Sunil Sethi

Proteomic Study Reveals That Proteins Involved in Metabolic and Detoxification Pathways Are Highly Expressed in HER-2/neu-positive Breast Cancer*

The receptor tyrosine kinase ErbB2 (HER-2/neu) is overexpressed in up to 30% of breast cancers and is associated with poor prognosis and an increased likelihood of metastasis especially in node-positive tumors. In this proteomic study, to identify the proteins that are associated with the aggressive phenotype of HER-2/neu-positive breast cancer, tumor cells from both HER-2/neu-positive and -negative tumors were procured by laser capture microdissection. Differentially expressed proteins in the two subsets of tumors were identified by two-dimensional electrophoresis and MALDI-TOF/TOF MS/MS. We found differential expression of several key cell cycle modulators, which were linked with increased proliferation of the HER-2/neu-overexpressing cells. Nine proteins involved in glycolysis (triose-phosphate isomerase (TPI), phosphoglycerate kinase 1 (PGK1), and enolase 1 (ENO1)), lipid synthesis (fatty acid synthase (FASN)), stress-mediated chaperonage (heat shock protein 27 (Hsp27)), and antioxidant and detoxification pathways (haptoglobin, aldo-keto reductase (AKR), glyoxalase I (GLO), and prolyl-4-hydrolase β-isoform (P4HB)) were found to be up-regulated in HER-2/neu-positive breast tumors. HER-2/neu-dependent differential expression of PGK1, FASN, Hsp27, and GLO was further validated in four breast cancer cell lines and 12 breast tumors by immunoblotting and confirmed by partially switching off the HER-2/neu signaling in the high HER-2/neu-expressing SKBr3 cell line with Herceptin treatment. Statistical correlations of these protein expressions with HER-2/neu status were further verified by immunohistochemistry on a tissue microarray comprising 97 breast tumors. Our findings suggest that HER-2/neu signaling may result, directly or indirectly, in enhanced activation of various metabolic, stress-responsive, antioxidative, and detoxification processes within the breast tumor microenvironment. We hypothesize that these identified changes in the cellular proteome are likely to drive cell proliferation and tissue invasion and that the key cell cycle modulators involved, when uncovered by future research, would serve as naturally useful targets for the development of therapeutic strategies to negate the metastatic potential of HER-2/neu-positive breast tumors.

GFR Estimating Equations in a Multiethnic Asian Population

BACKGROUND Clinical practice guidelines recommend using equations for estimating glomerular filtration rate (GFR) in chronic kidney disease (CKD) management and research. The MDRD (Modification of Diet in Renal Disease) Study and CKD-EPI (CKD Epidemiology Collaboration) equations originally were derived from a North American population and had an ethnic coefficient adjustment for African Americans. A Chinese coefficient for the MDRD Study equation subsequently was determined, but this has not been externally validated. We compared the accuracy of the equations, evaluated the ethnic coefficients, and assessed the equations for disease staging in a multiethnic Asian population with CKD. STUDY DESIGN A diagnostic test study comparing the Asian coefficient (and subgroups)-modified MDRD Study and CKD-EPI equations and a cross-sectional study assessing disease staging. SETTING & PARTICIPANTS 232 outpatients (52% men; 40.5% Chinese, 32% Malay, and 27.5% Indian/other) with stable CKD. INDEX TEST Asian and ethnicity-based modifications of the MDRD Study and CKD-EPI equations. REFERENCE TEST Measured GFR using 3-sample plasma clearance of technetium-99m diethylenetriaminepentaacetic acid ((99m)Tc-DTPA), calculated using the slope-intercept method, with body surface area normalization (du Bois) and Brochner-Mortensen correction. RESULTS Overall, the CKD-EPI equation is more accurate than the MDRD Study equation throughout the GFR range, with improved bias (median difference of estimated GFR - measured GFR) and root mean square error (P <0.001). CKD-EPI versus MDRD Study equation: bias, 1.1 ± 13.8 vs -1.0 ± 15.2 mL/min/1.73 m(2); precision, 12.1 vs 12.2 mL/min/1.73 m(2). Ethnic coefficients did not improve estimates of GFR significantly. The correctness of staging was improved using the CKD-EPI equation. LIMITATIONS All participants had CKD, but few were of European descent. The reference GFR technique was different from the original studies. CONCLUSIONS The CKD-EPI is more accurate than the MDRD Study equation, particularly at higher GFRs. Therefore, we recommend adopting the CKD-EPI equation without ethnic adjustment for estimating GFR in multiethnic Asian patients with CKD.

Local inflammation induces complement crosstalk which amplifies the antimicrobial response.

