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.

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.

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.

Tissue localization and the establishment of a sensitive immunoassay of the newly discovered human phospholipase B-precursor (PLB-P).

Human phospholipase B-precursor (PLB-P) is a newly identified and purified protein from human neutrophils. The precise function of PLB-P in vivo is not yet known. Its existence in neutrophils and the enzymatic activity against phospholipids imply a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation. We describe here the generation of specific antibodies against PLB-P, the tissue localizations of PLB-P and the establishment of an accurate, specific, and reproducible radioimmunoassay (RIA). A survey of normal and malignant tissues showed strong immunostaining of PLB-P in neuronal and myeloid cells and in adrenal glands. Elevated levels were found in sera of patients with influenza A infection i.e. >1 microg/L and in gut fluids of patients with inflammatory bowel disease i.e. >20 microg/L. The levels correlated to markers of neutrophil activation, suggesting a neutrophil origin of PLB-P in these conditions. The antibodies and the assay will be useful in the future basic and clinical investigations of PLB-P.

Lipid rafts in Cryptococcus neoformans concentrate the virulence determinants phospholipase B1 and Cu/Zn superoxide dismutase.

Lipid rafts have been identified in the membranes of mammalian cells, the yeast Saccharomyces cerevisiae, and the pathogenic fungus Candida albicans. Formed by a lateral association of sphingolipids and sterols, rafts concentrate proteins carrying a glycosylphosphatidylinositol (GPI) anchor. We report the isolation of membranes with the characteristics of rafts from the fungal pathogen Cryptococcus neoformans. These characteristics include insolubility in Triton X-100 (TX100) at 4 degrees C, more-buoyant density within a sucrose gradient than the remaining membranes, and threefold enrichment with sterols. The virulence determinant phospholipase B1 (PLB1), a GPI-anchored protein, was highly concentrated in raft membranes and could be displaced from them by treatment with the sterol-sequestering agent methyl-beta-cyclodextrin (MbetaCD). Phospholipase B enzyme activity was inhibited in the raft environment and increased 15-fold following disruption of rafts with TX100 at 37 degrees C. Treatment of viable cryptococcal cells in suspension with MbetaCD also released PLB1 protein and enzyme activity, consistent with localization of PLB1 in plasma membrane rafts prior to secretion. The antioxidant virulence factor Cu/Zn superoxide dismutase (SOD1) was concentrated six- to ninefold in raft membrane fractions compared with nonraft membranes, whereas the cell wall-associated virulence factor laccase was not detected in membranes. We hypothesize that raft membranes function to cluster certain virulence factors at the cell surface to allow efficient access to enzyme substrate and/or to provide rapid release to the external environment.

An ultrastructural and a cytochemical study of candidal invasion of reconstituted human oral epithelium.

BACKGROUND: Opportunistic yeast, Candida albicans causes superficial and systemic mycoses in compromised patients. Adhesion to host tissues, morphogenesis and extracellular phospholipases (PL) are thought to contribute to its virulence. The nature of numerous host-parasite interactions at the invasive phase of oral candidiasis is not fully understood. Hence in this study, we explore the ultrastructural features of oral candidiasis using a tissue culture model based on reconstituted human oral epithelium (RHOE). METHODS: Reconstituted human oral epithelium (Skinethic Laboratory, Nice, France) was inoculated with C. albicans SC5314 and incubated up to 48 h. The infected tissue was harvested at 12, 24 and 48 h and examined using light, scanning (SEM) and transmission electron microscopy (TEM). Localized activity of PLs of C. albicans during tissue invasion was also examined using a cytochemical method. RESULTS: Over a period of 48 h C. albicans invaded the RHOE, and histological examination revealed characteristic hallmarks of pathological tissue invasion. Hyphal penetration into the superficial epithelium, particularly at cell junctions, together with features of cellular internalization of yeasts was noted. Phospholipase activity was visible at the tips of hyphae and initial sites of bud formation. Further, SEM studies revealed cavitations on the surface epithelial cells particularly pronounced at the sites of hyphal invasion. Hyphal invasion was seen both at cell surfaces and intercellular cell junctions of the epithelium, the latter resembling thigmotropic behaviour. CONCLUSIONS: Our findings confirm that multiple cellular interactions such as internalization, thigmotropism and extracellular PLs contribute to invasive candidiasis. The RHOE model, described here, appears to be a satisfactory model for the investigation of ultrastructural and histochemical features of invasive candidiasis in humans.

Pseudomonas aeruginosa causes acute lung injury via the catalytic activity of the patatin-like phospholipase domain of ExoU.

OBJECTIVE: Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU toxin being delivered directly into the eukaryotic cell cytosol through the type III secretion system. The amino-acid sequence of ExoU has a potato patatin-like phospholipase domain, similar to the sequence of mammalian Ca-independent phospholipase A2. We examined whether the acute lung injury caused by cytotoxic P. aeruginosa was dependent on the patatin-like phospholipase domain of ExoU. DESIGN: Laboratory investigation using an established mouse model for P. aeruginosa pneumonia with quantitative measurements of acute lung injury and mortality. SETTING: University experimental research laboratory. SUBJECTS: Balb/c mice. INTERVENTIONS: First, a site-directional mutation was introduced in the predicted catalytically active site of the patatin-like phospholipase domain of recombinant ExoU protein. The effect of the mutation on the catalytic activity of ExoU was tested by the in vitro lysophospholipase A assay. Second, the same site-directional mutation was introduced into the exoU gene of P. aeruginosa PA103. Mice were intratracheally infected with either a wild-type P. aeruginosa strain PA103 or an isogenic mutant containing the mutation in exoU. Acute epithelial lung injury, lung edema, bacteremia, and mortality were evaluated quantitatively. MEASUREMENTS AND MAIN RESULTS: Recombinant ExoU had lysophospholipase A activity. Site-directional mutations in the predicted catalytic site of ExoU caused a loss of the lysophospholipase A activity. Whereas the airspace instillation of PA103 caused acute lung injury and death of the infected mice, the airspace instillation of isogenic mutants secreting catalytically inactive ExoU were noncytotoxic and did not cause acute lung injury or death of the infected mice. CONCLUSION: Virulent P. aeruginosa causes acute lung injury and death by the cytotoxic activity derived from the patatin-like phospholipase domain of ExoU.

Differential expression of Candida albicans secreted aspartyl proteinase and phospholipase B genes in humans correlates with active oral and vaginal infections.

The in vivo expression of Candida albicans secreted aspartyl proteinase (SAP1-SAP8) and phospholipase B (PLB1 and PLB2) genes was analyzed in 137 human subjects with oral and vaginal candidiasis or carriage. Total RNA was isolated from whole unstimulated saliva or vaginal swabs, and the expression of SAP1-8 and PLB1-2 was evaluated by reverse-transcriptase polymerase chain reaction using specific primer sets. A spectrum of SAP gene expression profiles was obtained from different C. albicans strains during symptomatic disease and asymptomatic carriage. SAP2 and SAP5 were the most common genes expressed during both infection and carriage. SAP1, SAP3, SAP4, SAP7, SAP8, and PLB1 expression was correlated with oral disease, whereas SAP1, SAP3, and SAP6-SAP8 expression was correlated with vaginal disease. Furthermore, SAP1, SAP3, and SAP8 were preferentially expressed in vaginal, rather than oral, infections. This study demonstrates the differential expression of the hydrolytic enzyme genes in humans and correlates the expression of specific Candida species virulence genes with active disease and anatomical location.

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.

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.