Multi-Omic blood analysis reveals differences in innate inflammatory sensitivity between species.

Vertebrates differ greatly in responses to pro-inflammatory agonists such as bacterial lipopolysaccharide (LPS), complicating use of animal models to study human sepsis or inflammatory disorders. We compared transcriptomes of resting and LPS-exposed blood from six LPS-sensitive species (rabbit, pig, sheep, cow, chimpanzee, human) and four LPS-resilient species (mice, rats, baboon, rhesus), as well as plasma proteomes and lipidomes. Unexpectedly, at baseline, sensitive species already had enhanced expression of LPS-responsive genes relative to resilient species. After LPS stimulation, maximally different genes in resilient species included genes that detoxify LPS, diminish bacterial growth, discriminate sepsis from SIRS, and play roles in autophagy and apoptosis. The findings reveal the molecular landscape of species differences in inflammation, and may inform better selection of species for pre-clinical models.

Compact capillary high performance liquid chromatography system for pharmaceutical on-line reaction monitoring.

Due to their size, conventional high performance liquid chromatographs (HPLCs) are difficult to place close to a reaction vessel within a pharmaceutical manufacturing or development site. Typically, long transfer lines are required to move sample from the reactor to the HPLC for analysis and high solvent usage is required. However, herein a compact and modular separation system has been developed to enable co-location of an HPLC with a small-scale reactor for reaction monitoring in the synthesis of active pharmaceutical ingredients. Using a framework based on capillary HPLC, a compact gradient separation system with a fully modular architecture is described. A custom miniature diode-array detector with a linear dynamic range (up to 1500 mAU at 210 nm) was integrated and evaluated for on-line reaction monitoring. In evaluating system suitability, average peak area %RSD of <3%, and an average retention time %RSD of <0.7%, were achieved. To demonstrate practical utility, the compact system was coupled directly to an on-line lab-scale flow through reactor for continuous reaction monitoring in the laboratory fume hood, where a study of the 3rd Bourne reaction was used to compare the performance of the compact system with a commercially available process HPLC instrument (Waters PATROL UPLC). Further, 33 off-line samples from a continuous crystallization reactor were analysed and it was found that the developed compact HPLC system showed equivalent quantitative performance to an Agilent 1290 Infinity II HPLC system.

Small footprint liquid chromatography-mass spectrometry for pharmaceutical reaction monitoring and automated process analysis.

Liquid chromatography (LC) has broad applicability in the pharmaceutical industry, from the early stages of drug discovery to reaction monitoring and process control. However, small footprint, truly portable LC systems have not yet been demonstrated and fully evaluated practically for on-line, in-line or at-line pharmaceutical analysis. Herein, a portable, briefcase-sized capillary LC fitted with a miniature multi-deep UV-LED detector has been developed and interfaced with a portable mass spectrometer for on-site pharmaceutical analysis. With this configuration, the combined small footprint portable LC-UV/MS system was utilized for the determination of small molecule pharmaceuticals and reaction monitoring. The LC-UV/MS system was interfaced directly with a process sample cart and applied to automated pharmaceutical analysis, as well as also being benchmarked against a commercial process UPLC system (Waters PATROL system). The portable system gave low detection limits (∼3 ppb), a wide dynamic range (up to 200 ppm) and was used to confirm the identity of reaction impurities and for studying the kinetics of synthesis. The developed platform showed robust performance for automated process analysis, with less than 5.0% relative standard deviation (RSD) on sample-to-sample reproducibility, and less than 2% carryover between samples. The system has been shown to significantly increase throughput by providing near real-time analysis and to improve understanding of synthetic processes.

Simulated microgravity in the ring-sheared drop.

