A pilot randomized clinical trial assessing the effect of cricoid pressure on risk of aspiration.

INTRODUCTION: Patients at risk for microaspiration during elective intubation often receive cricoid pressure in the hopes of mitigating such risk. However, there is scarce evidence to either support or reject this practice. The objective of this investigation was to assess the effect of cricoid pressure on microaspiration and to inform the potential feasibility of conducting a larger, more definitive clinical trial. METHODS: This was a pilot randomized clinical trial set in the operating rooms of a tertiary referral hospital between August and October of 2014. Patients with risk factors for microaspiration (obesity, gastroesophageal reflux disease, or diabetes) were enrolled. The patients were randomized to either cricoid pressure or no cricoid pressure during induction of anesthesia with endotracheal intubation. Immediately after intubation, a sample of lower airway secretions was collected and analyzed for pepsin A. MAIN RESULTS: A total of 95 patients were evaluated, randomized and completed the study protocol. 46 were randomized to cricoid pressure and 49 to no cricoid pressure. Seven patients crossed-over treatment arms. A total of 18 (19.6%) patients met the pre-defined criteria for microaspiration. In both the intention-to-treat and per-protocol analyses, there were no statistically significant differences in the rate of microaspiration [OR (95% CI)] = 1.39 (0.49-3.92) and 1.30 (0.44-3.86), respectively. CONCLUSIONS: Utilizing pepsin A as a biomarker of aspiration, this pilot clinical trial did not find evidence for a reduced rate of aspiration or adverse clinical events with the administration of cricoid pressure during elective endotracheal intubation of patients with risk factors for microaspiration.

Detection of gastric pepsin in middle ear fluid of children with otitis media.

OBJECTIVE: We sought to confirm the finding of pepsin/pepsinogen in the middle ear fluid of children with otitis media in a larger sample size using a sensitive and specific pepsin assay. STUDY DESIGN AND SETTING: We evaluated 152 children (225 ear samples) in a prospective study at a tertiary care children's hospital. The presence of pepsin in middle ear aspirates was determined using enzymatic assay. RESULTS: Of the patients, 14.4 percent (22 of 152) had detectable pepsin activity in one or both of the ear samples with no pepsin activity detected in control serum. Average pepsin concentration in the samples was 96.6 +/- 170.8 ng/ml, ranging from 13 to 687 ng/ml. Pepsin concentration in the middle ear of children younger than 1.0 year was significantly higher than in older age groups. CONCLUSION AND SIGNIFICANCE: Results indicate that pepsin/pepsinogen is present in the middle ears of children with otitis media, although not at the high rate previously reported. Gastric reflux may be one causative factor in the pathogenesis of otitis media.

The role of gastric pepsin in the inflammatory cascade of pediatric otitis media.