By eliciting inflammatory responses, the human immunosurveillance system notably combats invading pathogens, during which acute phase proteins (CRP and cytokines) are elevated markedly. However, the Pseudomonas aeruginosa is a persistent opportunistic pathogen prevalent at the site of local inflammation, and its acquisition of multiple antibiotic-resistance factors poses grave challenges to patient healthcare management. Using blood samples from infected patients, we demonstrate that P. aeruginosa is effectively killed in the plasma under defined local infection-inflammation condition, where slight acidosis and reduced calcium levels (pH 6.5, 2 mM calcium) typically prevail. We showed that this powerful antimicrobial activity is provoked by crosstalk between two plasma proteins; CRP∶L-ficolin interaction led to communication between the complement classical and lectin pathways from which two amplification events emerged. Assays for C4 deposition, phagocytosis, and protein competition consistently proved the functional significance of the amplification pathways in boosting complement-mediated antimicrobial activity. The infection-inflammation condition induced a 100-fold increase in CRP∶L-ficolin interaction in a pH- and calcium-sensitive manner. We conclude that the infection-induced local inflammatory conditions trigger a strong interaction between CRP∶L-ficolin, eliciting complement-amplification pathways which are autonomous and which co-exist with and reinforce the classical and lectin pathways. Our findings provide new insights into the host immune response to P. aeruginosa infection under pathological conditions and the potential development of new therapeutic strategies against bacterial infection.

Natriuretic Peptides in Chronic Kidney Disease

BACKGROUND AND OBJECTIVES B-type natriuretic peptide (BNP) and amino-terminal pro-B-type natriuretic peptide (NT-proBNP) are biomarkers of cardiovascular disease that is common in patients with chronic kidney disease (CKD). Conflicting data on the influence of glomerular filtration rate (GFR) on BNP and NT-proBNP levels in CKD may stem from failure to account fully for the effects of coexistent cardiac disease, dysfunction, and volume overload. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Prospective head-to-head comparison of plasma BNP and NT-proBNP in ambulatory euvolemic CKD patients with normal LV ejection fraction and no manifest cardiac or vascular disease. GFR was estimated by the Modification of Diet in Renal Disease formula, BNP and NT-proBNP measured using Abbott AxSYM and Roche Elecsys assays, respectively, and cardiac morphology and function assessed by transthoracic echocardiography. RESULTS In 142 patients (42% female) of mean age 60 +/- 11 yr, mean left ventricular ejection fraction was 71% +/- 6%, GFR 38 +/- 14 ml/min per 1.73 m(2), and median BNP and NT-proBNP level 59 and 311 pg/ml, respectively. Multivariate predictors of NT-proBNP level were GFR, beta-blocker usage, LV mass index, and hemoglobin level. Plasma BNP was independently predicted by LV mass index and beta-blocker usage but not GFR. In the 74 patients without diastolic dysfunction, there was a significant rise in NT-proBNP but not BNP as GFR declined. CONCLUSIONS Unlike NT-proBNP, plasma BNP level is relatively independent of GFR. BNP may therefore be the more appropriate biomarker to screen for cardiac dysfunction in CKD.

Increasing Trends of Leptospirosis in Northern India: A Clinico-Epidemiological Study

Background Leptospirosis, a zoonosis associated with potentially fatal consequences, has long been a grossly underreported disease in India. There is no accurate estimate of the problem of leptospirosis in non-endemic areas such as north India. Methods/Principal Findings In order to understand the clinical spectrum and risk factors associated with leptospirosis, we carried out a retrospective study in patients with acute febrile illness in north India over the last 5 years (January 2004 to December 2008). There was increased incidence of leptospirosis (11.7% in 2004 to 20.5% in 2008) as diagnosed by IgM ELISA and microscopic agglutination titer in paired acute and convalescent sera. The disease showed a peak during the rainy season (August and September). We followed up 86 cases of leptospirosis regarding their epidemiological pattern, clinical features, laboratory parameters, complications, therapy, and outcome. Mean age of patients was 32.6 years (2.5 years to 78 years) and males (57%) outnumbered females (43%). Infestation of dwellings with rats (53.7%), working in farm lands (44.2%), and contact with animals (62.1%) were commonly observed epidemiological risk factors. Outdoor workers including farmers (32.6%), labourers (11.6%), para-military personnel (2.3%), and sweepers (1.2%) were commonly affected. Modified Faines criteria could diagnose 76 cases (88.3%). Renal failure (60.5%), respiratory failure (20.9%), the neuroleptospirosis (11.6%), and disseminated intravascular coagulation (DIC) (11.6%) were the commonest complications. Five patients died, giving a case fatality rate of 5.9%. Conclusions/Significance There has been a rapid rise in the incidence of leptospirosis in north India. Severe complications such as renal failure, respiratory failure, neuroleptospirosis, and DIC are being seen with increasing frequency. Increased awareness among physicians, and early diagnosis and treatment, may reduce mortality due to leptospirosis.

