Cytosolic phospholipase A2 (cPLA2) IVA as a potential signature molecule in cigarette smoke condensate induced pathologies in alveolar epithelial lineages.

BACKGROUND: Smoking is one of the leading causes of millions of deaths worldwide. During cigarette smoking, most affected and highly exposed cells are the alveolar epithelium and generated oxidative stress in these cells leads to death and damage. Several studies suggested that oxidative stress causes membrane remodeling via Phospholipase A2s but in the case of cigarette smokers, mechanistically study is not yet fully defined. In view of present perspective, we evaluated the involvement of cytosolic phospholipase A2 (cPLA2) IVA as therapeutic target in cigarette smoke induced pathologies in transformed type I and type II alveolar epithelial cells. METHODS: Transformed type I (WI26) and type II (A549) alveolar epithelial cells were used for the present study. Cigarette smoke condensate (CSC) was prepared from most commonly used cigarette (Gold Flake with filter) by the Indian population. CSC-induced molecular changes were evaluated through cell viability using MTT assay, reactive oxygen species (ROS) measurement using 2,7 dichlorodihydrofluorescin diacetate (DCFH-DA), cell membrane integrity using fluorescein diacetate (FDA) and ethidium bromide (EtBr) staining, super oxide dismutase (SOD) levels, cPLA2 activity and molecular involvement of specific cPLA2s at selected 24 h time period. RESULTS: CSC-induced response on both type of epithelial cells shown significantly reduction in cell viability, declined membrane integrity, with differential escalation of ROS levels in the range of 1.5-15 folds and pointedly increased cPLA2 activity (p < 0.05). Likewise, we observed distinction antioxidant potential in these two types of lineages as type I cells had considerably higher SOD levels when compared to type II cells (p < 0.05). Further molecular expression of all cPLA2s increased significantly in a dose dependent manner, specifically cytosolic phospholipase A2 IVA with maximum manifestation of 3.8 folds. Interestingly, CSC-induced ROS levels and cPLA2s expression were relatively higher in A549 cells as compared to WI26 cells. CONCLUSIONS: The present study indicates that among all cPLA2s, specific cPLA2 IVA are the main enzymes involved in cigarette smoke induced anomalies in type I and type II lung epithelial cells and targeting them holds tremendous possibilities in cigarette smoke induced lung pathologies.

Comparison of encapsulated versus nonencapsulated (14) C-urea breath test for the detection of Helicobacter pylori infection: a scintigraphy study.

BACKGROUND AND AIMS: (14) C-urea breath test ((14) C-UBT) is considered as "gold standard" for detection of active gastric H. pylori infection. However, till date no comparative study using encapsulated and non-encapsulated (14) C-UBT protocols has been conducted in same subjects in identical conditions. We monitored gastric fate of capsule containing (14) C-urea with real time display and compared sensitivities of these protocols at different time points of breath collection. METHODS: Non-encapsulated (14) C-UBT was performed using 74 kBq of (14) C-urea in 100 dyspeptic patients by collecting breath samples at 10, 15 and 20 minutes. Thereafter, within 2 days a gelatin capsule containing (14) C-urea along with 6.0 MBq of (99m) Tc-diethylene triamine penta-acetic acid was administered to each patient for real time display of capsule movement and its fate in gastrointestinal tract by gamma camera. Simultaneously, breath samples were collected for (14) CO2 measurement during image acquisition. RESULTS: Employing non-encapsulated (14) C-UBT, 74 out of 100 dyspeptic patients were found to be H. pylori positive. Discordant (14) C-UBT results were obtained in 4/74 (5.4%) cases using these two protocols. By employing encapsulated and nonencapsulated (14) C-UBT protocols, sensitivities of (14) C-UBT were found to be 90.5 versus 98.6% at 10 and 91.8 versus 97.2% at 15 minutes respectively; while these were 94.6 versus 100, 90.7 versus 98.6 and 83.7 versus 93.2% considering any one, two or all three positive values respectively. CONCLUSIONS: Incomplete/non-resolution of (14) C-urea capsule in stomach during the phase of breath collections appears to decrease sensitivity of encapsulated (14) C-UBT as compared to nonencapsulated protocol for detection of H. pylori infection.

Superiority of non-capsulated 14C-urea breath test over capsule based method for detection of Helicobacter pylori infection - a preliminary report.