IMPORTANCE: Otitis media is characterized as an ongoing inflammation with accumulation of an effusion in the middle ear cleft. The molecular mechanisms underlying the pathogenesis, particularly the inflammatory response, remain largely unknown. We hypothesize that aspiration of gastric contents into the nasopharynx may be responsible for the initiation of the inflammatory process or aggravate a preexisting condition. OBJECTIVE: To investigate the correlation of gastric pepsin A with inflammatory cytokines, bacterial infection, and clinical outcomes. DESIGN, SETTING, AND PARTICIPANTS: Prospective study of 129 pediatric patients undergoing myringotomy with tube placement for otitis media at a tertiary care pediatric hospital. MAIN OUTCOMES AND MEASURES: Ear samples were tested for pepsin A; cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor; and bacterial culture inoculation. Data were analyzed by descriptive statistics and regression analysis to identify risk factors for the presence of pepsin A and to correlate pepsin A levels with cytokine levels, infection status, and clinical outcomes. RESULTS: Of the 129 patients, 199 ear samples were obtained; 82 samples (41%) and 64 patients (50%) were positive for pepsin A as measured by immunoassay. Pepsin A positivity correlated with age younger than 3.0 years (mean [SD], 2.3 [2.1] years in the positive group vs 3.3 [3.0] years in the negative group) and with all 3 cytokine levels (mean [SD] tumor necrosis factor, 29.5 [45.9] pg/mL in the positive group vs 13.2 [21.6] pg/mL in the negative group; IL-6, 6791.7 [9389.1] pg/mL in the positive group vs 2849.9 [4066.3] pg/mL in the negative group; and IL-8, 6828.2 [8122.3] pg/mL in the positive group vs 2925.1 [3364.5] pg/mL in the negative group [all P < .05]); however, logistic regression analysis showed that only IL-8 (odds ratio, 3.96; 95% CI, 1.3-12.0; P = .02) and age (odds ratio, 3.83; 95% CI, 1.2-12.7; P = .03) were significant independent variables. No statistically significant association was found with other parameters. Multiple linear regressions revealed that the levels of pepsin A were correlated with IL-8 levels (R2 = 0.248; P < .001) and the need for second or third tubes 6 to 12 months after the first (R2 = 0.102; P = .006). The presence of pepsin A in the middle ear was not associated with increased bacterial infection. Interleukin 8 was independent and significantly associated with both pepsin A levels and bacterial infection (R2 = 0.144 and 0.263, respectively; P = .001 for both). CONCLUSIONS AND RELEVANCE: Extraesophageal reflux as indicated by the presence of pepsin A is closely involved in the middle ear inflammatory process and may worsen the disease in some children; however, a proof of cause and effect between extraesophageal reflux and middle ear inflammation requires further investigation.

Super gas barrier and selectivity of graphene oxide-polymer multilayer thin films.

Super gas barrier thin films, fabricated with layer-by-layer assembly of polyethylenimine and graphene oxide, exhibit significantly reduced oxygen and carbon dioxide transmission rates. This thin film's nanobrick wall structure also provides high gas selectivity for hydrogen.

Assessment of the prevalence of microaspiration by gastric pepsin in the airway of ventilated children.

AIM: Mechanically ventilated patients are at risk for aspiration of gastric contents. The aim of this observational study was to determine the prevalence of micro-aspiration in children with cuffed and uncuffed endotracheal (ET) tubes and with tracheostomies and to assess the effect of feeding status on aspiration. Micro-aspiration was determined by measuring gastric pepsin in tracheal aspirates. METHODS: We studied 27 children on ventilators in paediatric intensive care unit (PICU) and 10 children undergoing elective surgeries for various indications. Tracheal aspirates were collected from children on ventilatory support in the intensive care unit during medically indicated suctioning and from the group of children undergoing elective surgery in the operation room. Pepsin was detected by enzymatic assay. RESULTS: Overall 70% of cases in PICU were positive for pepsin in at least one of the aspirates. Pepsin positivity was significantly lower in the cuffed group than in the uncuffed and tracheostomy groups. Tube feedings did not significantly influence the prevalence of pepsin positivity. CONCLUSIONS: Measurement of gastric pepsin in tracheobronchial fluid is a sensitive tool to detect aspirations in mechanically ventilated children and to assess the efficacy of preventive measures in PICU settings.

The role of extraesophageal reflux in otitis media in infants and children.

