Approaches for targeted proteomics and its potential applications in neuroscience.

An extensive guide on practicable and significant quantitative proteomic approaches in neuroscience research is important not only because of the existing overwhelming limitations but also for gaining valuable understanding into brain function and deciphering proteomics from the workbench to the bedside. Early methodologies to understand the functioning of biological systems are now improving with high-throughput technologies, which allow analysis of various samples concurrently, or of thousand of analytes in a particular sample. Quantitative proteomic approaches include both gel-based and non-gel-based methods that can be further divided into different labelling approaches. This review will emphasize the role of existing technologies, their advantages and disadvantages, as well as their applications in neuroscience. This review will also discuss advanced approaches for targeted proteomics using isotope-coded affinity tag (ICAT) coupled with laser capture microdissection (LCM) followed by liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis. This technology can further be extended to single cell proteomics in other areas of biological sciences and can be combined with other ‘omics’ approaches to reveal the mechanism of a cellular alterations. This approach may lead to further investigation in basic biology, disease analysis and surveillance, as well as drug discovery. Although numerous challenges still exist, we are confident that this approach will increase the understanding of pathological mechanisms involved in neuroendocrinology, neuropsychiatric and neurodegenerative disorders by delivering protein biomarker signatures for brain dysfunction.

Association between gastric mucosal glutathione-S-transferase activity, glutathione-S-transferase gene polymorphisms and Helicobacter pylori infection in gastric cancer.

Aim Helicobacter pylori infection, though common, leads to gastric cancer (GC) in less than 1% individuals, suggesting the role of host factors. We previously reported the role of glutathione–S–transferase (GST) polymorphisms, the gene encoding a carcinogen–detoxifying enzyme, in GC. This study was aimed to evaluate GST enzyme activity, GST polymorphism, glutathione (GSH) levels and H. pylori in patients with GC. Methods GST and GSH levels were estimated in gastric biopsies of 52 patients with GC, 37 functional dyspepsia (FD) and 39 peptic ulcer (PU), and correlated with H. pylori (ELISA) infection and GST polymorphisms. GST polymorphisms were separately analyzed in relationship to H. pylori in 82 GC, 72 FD, 53 PU and 89 healthy controls (HC). Results GST activity was lower in patients with GC in comparison to PU (p=0.03), but GSH levels were comparable. GSTT1 null genotype (GSTT1*0) and simultaneous deletion of both GSTT1 and GSTM1 genes was associated with lower enzyme activity (p=0.02 and 0.01, respectively). GST and GSH levels in H. pylori positive and negative patients with GC, FD and PU were comparable. Presence of H. pylori infection along with GSTT1*0 (p= 0.006) and GSTM1*0 (p=0.05) was associated with lower enzyme activity. GSTT1*0 was associated with higher odds ratio (OR) of GC in presence of H. pylori (GC vs. HC: p=0.02, OR 2.6 [95% CI=1–6] vs. p=0.7, 1.3 [0.4– 5.0]; GC vs. PU: p=0.04, OR 3 [95% CI=1–9] vs. not applicable (OR could not be computed as frequency of GSTT1*0 in H. pylori negative patients with PU was zero)]. Conclusions GC is associated with reduced GST activity. Odds ratio of GC associated with GSTT1*0 is enhanced in presence of H. pylori probably due to combined effect of both on enzyme activity.

Serological assessment of gastric intestinal metaplasia and atrophy using pepsinogen-I, pepsinogen-II and gastrin-17 levels in a low incidence area of gastric cancer endemic for H. pylori infection.

Background: Intestinal metaplasia (IM), a precursor of gastric cancer (GC), may be amenable to non-invasive assessment. Aims:We evaluated the diagnostic utility of serum PG-I, PG-II, PG-I/PG-II ratio and gastrin-17 (G-17) to detect IM and atrophy. Methods: The study was conducted at a tertiary care center located in a low-incidence area of GC, endemic for H. pylori infection. Patients with GC and dyspepsia were evaluated by endoscopy, histology for IM (H&E, PAS and Alcian blue stains), gastritis and H. pylori (H&E and Giemsa stains) infection, which was considered to be present if two of three tests (rapid urease test, IgG antibody and histology) were positive. Serum levels of PG-I, PG-II and G-17 were estimated using ELISA. Results: Of 98 patients with GC and 62 with dyspepsia, 35 (36%) and 9 (14%) had IM, respectively (p=0.004). Patients with IM (n=44) had lower PG-I/PG-II ratio than those without IM (n=116; median 4.4, 0.37-23.6 vs. 6.3, 0.19-38.6, respectively; p=0.005). A cut-off value of PG-I/PG-II ratio of 6.0 had 64% sensitivity and 52% specificity for detecting IM (area under ROC curve 0.64). 26/44 (60%) patients with IM and 52/98 (53%) with GC had PG-I/PG-II ratio <6. Serum G-17 was comparable among patients with and without IM. Conclusions: Though PG-I/PG-II ratio was lower in patients with IM, only 60% had a lower ratio suggesting that this test and G-17 may not be useful to detect IM in a low-incidence area of GC, endemic for H. pylori infection.

