Clozapine-Related Diarrhea and Colitis: Report of 4 Cases.

BACKGROUND: During clozapine treatment, diarrhea is a rare but clinically relevant adverse effect. Cases of microscopic colitis and eosinophilic colitis have been previously reported. PROCEDURES: We present 4 patients who developed severe diarrhea in early weeks of clozapine therapy. FINDINGS: Two patients had significant peripheral eosinophilia 1 week after diarrhea symptoms. One of these patients also had Charcot-Leyden crystals in stool afterward, confirming the presence of eosinophils in the gut lumen. One of our patients had a confirmed microscopic colitis and later also neutropenia, which required treatment. CONCLUSIONS: Charcot-Leyden crystals in stool may be associated with concurrent diarrhea and eosinophilia during clozapine treatment, which is a previously unreported finding. Occurrence of blood dyscrasias with diarrhea symptoms during clozapine treatment needs further investigation to understand the possible shared mechanisms.

More similar than different: Host cell protein production using three null CHO cell lines.

To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell-specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time-course profiles of HCP (as measured by multi-analyte ELISA) and those of two intracellular HCP species, phospholipase B-like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC-MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from ∼80% to <20% and extracellular LDH levels increased several-fold. Our findings indicate that the CHO-derived HCPs in the feedstock for downstream processing may not be as diverse across cell lines and upstream processes, or change as dramatically upon viability decline as originally expected. In addition, our findings show that high density CHO cultures (>10(7) cells/mL)-operated in fed-batch mode and exhibiting high viabilities (>70%) throughout the culture duration-can accumulate a considerable amount of immunogenic HCP (∼1-2 g/L) in the extracellular environment at the time of harvest (day 14). This work also demonstrates the potential of using LC-MS/MS to overcome the limitations associated with ELISA and 2D-PAGE for HCP analysis.

Charcot-Leyden crystal protein/galectin-10 is a surrogate biomarker of eosinophilic airway inflammation in asthma.

Aim: Eosinophilic asthma is associated with more exacerbations and differential responses to treatment. The aim of this study was to assess if CLC/Gal-10 and MBP-1 are surrogate biomarkers of eosinophilic inflammation in asthma. Methods & results: Sputum induction was performed in patients with asthma and in healthy controls. Sputum analysis revealed higher (p < 0.001) levels of CLC/Gal-10 and MBP-1 in asthmatics versus healthy controls. CLC/Gal-10 levels were highly correlated (rs = 0.74; p < 0.001) with sputum eosinophils; MBP-1 approached significance (r = 0.44; p = 0.07). Conclusion: Increased CLC/Gal-10 and MBP-1 levels in the sputum were strongly correlated with sputum eosinophils in patients with asthma. CLC/Gal-10 and MBP-1 may be useful biomarkers for differentiation of eosinophilic airway inflammation in asthma.

Superior turbinate eosinophilia correlates with olfactory deficit in chronic rhinosinusitis patients.

OBJECTIVE: To evaluate if molecular markers of eosinophilia in olfactory-enriched mucosa are associated with olfactory dysfunction. STUDY DESIGN: Cross-sectional study of tissue biopsies from 99 patients, and an additional 30 patients who underwent prospective olfactory testing prior to sinonasal procedures. METHODS: Tissue biopsies were processed for analysis of inflammatory markers using quantitative real time polymerase chain reaction (qRT-PCR). Ipsilateral olfactory performance was assessed using the Sniffin' Sticks (Burghart, Wedel, Germany) threshold component and the University of Pennsylvania Smell Identification Test (Sensonics, Haddon Heights, NJ). Age-adjusted data was correlated with inflammatory marker expression and clinical measures of obstruction from computed tomography and endoscopy. RESULTS: Gene expression of the eosinophil marker CLC (Charcot Leyden crystal protein) was elevated in superior turbinate (ST) tissue in chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) compared to ST and inferior turbinate tissue in CRS without nasal polyps (CRSsNP) and control patients (all P < 0.001, respectively). CLC in ST tissue was correlated with IL-5 and eotaxin-1 expression (all P < 0.001; P = 0.65, and 0.49, respectively). CLC expression was strongly correlated with eosinophilic cationic protein levels (P < 0.001; r = -0.76), and ST CLC expression was inversely related to olfactory threshold (P = 0.002, r = -0.57) and discrimination scores (P = 0.05, r = -0.42). In multiple linear regression of CLC gene expression, polyp status, and radiographic and endoscopic findings with olfactory threshold, CLC was the only significantly correlated variable (P < 0.05). CONCLUSION: Markers of eosinophils are elevated in the ST of patients with CRSwNP and correlate with olfactory loss. These findings support the hypothesis that olfactory dysfunction in CRS correlates local eosinophil influx into the olfactory cleft. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:2210-2218, 2017.

Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles.

This study investigated the root cause behind an observed free fatty acid particle formation and resulting Polysorbate 20 (PS20) loss for a sulfatase drug product upon long-term storage at 5 ± 3°C. Reversed- phase chromatography with mass spectrometric analysis as well as charged aerosol detection was used to characterize the peaks associated with the intact and degraded PS20. Additionally, a proteomics study was undertaken to identify the residual host cell proteins in the sulfatase drug substance. PS20 stability studies were conducted in the presence of sulfatase, a sulfatase inhibitor, putative phospholipase B-like 2, and mock drug substance produced using a null cell line vector under experimental conditions optimized for PS20 degradation. This study provides the first published evidence where the residual host cell protein present in the drug substance was identified and experimentally shown to catalyze the breakdown of PS20 in a protein formulation over time, resulting in free fatty acid particles and PS20 loss. This study demonstrates the importance of early detection of potential impurities in the protein drug substance that may contribute to polysorbate degradation to make a judicious selection of the surfactant and its optimized concentration for the final drug product.

Subcellular localization and PKC-dependent regulation of the human lysophospholipase A/acyl-protein thioesterase in WISH cells.

Lysophospholipases play essential roles in keeping their multi-functional substrates, the lysophospholipids, at safe levels. Recently, a 25 kDa human lysophospholipase A (hLysoPLA I) that is highly conserved among rat, mouse, human and rabbit has been cloned, expressed and characterized and appears to hydrolyze only lysophospholipids among the various lipid substrates. Interestingly, this enzyme also displays acyl-protein thioesterase activity towards a G protein alpha subunit. To target the subcellular location of this hLysoPLA I, we have carried out immunocytochemical studies and report here that hLysoPLA I appears to be associated with the endoplasmic reticulum (ER) and nuclear envelope in human amnionic WISH cells and not the plasma membrane. In addition, we found that the hLysoPLA I can be up-regulated by phorbol 12-myristate 13-acetate (PMA) stimulation, a process in which phospholipase A(2) is activated and lysophospholipids are generated in WISH cells. Furthermore, the PMA-induced hLysoPLA I expression can be blocked by the protein kinase C (PKC) inhibitor Gö6976. The regulated expression of the LysoPLA/acyl-protein thioesterase by PKC may have important implications for signal transduction processes.

Partial characterization of rat heart cytosolic phospholipase A1 and demonstration of essential sulfhydryl groups.

The cytosol of rat heart has been previously shown to contain phospholipase A activity in substantial amounts. This paper describes the isolation and partial purification of rat heart cytosolic phospholipase A. After homogenization of rat heart followed by centrifugation to remove membraneous material, the phospholipase A activity was isolated by ammonium sulfate precipitation and further purified by gel permeation chromatography with Sephadex G-100 in the presence of 5 mM taurodeoxycholate. Two peaks were isolated: a minor peak at the void volume and major peak corresponding to a molecular weight of 45,000. The molecular weight observed in HPLC gel permeation chromatography experiments was also Mr 45,000 and was not significantly affected by the nature of the detergent used. Phospholipase A was purified 77-fold over the crude cytosol. Further purification could not be attained due to lability of the phospholipase A activity. The enzyme is a phospholipase of the A1 type which does not require Ca2+ and lacks lipase or transacylase activity. It is unusual for the phospholipases A described to date, since it is inhibited by thiol reagents and is protected by beta-mercaptoethanol, suggesting the presence of essential sulfhydryl residues.

The gene for human eosinophil Charcot-Leyden crystal protein directs expression of lysophospholipase activity and spontaneous crystallization in transiently transfected COS cells.

