4-fold Rise Research Articles

Bioinspired Calcium Phosphate Nanoparticles Featuring as Efficient Carrier and Prompter for Macrophage Intervention in Experimental Leishmaniasis.

To develop a biocompatible and bioresorbable calcium phosphate (CaP) nanoparticles (NPs) bearing Amphotericin B (AmB) with an aim to provide macrophage specific targeting in visceral leishmaniasis (VL). CaP-AmB-NPs were architectured through emulsion precipitation method. The developed formulation was extensively characterized for various parameters including in-vitro and in-vivo antileishmanial activity. Moreover, plasma pharmacokinetics, tissue biodistribution and toxicity profile were also assessed. Optimized CaP-AmB-NPs exhibited higher entrapment (71.1 ± 6.68%) of AmB. No trend related to higher hemolysis was apparent in the developed formulation as evidenced in commercially available colloidal and liposomal formulations. Cellular uptake of the developed CaP-AmB-NPs was quantified through flow cytometry in J774A.1 cell line, and a 23.90 fold rise in uptake was observed. Fluorescent microscopy also confirmed the time dependent rise in uptake. In-vivo multiple dose toxicity study demonstrated no toxicity upto 5mg/kg dose of AmB. Plasma kinetics and tissue distribution studies established significantly higher concentration of AmB in group treated with CaP-AmB-NPs in liver and spleen as compared to CAmB, LAmB and AmB suspension group. In-vivo animal experimental results revealed that the CaP-AmB-NPs showed higher splenic parasite inhibition compared to CAmB and LAmB in leishmania parasite infected hamsters. The investigated CaP-AmB-NPs are effective in provoking macrophage mediated uptake and collectively features lower toxicity and offers a suitable replacement for available AmB-formulations for the obliteration of intra-macrophage VL parasite.

The clinical significance of ABCB1 overexpression in predicting outcome of CML patients undergoing first-line imatinib treatment.

Tyrosine kinase inhibitor (TKI) therapy results in excellent responses in the majority of patients with chronic myeloid leukaemia. First-line imatinib treatment, with selective switching to nilotinib when patients fail to meet specific molecular targets or for imatinib intolerance, results in excellent overall molecular responses and survival. However, this strategy is less effective in cases of primary imatinib resistance; moreover, 25% of patients develop secondary resistance such that 20-35% of patients initially treated with imatinib will eventually experience treatment failure. Early identification of these patients is of high clinical relevance. Since the drug efflux transporter ABCB1 has previously been implicated in TKI resistance, we determined if early increases in ABCB1 mRNA expression (fold change from diagnosis to day 22 of imatinib therapy) predict for patient response. Indeed, patients exhibiting a high fold rise (⩾2.2, n=79) were significantly less likely to achieve early molecular response (BCR-ABL1IS ⩽10% at 3 months; P=0.001), major molecular response (P<0.0001) and MR4.5 (P<0.0001). Additionally, patients demonstrated increased levels of ABCB1 mRNA before the development of mutations and/or progression to blast crisis. Patients with high fold rise in ABCB1 mRNA were also less likely to achieve major molecular response when switched to nilotinib therapy (49% vs 82% of patients with low fold rise). We conclude that early evaluation of the fold change in ABCB1 mRNA expression may identify patients likely to be resistant to first- and second-generation TKIs and who may be candidates for alternative therapy.

The immunogenicity of the intradermal injection of seasonal trivalent influenza vaccine containing influenza A(H1N1)pdm09 in COPD patients soon after a pandemic

ABSTRACTThe antibody responses of a reduced-dose intradermal seasonal influenza vaccination have never been studied in COPD patients soon after a pandemic. A total of 149 COPD patients (60 y of age or older) were randomized to receive trivalent influenza vaccine (Sanofi-Pasteur, France) either 9 µg of hemagglutinin (HA) per strain split into 2-site intradermal (ID) injections via the Mantoux technique or one intramuscular (IM) injection of 15 µg of HA per strain. The geometric mean titers, seroconversion factors, seroconversion rates and seroprotection rates for influenza A(H3N2) and B administered through the ID injection (n = 75) were similar to those obtained with the IM injection (n = 74) 4 weeks post-vaccination. The antibody responses for influenza A(H1N1)pdm09 administered through the ID injection were lower than those obtained with the IM injection, but all of these responses met the 3 criteria proposed by the Committee for Proprietary Medicinal Products (CPMP) for annual re-licensure. The seroprotection rates 4 weeks post-vaccination for influenza A(H1N1)pdm09 were 64.0% (95%CI 52.7-74.0%) in the ID group vs. 78.4% (95% CI 67.6-86.3%) in the IM group (p = 0.053). Influenza-related acute respiratory illness (ARI), diagnosed as a 4-fold rise in HI titers with a convalescent titer > 1:40, and/or the RT-PCR between the ID group (5.3%) and the IM group (8.1%) were not significantly different. The reduced-dose intradermal influenza vaccine may expand vaccine coverage in cases of vaccine shortage.