Genetic factors in the pathogenesis of gastroesophageal reflux disease.

Multiple factors play a role in the pathogenesis of gastroesophageal reflux disease (GERD). Two landmark studies showing higher concordance of disease in monozygotic than dizygotic twin pairs suggested the role of host genetic factors in its pathogenesis. Recent studies have shown that genetic polymorphism in genes influencing host’s inflammatory response, drug metabolism, cell cycle regulation, xenobiotic pathways, DNA repair, mutagenesis, esophageal sensory function and gene silencing are associated with risk of GERD and its sequelae—Barrett’s esophagus and esophageal adenocarcinoma. However, more studies on larger sample size are needed before reaching a definite conclusion on the role of an individual gene.

Patients with Helicobacter pylori infection have less severe gastroesophageal reflux disease: a study using endoscopy, 24-hour gastric and esophageal pH metry.

Background and aim The relationship between gastroesophageal reflux disease (GERD) and Helicobacter pylori is controversial. We evaluated endoscopic, 24-h gastric and esophageal acid profile among patients with GERD in relation to H. pylori, as the latter might alter gastric acid secretion. Methods Patients with GERD (n=123), who were not on acid-suppressive drugs, and had not received anti-H. pylori therapy, underwent gastroduodenoscopy and tests for H. pylori detection. Esophageal manometry, 24-h pH metry, serum pepsinogen-I (PG-I), PG-II and gastrin-17 ELISA were done in all these patients. Univariate and multivariate analyses were performed to assess independent predictors for erosive esophagitis (EE). Results Of 123 patients (mean age 40.5 [13.1] years, 85 [69.1%] men), 59 (47.9%) had H. pylori infection. EE was more common in H. pylori non-infected than infected (49 vs. 32, p<0.001). Among patients older than 40 years, absence of H. pylori was associated with lower esophageal pH and longer reflux (p=0.02 and p<0.001, respectively). PG-I/PG-II ratio was lower in H. pylori infected subjects (p <0.001). In patients with higher LA grade of esophagitis, elevated PG-I levels and PG-I/PG-II ratio were associated with more acidic stomach (p=0.04 and p=0.01, respectively). Multivariate analyses showed low gastrin-17 (p=0.016), higher age (p=0.013), hiatus hernia (p=0.004) and absence of H. pylori (p=0.03) were independent predictors for risk of EE. Conclusion H. pylori infection is associated with less acidic stomach and less severe GERD. Low gastrin-17, higher age, hiatus hernia and absence of H. pylori were the best predictors for EE risk.

Manometric and symptomatic spectrum of motor dysphagia in a tertiary referral center in northern India.

Background We studied the spectrum of motor dysphagia in a northern Indian tertiary referral center. Methods In this retrospective study, consecutive patients with motor dysphagia referred to the Gastrointestinal Pathophysiology and Motility Laboratory from 2002 to 2007 were evaluated clinically and with eight-channel water-perfusion manometry. Causes of dysphagia were diagnosed using standard criteria. Results Of 250 patients (age 41.3 [15.0] years, 146 men), 193 (77%) had achalasia cardia (AC) and 57 (23%) had other causes (11, 4.4%: diffuse esophageal spasm [DES]; 9, 3.6%: hypertensive lower esophageal sphincter [Hy LES]); manometry was normal in 37 patients. Twenty-seven patients (14%) had vigorous AC. Duration of dysphagia at presentation was longer in those with AC and Hy LES than in normal manometry (NM) (21 months [1–180] vs. 6 [1–360], p = 0.000; 24 months [7–48] vs. 6 [1–360], p = 0.015). Regurgitation and bolus obstruction were more frequent in those with AC than in NM (89/154, 57.79% vs. 3/27, 11.11%, p = 0.000001). Heartburn was less frequent in patients with AC than in others (AC: 4/146, 2.73% vs. normal: 4/27, 14.8% [p = 0.02] and others: 3/15, 20% [p = 0.018]). Chest pain was reported by 74/135 (54.8%) classic and 12/19 (63.2%) vigorous AC (p = NS). Patients with NM had lower LES pressure than those with classic AC, Hy LES and vigorous AC (p < 0.0001 in each case). Patients with DES had lower LES pressure than in classic AC, Hy LES and vigorous AC (p = 0.043, p < 0.0001, and p = 0.002, respectively). Patients with classic AC had lower LES pressure than in Hy LES and vigorous AC (p = 0.024, p = 0.001, respectively). Conclusion Classic AC was the commonest cause of motor dysphagia in our center. AC was associated with higher LES pressure, longer duration of dysphagia, frequent regurgitation and bolus obstruction.