BACKGROUND: 14C-urea breath test (14C-UBT) is employed as a 'gold standard' technique for the detection of active gastric Helicobacter pylori infection and is recommended as the best option for "test-and-treat" strategy in primary health care centers. AIM: To compare the performance of capsulated and non-capsulated 14C-UBT protocols for the detection of H. pylori infection in patients. METHODS: Fifty eight H. pylori infected patients underwent routine upper GI endoscopy and biopsies were processed for rapid urease test (RUT) and histopathology examination. Capsulated 14C-UBT was done in a novel way by using 74 kBq of 14C-urea along with 6.0 MBq of 99mTc-diethylene triamine penta-acetic acid (99mTc-DTPA) to simultaneously monitor the movement and the fate of ingested capsule after delineating the stomach contour by using 20.0 MBq of 99mTechnetium pertechnetate (99mTcO4-) under dual head gamma camera. Non-capsulated 14C-UBT was performed within 2 days of the previous test and the results of these protocols were compared. RESULTS: In 3 out of 58 H. pylori positive cases (5.17%), 14C-UBT results were found to be negative by using the capsulated method. Interestingly, on monitoring the real time images of the capsule in these cases it was found that misdiagnosis of H. pylori infection occurred mainly due to either rapid transit of the 14C-urea containing capsule from the upper gastric tract or its incomplete resolution in the stomach during the phase of breath collection. CONCLUSION: Use of non-capsulated '4C-UBT protocol appears to be a superior option than the conventional capsule based technique for the detection of H. pylori infection.

14C-urea breath test is safe for pediatric patients.

Helicobacter pylori (H. pylori) infection, quite prevalent in the developing countries, is considered to be one of the causative factors for various gastric pathologies and other nongastric diseases. It is believed that H. pylori infection is almost always acquired in early childhood and persists throughout life unless specific treatment is given. The (13/14)C-urea breath test (UBT) is now considered to be a 'gold standard' technique for the detection of H. pylori infection. However, because of the lack of facilities and high cost, the preferred nonradioactive ¹³C-UBT cannot be performed on pediatric patients in developing countries, whereas the radioactive ¹4C-UBT is not used on children because of the fear of radiation exposure. When using 37 kBq (1 µCi) of ¹4C-urea for the ¹4C-UBT, the patient is not exposed to more radiation than is acquired from the natural environment in one day, as almost all the ingested radioactivity is excreted from the body (urine and breath) within 72-120 h. This article reviews the importance of the ¹4C-UBT for the detection of H. pylori and justifies the radiation safety aspects of its use in children without any fear of 'radiation phobia' where the facility for ¹³C-UBT is lacking.

Involvement of various molecular events in cellular injury induced by smokeless tobacco.

Smokeless tobacco (ST) consumption is implicated in the pathogenesis of oral diseases, including cancer. However, its pathological effect in other organs is not well understood. In the present study, the effect of aqueous extract of smokeless tobacco (AEST) prepared from "gutkha" (a form of ST) on the xenobiotic drug-metabolizing enzymes, histopathological changes, and damage to the genetic material in lung, liver, and kidney of rats was evaluated. Animals were orally administered AEST at a low dose (L-AEST, 96 mg/kg body wt/day) for 2 (L-AEST(2)) and 28 weeks (L-AEST(28)) and at a high dose (H-AEST, 960 mg/kg body wt/day) for 2 weeks (H-AEST(2)). Real-time PCR and immunohistological studies showed that administration of L-AEST(2) did not induce the expression of phase I cytochrome P450s (CYP1A1, 1A2, and 2E1) and phase II mu-glutathione-s-transferase (GST-mu) drug-metabolizing enzymes in lung, liver, and kidney. Although H-AEST(2) administration significantly induced both gene and protein expression of CYP1A1, 1A2, and 2E1 in all of the above organs, it mildly expressed the phase II detoxifying enzyme, GST-mu, in type I and type II epithelial cells of lung and in proximal tubular cells of kidney. L-AEST(28) enhanced the gene and protein expression of CYP1A1, 1A2, and 2E1 in lung, liver, and kidney in a differential manner and induced the expression of GST-mu in lung and kidney. L-AEST(28) induced the micronuclei formation in the peripheral blood mononuclear cells, TNF-alpha in plasma, and myeloperoxidase activity in the organs. L-AEST(28) significantly enhanced Bax, p53, and NF-kappaB and decreased Bcl-2 gene expressions differentially in an organ-specific manner. The differential changes in these organs due to AEST might be due to their different physiological functions and variable sensitivities toward the metabolites of AEST, which create a microenvironment favorable for AEST-induced pathogenesis. This study broadens the insight into the different molecular mechanisms in various organs, which appear to be deregulated due to AEST. Understanding these processes may help in clinical treatment planning strategies for tobacco-related diseases.

Targeting the vicious inflammation-oxidative stress cycle for the management of heart failure.