Expression of the gene encoding human eosinophil lysophospholipase, the Charcot-Leyden crystal (CLC) protein, was studied in transiently transfected COS cells. Recombinant CLC (rCLC) protein expression was demonstrated both by Western blot and radioimmunoassay inhibition analyses of transfected COS cell extracts and by immunofluorescent staining and ultrastructural immunogold analyses of intact cells. The rCLC protein was immunochemically indistinguishable from native eosinophil-derived CLC protein, and each transfected COS cell expressed approximately 11 pg of rCLC protein as determined by radioimmunoassay and assessment of transfection efficiency. Immunofluorescent microscopy and ultrastructural immunogold analyses localized rCLC protein to the nucleus, cytoplasm, and plasma membrane of COS cells. Lysates from transfected COS cells producing CLC protein expressed significant lysophospholipase activity. Furthermore, rCLC protein expressed in COS cells spontaneously formed the distinctive intracytoplasmic and intranuclear hexagonal bipyramidal crystals characteristic of the native eosinophil and basophil-derived protein. Expression of the CLC gene confirms the authenticity of the CLC cDNA, the expression of lysophospholipase activity by this unique eosinophil and basophil constituent, and will facilitate the routine purification of the active enzyme for in vitro and animal model studies of its role (or roles) in eosinophil and basophil associated allergic inflammation and eosinophil-parasite interactions.

The effect of salmeterol on nocturnal symptoms, airway function, and inflammation in asthma.

STUDY OBJECTIVE: To determine the efficacy of salmeterol alone in a group of patients with moderate asthma with nocturnal worsening of symptoms. DESIGN: Double-blind, randomized, placebo-controlled crossover study. SETTING: Tertiary care hospital specializing in respiratory diseases. PARTICIPANTS: Ten patients with nocturnal asthma. INTERVENTIONS: Subjects were randomized to salmeterol, 100 micrograms twice daily, or placebo for 6 weeks with a 1-week washout between treatment periods. Symptoms, nocturnal awakenings, and beta 2-agonist use were recorded daily. Spirometry was performed at weeks 1 and 6 of each period at bedtime and at 4 AM, and methacholine challenge was performed at 4 AM followed by bronchoscopy with BAL. BAL fluid analysis included cell count and differential count, eosinophil cationic protein, Charcot-Leyden crystal protein, leukotriene B4, and thromboxane B2. RESULTS: The percentage of nights with awakenings decreased significantly with salmeterol (69.8 +/- 8.7% vs 30.6 +/- 10.8% for placebo and salmeterol, respectively; p = 0.02). The percentage of 24-h days with supplemental inhaled beta 2-agonist use significantly decreased with salmeterol (85.9 +/- 9.4% vs 70.4 +/- 10.1% for placebo and salmeterol, respectively; p = 0.04). There were no significant differences in bronchial reactivity, 4 AM FEV1, overnight percentage change in FEV1, or indexes of airway inflammation. CONCLUSIONS: Salmeterol alone improves the number of nocturnal awakenings and supplemental 24-h beta 2-agonist use in nocturnal asthma without significantly altering lung function and airway inflammation.

Purification and some properties of membrane-bound phospholipase B from Torulaspora delbrueckii.

Membrane-bound phospholipase B was purified to a homogeneous state from Torulaspora delbrueckii cell homogenate. Cell homogenate was extracted with Triton X-100, and the enzyme was precipitated with acetone. The acetone powder was washed repeatedly with Tris-HCl buffer (pH 8.0) until no phospholipae B activity was detected in the soluble fraction. The enzyme was extracted with Triton X-100 from the final residue and purified about 1,390-fold by sequential chromatofocusing, Sepharose 6B, and DEAE-Sephadex A-50 column chromatography. The final preparation showed a single broad protein band on SDS-polyacrylamide gel electrophoresis when stained with silver stain reagent and PAS-reagent. The molecular weight of phospholipase B was about 390,000 and 140,000-190,000 as estimated by gel filtration on Sepharose 6B and SDS-polyacrylamide gel electrophoresis, respectively, suggesting that phospholipase B is an oligomeric protein. The isoelectric point was at pH 4.5. Phospholipase B has two pH optima, one acidic (pH 2.5-3.0) and the other alkaline (pH 7.2-8.0). At acidic pH the phospholipase B activity was greatly increased in the presence of divalent metal ions, although metal ions are not a factor for enzyme activity. On the other hand, at alkaline pH the enzyme required Ca2+ or Mn2+ for activity. The pH- and thermal-stabilities at both pHs were similar. The phospholipase B hydrolyzed all diacylphospholipids tested at acidic pH, but hydrolyzed only phosphatidylcholine at alkaline pH. The hydrolysis rates of lysophospholipids were much higher (about 10-fold) than those of diacylphospholipids at both pHs.