Lactose hydrogen breath test versus lactose tolerance test in the tropics: Does positive lactose tolerance test reflect more severe lactose malabsorption.

Background: We compared the lactose hydrogen breath (lactose HBT) and lactose tolerance tests (LTT) in their ability to diagnose lactose malabsorption (LM). Methods: Fasting and post-lactose (50 g) breath hydrogen and blood sugar were tested in patients with irritable bowel syndrome (IBS). Persistent rise in breath hydrogen by 20 ppm and failure of blood sugar to rise by >20 mg/dL above basal level, were considered positive lactose HBT and LTT, respectively. Symptoms of diarrhoea, bloating, abdominal pain and flatulence were noted. Results: Of 203 patients, 11 demonstrated high basal breath hydrogen and hence, 192 (age 37±14 years, 134 male) were included in the study. 125 (65%) and 137 (71%) were lactose HBT and LTT positive, respectively. 102/125 lactose HBT positive patients were LTT positive and 35/67 lactose HBT negative patients were LTT positive. 62/192 (32%) developed symptoms following lactose ingestion, which tended to be more in the LTT positive (49/137, 36% vs. 13/55, 24% p=0.07) but not in the lactose HBT positive patients (44/125, 35% vs. 18/ 67, 27% p=0.2). Peak breath hydrogen was higher (38±37 vs. 66±43; p<0.01) in LTT positive than negative patients. Peak level of breath hydrogen inversely correlated (58±43 vs. 10±23; p<0.001) with change in blood glucose following lactose ingestion. Conclusions: Positive LTT is associated with a higher breath hydrogen score than negative LTT. There was a trend towards more frequent symptom development following lactose load in LTT positive but not in lactose HBT positive patients. LTT is an easy and efficient test for diagnosis of LM.

Gastric atrophy and intestinal metaplasia in a patient on long-term proton pump inhibitor therapy.

We report the case of a patient with gastroesophageal reflux disease who developed gastric atrophy and intestinal metaplasia (IM) while on 20-year treatment with proton pump inhibitors. This is perhaps the first report in human beings. A 74-year-old man, who presented with heartburn, showed abnormally high gastric pH (average 6.57) on 24-hour dual channel pH-metry even after discontinuing acid suppressive drugs for one month. No significant esophageal acid exposure was noted, which may be related to an impairment of the acid secreting capacity of the stomach (percentage time esophageal pH<4 during 24-h period 0.3%). Upper gastrointestinal endoscopy was normal except for the prominent submucosal vessels in the body and fundus suggesting gastric atrophy. Histopathological examination of multiple biopsies from the body and antrum of stomach showed signs of gastric atrophy and IM. Rapid urease test and histopathology of gastric biopsies were negative for Helicobacter pylori. Anti-H.pylori IgG ELISA however, was positive. Patient was asked to stop all anti-secretory drugs and only prokinetics were prescribed following which his symptoms markedly improved. On follow-up, in April 2007, he developed symptoms of peripheral neuropathy; serum vitamin 812 level was low. He responded to parenteral vitamin 812 therapy. 24-h dual channel pH-metry repeated after one and a half years showed persistently high gastric pH (average pH 6.76). The patient remained well after discontinuing proton pump inhibitors and continuing prokinetics and vitamin B12 injections.

Pathogenesis of gastro-oesophageal reflux disease: what role do Helicobacter pylori and host genetic factors play?

Gastro-oesophageal reflux disease is a multifactorial disease. The roles of environmental, dietary, and host physiological factors are well established. However, the plausible role of Helicobacter pylori infection in gastro-oesophageal reflux disease is still controversial. Furthermore, the role of host genetic factors remains unidentified. Extensive PubMed review of the previous literature has revealed that H. pylori may be negatively associated with gastro-oesophageal reflux disease. Ethnic or inter-individual variations in response to H. pylori infection may also determine disease outcome. Thus, host genetic factors may play an important role in deciding the final outcome of disease. Limited studies have shown that homEM ofCYP2C19, b allele (val105) ofGSTP1, T allele of IL1B-31, 2/2 genotype of IL1RN +2018, 2/2 genotype of IL-10-1082, A/A genotype of CCND1 G870A, and homozygous variant of XPC PAT gene are potential risk factors for the development of gastro-oesophageal reflux disease or its complications such as Barrett's oesophagus and oesophageal adenocarcinoma. There is scant data on the relationship between gastro-oesophageal reflux disease and H. pylori in India, and therefore, further studies are directly required to explore this issue.