Oxidative stress and inflammation are each implicated independently in the development and progression of heart failure. Their interaction, however, is also evident throughout the process from initial injury to cardiac remodeling and failure. In the failing heart, the linkage between excessive reactive oxygen species (ROS) and the cytokine elaboration is manifested in shared elements and cross-promotion within downstream signaling pathways. In spite of this, the failure of anticytokine immunotherapy and antioxidant therapy, which had previously shown promise, suggests that a more complete perspective of ROS-cytokine interaction is required. The present review focuses on two of the major cytokines that are demonstrably connected to oxidative stress--the pro-inflammatory tumor necrosis factor-alpha (TNF-alpha) and the anti-inflammatory interleukin-10 (IL-10)--and their interactions in cardiac remodeling and failure. It is proposed that an optimal balance between TNF-alpha and IL-10 may be of crucial importance in mitigating both inflammation and oxidative stress processes leading to heart failure.

Kinetics of 14carbon dioxide excretion from 14C-urea by oral commensal flora.

BACKGROUND AND AIM: Previous studies have shown that while performing the (14)C-urea breath test ((14)C-UBT) for the detection of Helicobacter pylori (H. pylori), there is possibility of false-positive results due to the other urease producing bacteria present in oropharynx, if breath samples are obtained within 30 min after administration of non-capsulated (14)C-urea. Therefore, we have exclusively evaluated the kinetics of (14)carbon dioxide ((14)CO(2)) excretion by oral commensal flora to theoretically propose optimum breath collection timings for (14)C-UBT. METHODS: Multiple breath samples up to 15 min were collected in 0.25 mmol benzethonium hydroxide from 25 healthy volunteers after they withheld 37 kBq (1 muCi) of (14)C-urea in their mouths for 15 s and then expectorated the tracer. The test was repeated on the same subjects without and with mouth cleansing protocols. Breath (14)CO(2) content was measured by the Liquid Scintillation Counter (1409; Wallac, Turku, Finland) and results were expressed as (14)CO(2) excretion per mmol breath CO(2) (% administered dose). RESULTS: Peak breath radioactivity at 1 min in the former protocol was 3.53 times higher than the latter which declined subsequently with a half time of 1 min and 2.5 min, and reached baseline levels by 15 and 10 min, respectively. The peak radioactivity (100%) at 1 min declined by 94% and 97.8% in the former and later protocols, respectively, at 15 min. Although magnitude of the peak varied in different subjects, the shape of curve remained almost similar in all cases. CONCLUSIONS: Without mouth cleansing, oral micro flora excreted more (14)CO(2) up to 15 min after administration of non-capsulated (14)C-urea. Therefore, it is proposed that two breath samples may be obtained either at 15 and 20 min without or at 10 and 15 min with mouth cleansing protocols for reliable analysis of (14)C-UBT data for H. pylori detection.

Changes in gastric environment with test meals affect the performance of 14C-urea breath test.

BACKGROUND: (14)C-urea breath test (UBT) is considered to be an accurate diagnostic test for the detection of active Helicobacter pylori infection. Various test meals are used in (14)C-UBT to slow down gastric emptying, and to enhance the gastric distribution, in order to increase the time and area of contact between microorganisms and the tracer substrate. The aim of the present paper was to evaluate the effect of gastric environment on the performance of (14)C-UBT using an alkaline and an acidic liquid test meal having gastric emptying retardant effect. METHODS: The comparison of (14)C-UBT was done with liquid test meals (200 mL water) comprising (i) plain drinking water (PDW); (ii) 1.3 g or 3.0 g citric acid (CA); and (iii) 3.0 g trisodium citrate (TSC). Eighteen patients (37 +/- 12 years, range 18-57 years) with complaints of dyspepsia participated in the study. The status of H. pylori was confirmed by histology and rapid urease test. A total of 93 kBq of (14)C-urea (0.5 mL) in a gelatin capsule was orally administered along with liquid test meals to the overnight fasting subjects. Breath samples were collected and radioactivity measured. Results were expressed as (14)CO(2)/mmol exhaled CO(2) as percentage of administered radioactive urea. RESULTS: Higher acidic gastric environment (pH approx. 2.0) with CA was found to increase the exhaled (14)CO(2) level in a dose-dependent manner as compared to PDW and TSC meal (P < 0.05) at all time points. With TSC test meal, the expired (14)CO(2) level decreased in the lower acidic gastric environment (pH approx. 5.3). The peaks of exhaled (14)CO(2) with TSC test meal were observed at the same time points as that with PDW and CA test meals. The (14)C-UBT with TSC was found to be positive in 77% of patients (10/13). CONCLUSION: Better interaction between the microbial urease and (14)C-urea, caused by a test meal that retards gastric emptying and that changes gastric pH, plays an important role in hydrolysis of the administered (14)C-urea by H. pylori urease.