Current 25-hydroxyvitamin D assays: Do they pass the test?

BACKGROUND Vitamin D testing is becoming increasingly important with recent research demonstrating a correlation between vitamin D insufficiency and metabolic diseases, immunodeficiencies and other diseases. However, existing 25-hydroxyvitamin D (25OHD) assays lack comparability to the candidate reference method, causing difficulties in diagnosis and monitoring of vitamin D deficiency. METHODS We looked at the accuracy of 3 automated assays (Roche Diagnostics Elecsys® Total 25OHD assay, Abbott Architect® Total vitamin D assay, Advia Centaur® vitamin D Total assay) and Diasorin® Radioimmunoassay (RIA) compared to a routine laboratory Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). RESULTS The correlation based on Passing Bablok regression was good with the slopes between 0.95 and 1.31 and the intercepts between -3.24 and 3.68. However, a significant positive bias was observed using the Abbott Architect and the Diasorin RIA. Using published analytical goals of coefficient of variation (CV) <10% and bias <5%, most methods did not meet these criteria. Using measurement of uncertainty of 9%, most methods were able to meet criteria using quality control materials but not patient samples. CONCLUSION Inadequacies of these assay performances are contributed by differences in method of extraction of vitamin D from vitamin D binding protein, cross-reactivities to 25OHD(2), 25OHD(3) and other vitamin D metabolites, matrix interferences and a lack of standardization.

Characterisation of blaTEM genes and types of β-lactamase plasmids in Neisseria gonorrhoeae - the prevalent and conserved blaTEM-135 has not recently evolved and existed in the Toronto plasmid from the origin.

BackgroundAntimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major concern worldwide. It has been recently feared that the blaTEM-1 gene is, via blaTEM-135, evolving into an extended-spectrum β-lactamase (ESBL), which could degrade all cephalosporins including ceftriaxone. The aims of the present study were to characterize the blaTEM genes, types of β-lactamase plasmids, the degradation of ampicillin by TEM-135 compared to TEM-1, and to perform molecular epidemiological typing of β-lactamase-producing N. gonorrhoeae strains internationally.Methodsβ-lactamase producing N. gonorrhoeae isolates (n = 139) cultured from 2000 to 2011 in 15 countries were examined using antibiograms, blaTEM gene sequencing, β-lactamase plasmid typing, and N. gonorrhoeae multiantigen sequence typing (NG-MAST). Furthermore, the blaTEM gene was sequenced in the first described Toronto plasmid (pJD7), one of the first Asian plasmids (pJD4) and African plasmids (pJD5) isolated in Canada. The degradation of ampicillin by TEM-135 compared to TEM-1 was examined using a MALDI-TOF MS hydrolysis assay.ResultsSix different blaTEM sequences were identified (among isolates with 125 different NG-MAST STs), i.e. blaTEM-1 (in 104 isolates), blaTEM-135 (in 30 isolates), and four novel blaTEM sequences (in 5 isolates). The blaTEM-1 allele was only found in the African and Asian plasmids, while all Rio/Toronto plasmids possessed the blaTEM-135 allele. Most interesting, the first described gonococcal Toronto plasmid (pJD7), identified in 1984, also possessed the highly conserved blaTEM-135 allele. The degradation of ampicillin by TEM-135 compared to TEM-1 was indistinguishable in the MALDI-TOF MS hydrolysis assay.ConclusionsblaTEM-135, encoding TEM-135, is predominantly and originally associated with the Rio/Toronto plasmid and prevalent among the β-lactamase producing gonococcal strains circulating globally. blaTEM-135 does not appear, as previously hypothesized, to have recently evolved due to some evolutionary selective pressure, for example, by the extensive use of extended-spectrum cephalosporins worldwide. On the contrary, the present study shows that blaTEM-135 existed in the Toronto plasmid from its discovery and that blaTEM-135 is highly conserved (not further evolved in the past >30 years). Nevertheless, international studies for monitoring the presence of different blaTEM alleles, the possible evolution of the blaTEM-135 allele, and the types of β-lactamase producing plasmids, remain imperative.

Biomarkers for clinical and incipient tuberculosis: performance in a TB-endemic country.