OBJECTIVES/HYPOTHESIS: Gastroesophageal reflux disease (GERD) is common in children, and extraesophageal reflux disease (EORD) has been implicated in the pathophysiology of otitis media (OM). We sought to 1) determine the incidence of pepsin/pepsinogen presence in the middle ear cleft of a large sample of pediatric patients undergoing myringotomy with tube placement for OM; 2) compare this with a control population of pediatric patients undergoing middle ear surgery (cochlear implantation) with no documented history of OM; 3) analyze potential risk factors for OM in children with EORD demonstrated by the presence of pepsin in the middle ear cleft; and 4) determine if pepsin positivity at the time of myringotomy with tube placement predisposes to posttympanostomy tube otorrhea. STUDY GROUP: prospective samples of 509 pediatric patients (n = 893 ear samples) undergoing myringotomy with tube placement for recurrent acute OM and/or otitis media with effusion in a tertiary care pediatric hospital with longitudinal follow-up of posttympanostomy tube otorrhea. CONTROL GROUP: prospective samples of 64 pediatric patients (n = 74 ears) with negative history of OM undergoing cochlear implantation at one of the three tertiary care pediatric hospitals. A previously validated, highly sensitive and specific modified enzymatic assay was used to detect the presence of pepsin in the middle ear aspirates of study and control patients. Risk factors for OM and potentially associated conditions, including GERD, allergy, and asthma were analyzed for the study group through review of the electronic medical record and correlated topresence of pepsin in the middle ear space. Study patients were followed longitudinally postoperatively to determine the incidence of posttympanostomy tube otorrhea. RESULTS: The incidence of pepsin in the middle ear cleft of the study group was 20% of patients and 14% of ears, which is significantly higher than 1.4% of control patients and 1.5% of control ears (P < .05). Study patients younger than 1 year had a higher rate of purulent effusions and pepsin in the middle ear cleft (P < .05). Patients with pepsin in the middle ear cleft were more likely to have an effusion at the time of surgery than patients without pepsin in the middle ear cleft (P < .05). There was no statistical association found between the presence of pepsin and clinical history of GERD, allergy, asthma, or posttympanostomy tube otorrhea. CONCLUSIONS: Pepsin is detectable in the middle ear cleft of 20% of pediatric patients with OM undergoing tympanostomy tube placement, compared with 1.4% of controls; recovery of pepsin in the middle ear space of pediatric patients with OM is an independent risk factor for OM. Patients under 1 year of age have a higher incidence of purulent effusions and pepsin-positive effusions. Clinical history of GERD, allergy, and asthma do not seem to correlate with evidence of EORD reaching the middle ear cleft. The presence of pepsin in the middle ear space at the time of tube placement does not seem to predispose to posttympanostomy tube otorrhea.

An automated method for the determination of intestinal disaccharidase and glucoamylase activities.

Determination of disaccharidase and glucoamylase activities is important for the diagnosis of intestinal diseases. We adapted a widely accepted manual method to an automated system that uses the same reagents reaction volumes, incubation times, and biopsy size as the manual method. A dye was added to the homogenates as the internal quality control to monitor the pipetting precision of the automated system. When the automated system was tested using human intestinal homogenates, the activities of all the routinely tested disaccharidases, including lactase, maltase, sucrase, and palatinase, as well as the activity of glucoamylase, showed perfect agreement with the manual method and were highly reproducible. The automated analyzer can perform the same routine assays of disaccharidases and glucoamylase with high consistency and accuracy and reduce testing costs by performing a larger sample size with the same number of staff. Additional developments, such as barcoding and built-in plate reading, would result in a completely automated system.

Structural requirements for the inhibitory action of the CD9 large extracellular domain in sperm/oocyte binding and fusion.

CD9 has been shown to be essential for sperm/oocyte fusion in mice, the only non-redundant role found for a member of the tetraspanin family. CD9 can act in cis, reconstituting sperm/oocyte fusion when ectopically expressed in oocytes from CD9 null mice, or in trans, inhibiting sperm fusion when the large extracellular domain (LED) is added to CD9-positive oocytes as a soluble protein. In contrast to cis inhibition, the structural requirements of the trans inhibition by soluble CD9 LED are unknown. Here we show that human CD9 LED is as potent an inhibitor as mouse CD9 LED in mouse sperm/oocyte fusion assays and that CD9 LED can also inhibit sperm/oocyte binding. The two disulphide bridges that define membership of the tetraspanin family are critical for structure and function of human CD9 LED and mutation of a pentapeptide sequence in the hypervariable region further defines the critical region for trans inhibition.

SAMP32, a testis-specific, isoantigenic sperm acrosomal membrane-associated protein.

