16.2% to 59.6 +/- 3.3 ml . ml-1 . m-2, an increase identical to that seen in normothermic alkalosis. Thus, the mechanism of the alkalosis-induced increase in oxygen consumption is not suppressed by the decrease in VO2 seen in hypothermia, and the increase in VO2 appears to be a consequence of the change in relative alkalinity rather than the increase in pH.

A pharmacological study of the control of nasal cooling in the dog.

Air temperature differences between the internal and external nars (Ti-e), a qualitative measure of nasal blood flow, and the rate and ionic content of the mucous secretion of the lateral nasal gland, were determined in conscious, chronically instrumented dogs at both 25 degrees C and 42 degrees C environmental temperature, before and after administration of epinephrine, propanolol, phenoxybenzamine, acetylcholine and atropine. B. At 25 degrees C, epinephrine increased Ti-e. This was unaffected by propranolol, but returned to control values with phenoxybenzamine. Acetylcholine and atropine had no effect. At 42 degrees C, acetylcholine increased Ti-e; this was not reversed with atropine. Epinephrine caused a slight decrease in Ti-e at 42 degrees C. C. acetylcholine reduced mucous flow at 25 degrees C; atropine further decreased the flow. Changes in ionic content of the mucous suggest that cholinergic mechanisms affect pressure in the excretory duct of the gland. Epinephrine decreased the mucous flow at 42 degrees C; this was reversed with propranolol. D. It was concluded that nasal blood flow and lateral nasal gland mucous flow both possess sympathetic tone at 25 degrees C, the former being alpha-adrenergic, the latter beta-adrenergic. This tone appears important in reducing heat and water loss under normal conditions, and its reduction at high temperatures allows maximum efficiency of nasal cooling. The role of the parasympathetic system in respiratory heat and water exchange in the dog appears somewhat equivocal.

Physiological responses of dogs on exposure to hot, arid conditions. Acid-base status.

Acid-base parameters were determined in chronically cannulated dogs exposed to ambient temperatures increasing from 25-47 degrees C (with relative humidity below 30%). pH increased from 7.409 +/- 0.004 (S.E.M.) to 7.538 +/- 0.017, PaCO2 decreased from 33.0 +/- 0.5 to 20.9 +/- 1.2 torr, and [HCO3-] decreased from 20.9 +/- 0.3 to 17.2 +/- 0.4 mEq/l. Minimal base excess change, together with a rapid return to normal parameters upon recooling to 25 degrees C, suggests that the stress is almost exclusively respiratory, with little metabolic involvement. Analysis of serial exposures shows no acclimatization effect in acid-base status. This suggests the possible existence of natural acclimation to heat in dogs maintained in a warm climate, permitting excellent tolerance of hot, arid conditions with limited acid-base disturbance.

Physiological responses of dogs on exposure to hot, arid conditions. Serum constituents.

Serum electrolytes, metabolites and enzymes were determined in arterial blood of chronically cannulated dogs at room temperature and on exposure to 44-50 degrees C. These dogs were naturally acclimated to hot, arid conditions. In dogs maintaining their rectal temperatures (TR) below 40 degrees C, no significant changes were seen in the levels of Na+, Cl-, cholesterol, uric acid, alkaline phosphatase, lactic dehydrogenase or glutamic-pyruvic transaminase (SGPT). K+, CO2, glucose decreased significantly, and urea nitrogen (BUN) and glutamic-oxaloacetic transaminase (SGOT) showed small but significant increases. In several cases of excitable dogs, in which TR increased above 40 degrees C, we found large, significant increases in uric acid, SGPT and SGOT, and a decrease in cholesterol. The results suggest that in dogs maintaining their TR when exposed to high temperatures, changes in serum constituents indicate merely the presence of respiratory alkalosis and an increased energetic demand. When control of TR is lost, changes occur which suggest liver, and possibly cardiac, tissue damage.