The ring-sheared drop is a module for the International Space Station to study sheared fluid interfaces and their influence on amyloid fibril formation. A 2.54-cm diameter drop is constrained by a stationary sharp-edged ring at some latitude and sheared by the rotation of another ring in the other hemisphere. Shearing motion is conveyed primarily by the action of surface shear viscosity. Here, we simulate microgravity in the laboratory using a density-matched liquid surrounding the drop. Upon shearing, the drop's deformation away from spherical is found to be a result of viscous and inertial forces balanced against the capillary force. We also present evidence that the deformation increases with increasing surface shear viscosity.

Different Bactericidal and Inflammatory Activities of Human and Mouse Blood.

We performed side-by-side experiments to compare the behavior of four strains of Escherichia coli and one strain of Pseudomonas aeruginosa in fresh human and mouse blood. Bacteria were multiplied in mouse whole blood and plasma but were killed in human whole blood and plasma. The percentage of granulocytes associated with fluorescence-labeled heat-killed E coli relative to total leukocytes counted was higher in human compared to mouse blood as assessed by flow cytometry analysis. Concentrations of proinflammatory cytokines were high in human blood, but undetectable in mouse blood despite high concentrations of bacteria. We conclude that bacterial killing, phagocytosis, and cytokine induction in blood during human bacteremia with these organisms are probably not mimicked in mouse models of bacterial challenge. Understanding the mechanisms for low cytokine induction with high bacterial loads in mouse blood may be helpful to interpret murine models of bacteremia and develop new approaches for treating sepsis in humans.

Semi-preparative high-resolution recycling liquid chromatography.

A semi-preparative high-resolution system based on twin column recycling liquid chromatography was built. The integrated system includes a binary pump mixer, a sample manager, a two-column oven compartment, two low-dispersion detection cells, and a fraction manager (analytical). It addresses challenges in drug/impurity purification, which involve several constraints simultaneously: (1) small selectivity factors (α < 1.2, poor resolution), (2) mismatch of elution strength between the sample diluent and the eluent causing severe band fronting or tailing, (3) diluent-to-eluent mismatch of viscosity causing viscous fingering and unpredictable band deformation, (4) low abundance of the impurity relative to the active pharmaceutical ingredient (API) (<1/100), and (5) yield and purity levels to be larger than 99% and 90%, respectively. The prototype system was tested for the preparation of a trace impurity present in a concentrated solution of an API, estradiol. The ultimate goal was to collect ∼1 mg of impurity (>90% purity) for unambiguous structure elucidation by liquid state nuclear magnetic resonance (NMR 600 MHz and above). First, the particle size (3.5 µm) used to pack the 4.6 mm × 150 mm long twin columns is selected so that the speed-resolution of the recycling process is maximized at 4000 psi pressure drop. Next, the production rate of the process is also maximized by determining the optimum number (7) of cycles and the corresponding largest sample volume (160 µL) to be injected. Finally, the process is fully automated by programming the time events related to (1) sample cleaning, (2) transfer of the targeted impurity from one to the second twin column, and (3) impurity collection. The process was tested without interruption during one week for the collection of a trace impurity (α = 1.166, strong acetonitrile-methanol sample diluent, concentration ∼2 mg/L) from a concentrated (10 g/L) stock solution (60 mL total) of estradiol. The process enriches the impurity content relative to the API by about a factor ∼5000. For the lack of a sufficient collected amount (∼120 µg only) of the pure impurity (purity 50% only), NMR experiments could not provide reliable results. Instead, the combination of LC-MS (single ion monitoring) and UV absorption spectra (λmax shift) revealed that the targeted impurity was likely the low-abundant enol tautomeric form of the ketone estrone, a possible intermediate or by-product of the synthesis reaction of estradiol.

Haptoglobin or Hemopexin Therapy Prevents Acute Adverse Effects of Resuscitation After Prolonged Storage of Red Cells.

