Chris D. St. Laurent

VSL#3® probiotic therapy does not reduce portal pressures in patients with decompensated cirrhosis

In patients with decompensated cirrhosis, bacterial translocation can contribute to splanchnic vasodilatation, decreased effective circulating volume, and portal hypertension. The primary objective of this randomized, double blind placebo controlled trial was to evaluate the effect of the probiotic VSL#3® on the hepatic venous pressure gradient (HVPG).

Regulation of inflammation by Rac2 in immune complex-mediated acute lung injury

Acute lung injury (ALI) is an inflammatory disorder associated with recruitment and activation of neutrophils in lungs. Rac2, a member of the Rho GTPase subfamily, is an essential regulator of neutrophil degranulation, superoxide release, and chemotaxis. Here, we hypothesized that Rac2 is important in mediating lung injury. Using a model of IgG immune complex-mediated ALI, we showed that injury was attenuated in rac2(-/-) mice compared with wild-type (WT) mice undergoing ALI, with significant decreases in alveolar leukocyte numbers, vascular leakage, and the inflammatory mediators, myeloperoxidase (MPO) and matrix metalloproteinases (MMPs). Reduced injury in rac2(-/-) mice was not associated with diminished cytokine and chemokine production, since bronchoalveolar lavage (BAL) levels of IL-17, TNF, CCL3, CXCL1, and CXCL2 were similarly increased in WT and rac2(-/-) mice with ALI compared with sham-treated mice (no ALI). BAL levels of MMP-2 and MMP-9 were significantly decreased in the airways of rac2(-/-) mice with ALI. Immunohistochemical analysis revealed that MMP-2 and MMP-9 expression was evident in alveolar macrophages and interstitial neutrophils in WT ALI. In contrast, MMP-positive cells were less prominent in rac2(-/-) mice with ALI. Chimeric mice showed that Rac2-mediated lung injury was dependent on hematopoietic cells derived from bone marrow. We propose that lung injury in response to immune complex deposition is dependent on Rac2 in alveolar macrophages and neutrophils.

Limited replication of influenza A virus in human mast cells

Mast cells are important in innate immunity and protective against certain bacterial infections. However, there is limited evidence that mast cells respond to viruses. As mast cells are abundant in mucosal tissues of the lung, they are in a prime location to detect and respond to influenza virus. In this study, we characterized for the first time the replication cycle of influenza A virus in human mast cells by measuring influenza A virus transcription, RNA replication, protein synthesis, and formation of infectious virus as compared to the replication cycle in epithelial cells. We detected the presence of influenza A viral genomic RNA transcription, replication, and protein synthesis in human mast cells and epithelial cells. However, there was no significant release of infectious influenza A virus from mast cells, whereas epithelial cells produce ~100-fold virus compared with the inoculating dose. We confirmed that influenza A virus infects human mast cells, begins to replicate, but the production of new virus is aborted. Thus, mast cells may lack critical factors essential for productive infection or there are intrinsic or inducible anti-influenza A mechanisms in mast cells.

Expression of nitric oxide synthases in leukocytes in nasal polyps

BACKGROUND Nitric oxide (NO) has various roles in airway physiology and pathophysiology. Monitoring exhaled NO levels is increasingly common to measure airways inflammation and inhaled NO studied for its therapeutic value in premature infants and adult respiratory distress syndrome. NO is produced by 3 isoforms of NO synthase (NOS1, 2, 3), and each can play distinct and perhaps overlapping roles in the airways. However, the distribution, regulation, and functions of NOS in various cells in the upper airways, particularly in leukocytes, are incompletely understood. OBJECTIVE To characterize the expression of NOS isoforms in leukocytes in normal middle turbinate tissues (MT) and in inflammatory nasal tissue (nasal polyps, NP). METHODS Normal MT tissue was collected from surgical specimens that were to be discarded. The NP samples were from surgical tissue archives of 15 patients with chronic rhinosinusitis. Isoforms of NOS in cells were identified by double immunostaining using NOS isoform-specific and leukocyte-specific (mast cell, eosinophil, macrophage, neutrophil, or T cell) antibodies. RESULTS The proportion of total cells below the epithelium that were positive for each isoform of NOS was higher in NP than in MT. Each isoform of NOS was found in all leukocyte populations studied, and there were significant differences in the percentage of leukocytes expressing NOS isoforms between MT and NP. CONCLUSION All isoforms of NOS are expressed in leukocytes in MT and NP, and their expression varies among leukocyte types. Our data provide a basis to investigate the regulation, cell distribution, and distinct functions of NOS isoforms in normal and inflamed nasal tissues.

Metabolomic profiling of bronchoalveolar lavage fluids by isotope labeling liquid chromatography mass spectrometry: a promising approach to studying experimental asthma

Metabolomic analysis of bronchoalveolar lavage fluid (BALF) may help understand the pathophysiology of pulmonary diseases such as asthma. However, a major analytical challenge is that most metabolites in BALF are in low abundance and further diluted by the collection procedure. Here we report a sensitive metabolomic profiling method based on 12C-/13C-differential isotope dansylation that targets amine- and phenol-containing metabolites, solid phase extraction for analyte enrichment, and liquid chromatography Fourier transform ion cyclotron resonance-mass spectrometry (LC–FTICR-MS). This method detected 250 ion pairs or putative metabolites in rat BALF and 36 of them were positively identified. The majority were not reported in previous studies using NMR or conventional LC–MS. The developed isotope labeling method was then applied to investigate metabolomic changes in BALF samples from a model of allergic asthma in rats. Statistical analysis of the resultant data showed that there was distinct separation between normal and inflamed rats. Metabolic pathway analysis indicated that the arginine-proline metabolic pathway was dysregulated in rats with experimental asthma.