Purification and characterization of lysophospholipase released from rat platelets.

Lysophospholipase released from rat platelets upon activation with thrombin has been purified to near homogeneity by sequential column chromatography on heparin-Sepharose, CM-Sephadex C-50, and TSK gel G2000SW. The final preparation showed a single band with a molecular mass of 32,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The purified enzyme was heat-labile and inactivated after 5 min at 60 degrees C. It showed a broad pH optimum (pH 6-10) and required a divalent cation, such as Ca2+, for the optimal activity. Appreciable activity, however, was observed in the presence of EDTA. Lysophospholipase activity was inhibited by diisopropylfluorophosphate and dithiothreitol. This enzyme activity was retained by a concanavalin A-Sepharose column and eluted with methyl-alpha-D-mannoside. Treatment of lysophospholipase with peptide: N-glycosidase F gave degraded products, suggesting that this protein contain N-linked carbohydrate chains. The purified enzyme was specific to 1-acyl-sn-glycero-3-phospho-L-serine; none of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, and 1-acyl-sn-glycero-3-phospho-D-serine was hydrolyzed appreciably.

Effect of dietary zinc on larval burdens, tissue eosinophil numbers, and lysophospholipase activity of Ascaris suum infected mice.

The effect of dietary zinc on larval burdens, tissue eosinophil numbers, and lysophospholipase (LPL) activity in the lungs and livers of BALB/c mice infected with Ascaris suum was evaluated. A suum larval numbers were increased in both liver and lungs in low zinc groups during primary and secondary infection as assessed at days 2 and 7 after egg administration. In the same groups, LPL activity and eosinophil numbers were reduced at both time points and in both tissues, with the exception of lung eosinophils in nonimmunized mice, since these animals did not develop an eosinophil response during primary infection.

Phospholipases B from Japanese yellow hornet (Vespa xanthoptera) venom.

Two phospholipases B (Vx I and Vx II) were purified from the venom of the Japanese yellow hornet, Vespa xanthoptera, by sequential chromatography on Sephadex G-100, SP-Sephadex and Mono S columns. They are very similar to each other in molecular and enzymatic properties, though the specific activity of Vx I was one-fifth that of Vx II. They hydrolyze the acyl ester bonds at the 1-position of phosphatidylcholine and lysophosphatidylcholine, therefore, their enzymatic specificities were of the A1 and L1 types.

Lysophospholipase activity in rabbit skeletal muscle.

Skeletal muscle contains appreciable lysophospholipase activity which is differentiated by muscle type with red muscle subcellular fractions having greater activity than the corresponding white ones.

Charcot-Leyden crystals within a periapical lesion.

Transmission electron microscopy revealed the presence of Charcot-Leyden crystals within a periapical lesion, which was assessed histopathologically as consistent with a periapical granuloma that failed to resolve after conventional endodontic treatment. This paper presents the clinical, radiographic, histological, and ultrastructural findings of this case and discusses their potential clinical significance.

Renomedullary phospholipases in salt-sensitive and salt-resistant Dahl rats.

Renomedullary prostaglandin synthesis is lower in salt-sensitive (S) Dahl rats than in age-matched salt-resistant (R) rats on either 0.6% or 8.0% NaCl. The possible role of substrate availability to the cyclooxygenase in determining these differences was examined. S and R rats were started on diets with variable salt content (0.6%, 4.0% or 8.0% NaCl) at five weeks and were sacrified at either 11 or 16 weeks of age for comparisons of renomedullary phospholipase A1 and A2 activities (EC 3.1.1.4. and 3.1.1.32) and arachidonate incorporation into the lipids of renal medulla. Both phospholipase activities were higher in R rats at the two ages examined and for all levels of salt intake. These differences could not be accounted for by variable amounts of endogenous phospholipid substrate. High salt intake did not influence the in vitro A2 deacylating activities per mg protein. Arachidonate incorporation into renomedullary triglycerides remained constant with age in both strains while phospholipid labeling declined with age in S rats only so that, at 16 weeks of age, S rats had significantly lower incorporation into membrane phospholipids. This age-related decline in phospholipid labeling in S rats was prevented by 4.0% NaCl. These results suggest that low renomedullary phospholipase activities in S rats may restrict the release of substrate for prostaglandin synthesis and may also be related to reduced rates of fatty acid incorporation into membrane phospholipids.