BACKGROUND: Extracellular hemoglobin and cell-free heme are toxic breakdown products of hemolyzed erythrocytes. Mammals synthesize the scavenger proteins haptoglobin and hemopexin, which bind extracellular hemoglobin and heme, respectively. Transfusion of packed red blood cells is a lifesaving therapy for patients with hemorrhagic shock. Because erythrocytes undergo progressive deleterious morphological and biochemical changes during storage, transfusion of packed red blood cells that have been stored for prolonged intervals (SRBCs; stored for 35-40 days in humans or 14 days in mice) increases plasma levels of cell-free hemoglobin and heme. Therefore, in patients with hemorrhagic shock, perfusion-sensitive organs such as the kidneys are challenged not only by hypoperfusion but also by the high concentrations of plasma hemoglobin and heme that are associated with the transfusion of SRBCs. METHODS: To test whether treatment with exogenous human haptoglobin or hemopexin can ameliorate adverse effects of resuscitation with SRBCs after 2 hours of hemorrhagic shock, mice that received SRBCs were given a coinfusion of haptoglobin, hemopexin, or albumin. RESULTS: Treatment with haptoglobin or hemopexin but not albumin improved the survival rate and attenuated SRBC-induced inflammation. Treatment with haptoglobin retained free hemoglobin in the plasma and prevented SRBC-induced hemoglobinuria and kidney injury. In mice resuscitated with fresh packed red blood cells, treatment with haptoglobin, hemopexin, or albumin did not cause harmful effects. CONCLUSIONS: In mice, the adverse effects of transfusion with SRBCs after hemorrhagic shock are ameliorated by treatment with either haptoglobin or hemopexin. Haptoglobin infusion prevents kidney injury associated with high plasma hemoglobin concentrations after resuscitation with SRBCs. Treatment with the naturally occurring human plasma proteins haptoglobin or hemopexin may have beneficial effects in conditions of severe hemolysis after prolonged hypotension.

Human annulus progenitor cells: Analyses of this viable endogenous cell population.

Back pain and intervertebral disc degeneration have growing socioeconomic/health care impacts. Increasing research efforts address use of stem and progenitor cell-based replacement therapies to repopulate and regenerate the disc. Data presented here on the innate human annulus progenitor cells: (i) assessed osteogenic, chondrogenic and adipogenic potentials of cultured human annulus cells; and (ii) defined progenitor-cell related gene expression patterns. Verification of the presence of progenitor cells within primary human disc tissue also used immunohistochemical identification of cell surface markers and microarray analyses. Differentiation analysis in cell cultures demonstrated a viable progenitor cell pool within Thompson grades III-IV discs. Osteogenesis was present in 8 out of 11 cultures (73%), chondrogenesis in 8 of 11 (73%), and adipogenesis in 6 of 6 (100%). Immunolocalization was positive for CD29, CD44, CD105, and CD14 (mean values 80.2%, 81.5%, 85.1%, and 88.6%, respectively); localization of CD45 and CD34 was negative in disc tissue. Compared to controls, surgical discs showed significantly downregulated genes with recognized progenitor cell functions: TCF7L2 (2.7 fold), BMI1 (3.8 fold), FGF receptor 2 (2 fold), PAFAH1B1 (2.3 fold), and GSTP1 (9 fold). Compared to healthier grade I/II discs, grade III/IV discs showed significantly upregulated XRCC5 (3.6 fold), TCF7L2 (6 fold), GSTP1 (3.7 fold), and BMI1 (3 fold). Additional significant cell marker analyses showed expression of platelet-derived growth factor receptor alpha, CD90, CD73, and STRO-1. Statement of Clinical Significance: Findings provide the first identification of progenitor cells in annulus specimens from older, more degenerate discs (in contrast to earlier studies of healthier discs or nondegenerative specimens from teenagers). Findings also increase knowledge on progenitor cells present in the disc and suggest their value in potential future utilization for regeneration and disc cell therapy. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1351-1360, 2016.

Purified and Recombinant Hemopexin: Protease Activity and Effect on Neutrophil Chemotaxis.