Measurement of Nitric Oxide in Mast Cells with the Fluorescent Indicator DAF-FM Diacetate

The production of nitric oxide in mast cells has been difficult to measure due to the low amounts made by mast cells, as well as limitations in the specificity and sensitivity of the assays available. We present here a sensitive and specific 96-well plate-based method to directly measure NO using the cell-permeable fluorescent compound DAF-FM diacetate.

Calcium-Binding Protein, Spermatid-Specific 1 is Expressed in Human Salivary Glands and Contains an Anti-inflammatory Motif

Salivary glands are involved in the production and exocrine and endocrine secretion of biologically active proteins, polypeptides, and hormones involved in growth and differentiation, homeostasis, and digestion. We have previously studied the prohormone submandibular rat 1 (SMR1), product of the Vcsa1 gene, which is highly expressed in the testes and salivary glands of rats, and can be cleaved to produce polypeptides with analgesic, erectile function, and anti-inflammatory activities. Humans lack the Vcsa1 gene, but homologous sequences and functions for analgesia and erectile function exist in the human genes Prol1, SMR3a, and SMR3b located on the human chromosomal region close to where Vcsa1 lies in the rat. Here we show the human protein calcium-binding protein spermatid-specific 1 (CABS1) contains a similar sequence to the anti-inflammatory sequence in rat SMR1, thus CABS1 may be another human gene with homologous function to Vcsa1. Using Western blot and PCR, we discovered that the human protein CABS1, previously thought to only be expressed in the testes, is also expressed in the salivary glands and lung, in a tissue-specific manner. Peptides derived from CABS1 were tested in an in vivo mouse model of lipopolysaccharide (LPS)-induced neutrophilia and an ex vivo rat model of antigen-induced intestinal anaphylaxis and significantly reduced both neutrophil accumulation in bronchoalveolar lavage fluid and antigen-induced ileal contractions, respectively. Thus human CABS1 has a peptide motif homologous to the anti-inflammatory peptide sequence of rat SMR1. Whether this similarity of CABS1 extends to the neuroendocrine regulation of the anti-inflammatory activity seen for SMR1 remains to be determined.

A novel biomarker associated with distress in humans: calcium-binding protein, spermatid-specific 1 (CABS1)

Calcium-binding protein spermatid-specific 1 (CABS1) is expressed in the human submandibular gland and has an anti-inflammatory motif similar to that in submandibular rat 1 in rats. Here, we investigate CABS1 in human saliva and its association with psychological and physiological distress and inflammation in humans. Volunteers participated across three studies: 1) weekly baseline measures; 2) a psychosocial speech and mental arithmetic stressor under evaluative threat; and 3) during academic exam stress. Salivary samples were analyzed for CABS1 and cortisol. Additional measures included questionnaires of perceived stress and negative affect; exhaled nitric oxide; respiration and cardiac activity; lung function; and salivary and nasal inflammatory markers. We identified a CABS1 immunoreactive band at 27 kDa in all participants and additional molecular mass forms in some participants. One week temporal stability of the 27-kDa band was satisfactory (test-retest reliability estimate = 0.62-0.86). Acute stress increased intensity of 18, 27, and 55 kDa bands; 27-kDa increases were associated with more negative affect and lower heart rate, sympathetic activity, respiration rate, and minute ventilation. In both acute and academic stress, changes in 27 kDa were positively associated with salivary cortisol. The 27-kDa band was also positively associated with VEGF and salivary leukotriene B4 levels. Participants with low molecular weight CABS1 bands showed reduced habitual stress and negative affect in response to acute stress. CABS1 is readily detected in human saliva and is associated with psychological and physiological indicators of stress. The role of CABS1 in inflammatory processes, stress, and stress resilience requires careful study.

Expression of nitric oxide synthase in nasal polyps

Background Nitric oxide (NO) is a short-lived, reactive molecule generated by nitric oxide synthase (NOS). Three isoforms of NOS have been identified including: neuronal NOS (NOS1), endothelial NOS (NOS3) and inducible NOS (NOS2). Objective To identify expression of isoforms of NOS in nasal polyps (NP) and normal nasal tissue, and to determine if differences exist in NOS expression in NP rich in eosinophils (Eo-hi) or with few eosinophils (Eo-low). Methods NP were obtained after endoscopic surgery for chronic rhinosinusitis, and separated into two categories based on eosinophil density. Nasal middle turbinates (MT) were also collected as normal controls from patients who had undergone surgery for pituitary adenomas. To identify cell types expressing isoforms of NOS, double immunostaining was performed using anti-NOS and anti-leukocyte antibodies (e.g. mast cells [MC], eosinophils, T cells and macrophages). Results Expression of NOS isoforms in all cell types was greater in NP than in MT. Number of NOS2 positive cells in Eo-hi NP was higher than in Eo-low NP. However, there were no differences in the numbers of NOS1 and NOS3 positive cells between Eo-hi and Eo-low NP. Both NOS2 positive MC and NOS2 positive Eo were significantly greater in NP than in MT. In addition, the percentage of NOS2 positive MC in Eo-hi NP was significantly higher than in Eo-low NP. Conclusion Elevation in NOS2 expression in several cell types might be an important factor in the life history of NP, especially those with an abundance of eosinophils.