To identify novel human sperm membrane antigens, we analyzed two-dimensional gels of sperm extracts containing hydrophobic proteins that partitioned into Triton X-114. Four protein spots with isoelectric points (pIs) ranging from 4.5 to 5.5 and apparent molecular weights from 32 to 34 kDa were sequenced by mass spectrometry and found to contain common peptide sequences. Cloning the corresponding cDNA revealed that these protein spots were products of a single gene (SAMP32), encoding a protein of 32 kDa with a predicted pI of 4.57. SAMP32 has a potential transmembrane domain in the carboxyl terminus and is phosphorylated in vivo on serine 256. Northern blotting of eight human tissues and RNA dot blotting of 76 human tissues showed that SAMP32 expression was testis specific. SAMP32 contained an amino terminal domain homologous to the major malarial circumsporozoite surface protein and a domain similar to that of Krp1 from Schizosaccharomyces pombe in its carboxyl terminus. The SAMP32 locus consists of seven exons on chromosome 6q15-16.2. Antiserum against recombinant SAMP32 recognized protein spots originally cored from a two-dimensional gel. This antiserum strongly stained the equatorial segment and faintly stained the acrosome cap of ejaculated human spermatozoa by immunofluorescence. Immunoelectron microscopy showed that SAMP32 was associated with the inner acrosomal membrane in the principal and the equatorial segments of the sperm acrosome. By immunostaining enzyme-dissociated testicular cells, SAMP32 was localized to Golgi phase round spermatids and subsequent stages of acrosome biogenesis. Recombinant SAMP32 reacted with serum from an infertile man, suggesting that it is isoantigenic. Antibodies against recombinant SAMP32 inhibited both the binding and the fusion of human sperm to zona-free hamster eggs.

SLLP1, a unique, intra-acrosomal, non-bacteriolytic, c lysozyme-like protein of human spermatozoa.

We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by beta-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P < or = 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, approximately 1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.

Role of multiple beta1 integrins in cell adhesion to the disintegrin domains of ADAMs 2 and 3.

ADAM disintegrin domains can support integrin-mediated cell adhesion. However, the profile of which integrins are employed for adhesion to a given disintegrin domain remains unclear. For example, we suggested that the disintegrin domains of mouse sperm ADAMs 2 and 3 can interact with the alpha6beta1 integrin on mouse eggs. Others concluded that these disintegrin domains interact instead with the alpha9beta1 integrin. To address these differing results, we first studied adhesion of mouse F9 embryonal carcinoma cells and human G361 melanoma cells to the disintegrin domains of mouse ADAMs 2 and 3. Both cell lines express alpha6beta1 and alpha9beta1 integrins at their surfaces. Antibodies to the alpha6 integrin subunit inhibited adhesion of both cell lines. An antibody that recognizes human alpha9 integrin inhibited adhesion of G361 cells. VLO5, a snake disintegrin that antagonizes alpha4beta1 and alpha9beta1 integrins, potently inhibited adhesion of both cell lines. We next explored expression of the alpha9 integrin subunit in mouse eggs. In contrast to our ability to detect alpha6beta1, we were unable to convincingly detect alpha9beta1 integrin on the surface of mouse eggs. Moreover, treatment of mouse eggs with 250 nm VLO5, which is 250 fold over its approximately IC(50) for inhibition of somatic cell adhesion, had minimal effect on sperm-egg binding or fusion. We did detect alpha9 integrin protein on epithelial cells of the oviduct. Additional studies showed that antibodies to the alpha6 and alpha7 integrins additively inhibited adhesion of mouse trophoblast stem cells and that an antibody to the alpha4 integrin inhibited adhesion of MOLT-3 cells to these disintegrin domains: Our data suggest that multiple integrins (on the same cell) can participate in adhesion to a given ADAM disintegrin domain and that interactions between ADAMs and integrins may be important for sperm transit through the oviduct.

ePAD, an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets.

Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.