Infusion of the heme-binding protein hemopexin has been proposed as a novel approach to decrease heme-induced inflammation in settings of red blood cell breakdown, but questions have been raised as to possible side effects related to protease activity and inhibition of chemotaxis. We evaluated protease activity and effects on chemotaxis of purified plasma hemopexin obtained from multiple sources as well as a novel recombinant fusion protein Fc-hemopexin. Amidolytic assay was performed to measure the protease activity of several plasma-derived hemopexin and recombinant Fc-hemopexin. Hemopexin was added to the human monocyte culture in the presence of lipopolysaccharides (LPS), and also injected into mice intravenously (i.v.) 30 min before inducing neutrophil migration via intraperitoneal (i.p.) injection of thioglycolate. Control groups received the same amount of albumin. Protease activity varied widely between hemopexins. Recombinant Fc-hemopexin bound heme, inhibited the synergy of heme with LPS on tumor necrosis factor (TNF) production from monocytes, and had minor but detectable protease activity. There was no effect of any hemopexin preparation on chemotaxis, and purified hemopexin did not alter the migration of neutrophils into the peritoneal cavity of mice. Heme and LPS synergistically induced the release of LTB4 from human monocytes, and hemopexin blocked this release, as well as chemotaxis of neutrophils in response to activated monocyte supernatants. These results suggest that hemopexin does not directly affect chemotaxis through protease activity, but may decrease heme-driven chemotaxis and secondary inflammation by attenuating the induction of chemoattractants from monocytes. This property could be beneficial in some settings to control potentially damaging inflammation induced by heme.

Dysregulation of the haem-haemopexin axis is associated with severe malaria in a case-control study of Ugandan children.

BACKGROUND: Malaria is associated with haemolysis and the release of plasma haem. Plasma haem can cause endothelial injury and organ dysfunction, and is normally scavenged by haemopexin to limit toxicity. It was hypothesized that dysregulation of the haem-haemopexin pathway contributes to severe and fatal malaria infections. METHODS: Plasma levels of haemin (oxidized haem), haemopexin, haptoglobin, and haemoglobin were quantified in a case-control study of Ugandan children with Plasmodium falciparum malaria. Levels at presentation were compared in children with uncomplicated malaria (UM; n = 29), severe malarial anaemia (SMA; n = 27) or cerebral malaria (CM; n = 31), and evaluated for utility in predicting fatal (n = 19) vs non-fatal (n = 39) outcomes in severe disease. A causal role for haemopexin was assessed in a pre-clinical model of experimental cerebral malaria (ECM), following disruption of mouse haemopexin gene (hpx). Analysis was done using Kruskall Wallis tests, Mann-Whitney tests, log-rank tests for survival, and repeated measures ANOVA. RESULTS: In Ugandan children presenting with P. falciparum malaria, haemin levels were higher and haemopexin levels were lower in SMA and CM compared to children with UM (haemin, p < 0.01; haemopexin, p < 0.0001). Among all cases of severe malaria, elevated levels of haemin and cell-free haemoglobin at presentation were associated with subsequent mortality (p < 0.05). Compared to ECM-resistant BALB/c mice, susceptible C57BL/6 mice had lower circulating levels of haemopexin (p < 0.01), and targeted deletion of the haemopexin gene, hpx, resulted in increased mortality compared to their wild type littermates (p < 0.05). CONCLUSIONS: These data indicate that plasma levels of haemin and haemopexin measured at presentation correlate with malaria severity and levels of haemin and cell-free haemoglobin predict outcome in paediatric severe malaria. Mechanistic studies in the ECM model support a causal role for the haem-haemopexin axis in ECM pathobiology.

Automated solvent system screening for the preparative countercurrent chromatography of pharmaceutical discovery compounds.