Background Simple biomarkers are required to identify TB in both HIV−TB+ and HIV+TB+ patients. Earlier studies have identified the M. tuberculosis Malate Synthase (MS) and MPT51 as immunodominant antigens in TB patients. One goal of these investigations was to evaluate the sensitivity and specificity of anti-MS and –MPT51 antibodies as biomarkers for TB in HIV−TB+ and HIV+TB+ patients from a TB-endemic setting. Earlier studies also demonstrated the presence of these biomarkers during incipient subclinical TB. If these biomarkers correlate with incipient TB, their prevalence should be higher in asymptomatic HIV+ subjects who are at a high-risk for TB. The second goal was to compare the prevalence of these biomarkers in asymptomatic, CD4+ T cell-matched HIV+TB− subjects from India who are at high-risk for TB with similar subjects from US who are at low-risk for TB. Methods and Results Anti-MS and -MPT51 antibodies were assessed in sera from 480 subjects including PPD+ or PPD− healthy subjects, healthy community members, and HIV−TB+ and HIV+TB+ patients from India. Results demonstrate high sensitivity (∼80%) of detection of smear-positive HIV−TB+ and HIV+TB+ patients, and high specificity (>97%) with PPD+ subjects and endemic controls. While ∼45% of the asymptomatic HIV+TB− patients at high-risk for TB tested biomarker-positive, >97% of the HIV+TB− subjects at low risk for TB tested negative. Although the current studies are hampered by lack of knowledge of the outcome, these results provide strong support for the potential of these biomarkers to detect incipient, subclinical TB in HIV+ subjects. Conclusions These biomarkers provide high sensitivity and specificity for TB diagnosis in a TB endemic setting. Their performance is not compromised by concurrent HIV infection, site of TB and absence of pulmonary manifestations in HIV+TB+ patients. Results also demonstrate the potential of these biomarkers for identifying incipient subclinical TB in HIV+TB− subjects at high-risk for TB.

Diagnostic Testing for Pandemic Influenza in Singapore: A Novel Dual-Gene Quantitative Real-Time RT-PCR for the Detection of Influenza A/H1N1/2009

With the relative global lack of immunity to the pandemic influenza A/H1N1/2009 virus that emerged in April 2009 as well as the sustained susceptibility to infection, rapid and accurate diagnostic assays are essential to detect this novel influenza A variant. Among the molecular diagnostic methods that have been developed to date, most are in tandem monoplex assays targeting either different regions of a single viral gene segment or different viral gene segments. We describe a dual-gene (duplex) quantitative real-time RT-PCR method selectively targeting pandemic influenza A/H1N1/2009. The assay design includes a primer-probe set specific to only the hemagglutinin (HA) gene of this novel influenza A variant and a second set capable of detecting the nucleoprotein (NP) gene of all swine-origin influenza A virus. In silico analysis of the specific HA oligonucleotide sequence used in the assay showed that it targeted only the swine-origin pandemic strain; there was also no cross-reactivity against a wide spectrum of noninfluenza respiratory viruses. The assay has a diagnostic sensitivity and specificity of 97.7% and 100%, respectively, a lower detection limit of 50 viral gene copies/PCR, and can be adapted to either a qualitative or quantitative mode. It was first applied to 3512 patients with influenza-like illnesses at a tertiary hospital in Singapore, during the containment phase of the pandemic (May to July 2009).

Estimating Glomerular Filtration Rates by Use of Both Cystatin C and Standardized Serum Creatinine Avoids Ethnicity Coefficients in Asian Patients with Chronic Kidney Disease

BACKGROUND The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation is most accurate for estimating glomerular filtration rate (GFR) but requires an adjustment for African-American patients. Estimation equations are also improved with the use of serum cystatin C combined with standardized creatinine. Combination equations have been derived by the CKD-EPI and Chinese investigators. We investigated whether these cystatin C-based equations improve estimation adequately, so that adjustments for ethnicity are not required in a multiethnic Asian population with chronic kidney disease (CKD). METHODS This was a cross-sectional study of 232 stable CKD patients who underwent GFR measurements using 3-sample plasma clearances of (99m)Tc-DTPA, and for whom serum cystatin C and creatinine were quantified. RESULTS For all patients, the median biases with cystatin C equations were generally greater than with the CKD-EPI equation, and precision and root mean square error (RMSE) were not significantly better. However, the combination serum creatinine and cystatin C equation improved the precision, RMSE, and percentage of estimated GFR to within 15% and 30% of the measured GFR (57.3% vs 50.0%, 88.4% vs 82.8%, respectively). The derived ethnicity coefficients for the combination equation were all >1 (1.009-1.082) but small, suggesting that coefficients are not required. The Chinese-specific equations were more biased and performed more poorly than the CKD-EPI equation. CONCLUSIONS The use of a cystatin C and creatinine combination equation for estimating GFR in a multiethnic Asian population with CKD does not require ethnicity coefficients because the derived coefficients are very close to each other.