A fully automated countercurrent chromatography system has been constructed to rapidly screen the commonly used heptane/ethyl acetate/methanol/water solvent system series and translate the results to preparative scale separations. The system utilizes "on-demand" preparation of the heptane/ethyl acetate/methanol/water solvent system upper and lower phases. Elution-extrusion countercurrent chromatography was combined with non-dynamic equilibrium injection reducing the screening time for each heptane/ethyl acetate/methanol/water system to 17 min. The result enabled solvent system development to be reduced to under 2 h. The countercurrent chromatography system was interfaced with a mass spectrometer to allow selective detection of target components in crude medicinal chemistry reaction mixtures. Mass-directed preparative countercurrent chromatography purification was demonstrated for the first time using a synthetic tetrazole epoxide derived from a routine medicinal chemistry support workflow.

Chromatographic resolution of atropisomers for toxicity and biotransformation studies in pharmaceutical research.

Atropisomerism can be a complex concept for those who have not encountered it before. This paper discusses the experiments for identification, isolation, thermal stability, toxicity and biotransformation of various species. The identified atropisomers are a series of rotational hindered biaryl, rotational hindered amide, ring flip, and macrocycles atropisomers identified using supercritical fluid chromatography (SFC) and high performance liquid chromatography (HPLC). These technologies offered the advantage of separating various atropoenantiomers, atropdiastereomers and mixed atropisomers with other forms of stereoisomers in both analytical and preparative scales. With ultra-performance convergence chromatography (UPC(2)), the detection of N-oxide atropisomer metabolites can be obtained at very low level thus enabling the observation of conversion in human plasma possible. As the resolution of atropisomers are related to the energy barriers on the rotational axis, a calculated computational protocol was developed to predict the formation. A threshold of 10kcal/mol was established for possible detection of the atropisomers' existence with chromatographic technologies at room temperature or above. The atropisomer with higher energy barrier (>20kcal/mol) were isolated via preparative chromatography and the isolates studied in vitro and in vivo for evaluation of their stability in human plasma. The detailed analytical method development to analyze the biotransformation of the atropisomers in human plasma are also discussed in this paper.

Hemopexin in severe inflammation and infection: mouse models and human diseases.

INTRODUCTION: Cell-free plasma hemoglobin is associated with poor outcome in patients with sepsis. Extracellular hemoglobin and secondarily released heme amplify inflammation in the presence of microbial TLR ligands and/or endogenous mediators. Hemopexin, a plasma protein that binds heme with extraordinary affinity, blocks these effects and has been proposed as a possible treatment approach to decrease inflammation in critically ill patients. METHODS: We studied mouse models of endotoxemia, burn wound infections and peritonitis in order to assess if a repletion strategy for hemopexin might be reasonable. We also measured hemopexin in small numbers of three patient populations that might be logical groups for hemopexin therapy: patients with sepsis and ARDS, patients with severe burns, and premature infants. RESULTS: Despite severe disease, mean plasma hemopexin levels were increased above baseline in each murine model. However, plasma hemopexin levels were decreased or markedly decreased in many patients in each of the three patient populations. CONCLUSIONS: Potentially different behavior of hemopexin in mice and humans may be important to consider when utilizing murine models to represent acute human inflammatory diseases in which heme plays a role. The findings raise the possibility that decreased hemopexin could result in insufficiently neutralized or cleared heme in some patients with ARDS, burns, or in premature infants who might be candidates to benefit from hemopexin administration.

On the method development of immobilized polysaccharide chiral stationary phases in supercritical fluid chromatography using an extended range of modifiers.

Polysaccharide-derived selectors are often used in the separation of enantiomers by supercritical fluid chromatography (SFC). Their recognition patterns are normally investigated with alcohols and acetonitrile as modifiers. The present paper describes the results of a research program designed by Pfizer and Chiral Technologies Inc. to explore the potential of other solvents (i.e. ethyl acetate, tetrahydrofuran, dichloromethane) in SFC by using a series of polysaccharide-derived supports with broad solvent versatility (CHIRALPAK IA, IB, IC, ID, IE and IF). The contribution of such extended solvent range to the overall success rate, as well as to overcome racemization, solubility and stability issues was confirmed by using standard non-proprietary samples and research molecules. Elution patterns with such lower polarity solvents, compared to alcohols, and the role of the different additives were also investigated.

Mitochondrial bioenergetics, mass, and morphology are altered in cells of the degenerating human annulus.

Back pain and intervertebral disc degeneration have a growing socioeconomic healthcare impact. Information on mitochondrial function in human intervertebral disc cells, however, is surprisingly sparse. We assessed mitochondrial bioenergetics, mass, and ultrastructure in annulus cells cultured from human discs of varying degenerative stages. Citrate synthase activity (reflecting mitochondrial mass) declined significantly with increasing Thompson grade (p < 0.0001). Both mitochondrial (p = 0.009) and non-mitochondrial (p = 0.0029) respiration showed significant changes with increasing stages of disc degeneration. No significant relationships were found for the association of respiration data with herniated or non-herniated status, or with subject age. Examination of mitochondrial ultrastructure in cultured annulus cells revealed unusual features which included mitochondrial inclusion bodies, poorly defined cristae and dark staining. Findings reported here are novel and document biochemical, metabolic, and morphologic abnormalities in mitochondria in cells from more degenerated annulus cells. Data suggest that the disc degenerative, not age, is a major factor associated with mitochondrial impairment, and also implicate oxidative stress, driven by mitochondrial dysfunction, as a major component within the degenerating disc. Findings have relevance to advancements in cell-based therapies to treat disc degeneration.

Osteogenic and chondrogenic potential of biomembrane cells from the PMMA-segmental defect rat model.

A layer of cells (the "biomembrane") has been identified in large segmental defects between bone and surgically placed methacrylate spacers or antibiotic-impregnated cement beads. We hypothesize that this contains a pluripotent stem cell population with potential valuable applications in orthopedic tissue engineering. Objectives using biomembranes harvested from rat segmental defects were to: (1) Culture biomembrane cells in specialized media to direct progenitor cells along bone or cartilage cell differentiation lineages; (2) evaluate harvested biomembranes for mesenchymal stem cell markers, and (3) define relevant gene expression patterns in harvested biomembranes using microarray analysis. Culture in osteogenic media produced mineralized nodules; culture in chondrogenic media produced masses containing chondroitin sulfate/sulfated proteoglycans. Molecular analysis of biomembrane cells versus control periosteum showed significant upregulation of key genes functioning in mesenchymal stem cell differentiation, development, maintenance, and proliferation. Results identified significant upregulation of WNT receptor signaling pathway genes and significant upregulation of BMP signaling pathway genes. Findings confirm that the biomembrane has a pluripotent stem cell population. The ability to heal large bone defects is clinically challenging, and novel tissue engineering uses of the biomembrane hold great promise in treating non-unions, open fractures with large bone loss and/or infections, and defects associated with tumor resection.

N-(6,7-dichloro-2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-N-alkylsulfonamides as peripherally restricted N-methyl-D-aspartate receptor antagonists for the treatment of pain.

It has been hypothesized that peripherally restricted NMDA receptor antagonists may be effective analgesics for osteoarthritis pain. A class of novel quinoxalinedione atropisomers, first discovered for an NMDA receptor antagonist program for the treatment of stroke, was evaluated and further optimized with the goal of finding peripherally restricted NMDA receptor antagonists.

Synthesis of a spirocyclic indoline lactone.

Base-promoted cyclization of tert-butyl [2-(benzylideneamino)phenyl]acetate (13a) and subsequent C3-alkylation with allyl bromide affords 3-allyl-2-phenyl-2,3-dihydro-1H-indole-3-carboxylic acid, tert-butyl ester (15b) in high yield as a single diastereomer. This result is contrary to prior publications that describe failed cyclization of an analogous ethyl ester (ethyl [2-(4-methoxybenzylideneamino)phenyl]acetate) under strongly basic conditions. N-Acylation, olefin dihydroxylation, and tert-butyl ester cleavage affords the spirocyclic lactone 18 as a pair of diastereomers. Isolation and characterization of individual diastereomers 18a and 18b are described.