An integrated liquid chromatography–tandem mass spectrometry approach for the ultra-sensitive determination of catecholamines in human peripheral blood mononuclear cells to assess neural-immune communication

Catecholamines play a vital role in the interactions between the nervous and immune systems and their dysfunctions are implicated in various autoimmune and neurological diseases. However, accurate quantitation of catecholamines in the immune system presents a special analytical challenge. We proposed the first LC–MS/MS method for the determination of catecholamines in human peripheral blood mononuclear cells (PBMC) with significantly improved sensitivity, selectivity and throughput without requiring derivatization, evaporation and ion-pairing reagent. PBMC were separated by density gradient centrifugation and lysed with 0.2M acetic acid. The analytical novelty includes the first solid phase extraction on a 96-well hydrophilic-lipophilic-balanced (HLB) μElution plate upon complexation with phenylboronic acid (PBA), enabling specific clean-up and fivefold pre-concentration of catecholamines in a single extraction. LC chromatographic separation was obtained on a PFP column with 0.01% HCOOH as additive with enhanced signal response. Summation of five MRM transitions yielded three–four fold rise in sensitivity. The lower limit of quantification of 1pg/mL for epinephrine (E) and 5pg/mL for norepinephrine (NE) and dopamine (DA) represents a considerable sensitivity improvement over available methods. Less than 8.7% of intraday and interday precision, 91.8–111.3% of accuracy and successful assessment of reference intervals for 40 healthy donors suggested good reproducibility and reliability of the assay. The novel PBA-HLB-PFP-MRM summation approach allows rapid, sensitive and reliable determination of catecholamines in PBMC, which will facilitate better understanding of the new arena of neural-immune network. Additionally, the substantially improved method can be modified to quantify catecholamines and metabolites in other biological matrices.

Reliability of cold agglutinin test (CAT) for the detection of patients with Mycoplasma pneumoniae pneumonia

Introduction : M. pneumoniae is one of the causative agents of primary atypical pneumonia. This infection causes 20-40% of community acquired pneumonia and is associated with an array of extra-pulmonary manifestations. There is a need for a rapid diagnostic test in order to prescribe prompt and appropriate antibiotic therapy. Even though isotype specific antibody testing provides definitive diagnosis, paired sera testing does not help in real time diagnosis. Cold agglutinins detectable by the Cold Agglutination Test (CAT) appear and disappear early in infection compared to long lasting specific antibodies that are detectable by specific immunoassays. Although there are some reports suggesting CAT is unreliable, it is being often used to diagnose M. pneumoniae pneumonia in Sri Lankan clinical settings. The aim of the current study was to evaluate the use of CAT as a bed-side screening test for early diagnosis of M. pneumoniae pneumonia compared to ELISA for detection of specific antibodies in the Sri Lankan context. Methods: Ninety seven clinically and radiologically confirmed patients with pneumonia were enrolled in the study. CAT was performed on acute stage sera. A CAT titer ≥1/32 was considered as positive. Isotype specific M. pneumoniae ELISA with paired sera was compared with CAT results. Results: Mycoplasma pneumonia was confirmed in 15 of the 97 patients in the study using Mycoplasma specific IgM and 4 fold rise in titre. Of these, 3 were positive by the CAT. The sensitivity and specificity of the CAT compared to IgM/4fold rise in IgG detection were 20% (3/15) and 81.7% (67/82) respectively. Negative and positive predictive values of the CAT compared to ELISA were 84.8% (67/79) and 16.7% (3/18) respectively. Conclusion: CAT is not a reliable screening test compared to specific antibody detection by isotype ELISA for the detection of M. pneumoniae pneumonia due to its low sensitivity and positive predictive values.

Diagnostic Accuracy of PCR Alone and Compared to Urinary Antigen Testing for Detection of Legionella spp.: a Systematic Review.

The diagnosis of Legionnaires' disease (LD) is based on the isolation of Legionella spp., a 4-fold rise in antibodies, a positive urinary antigen (UA), or direct immunofluorescence tests. PCR is not accepted as a diagnostic tool for LD. This systematic review assesses the diagnostic accuracy of PCR in various clinical samples with a direct comparison versus UA. We included prospective or retrospective cohort and case-control studies. Studies were included if they used the Centers for Disease Control and Prevention consensus definition criteria of LD or a similar one, assessed only patients with clinical pneumonia, and reported data for all true-positive, false-positive, true-negative, and false-negative results. Two reviewers abstracted data independently. Risk of bias was assessed using Quadas-2. Summary sensitivity and specificity values were estimated using a bivariate model and reported with a 95% confidence interval (CI). Thirty-eight studies were included. A total of 653 patients had confirmed LD, and 3,593 patients had pneumonia due to other pathogens. The methodological quality of the studies as assessed by the Quadas-2 tool was poor to fair. The summary sensitivity and specificity values for diagnosis of LD in respiratory samples were 97.4% (95% CI, 91.1% to 99.2%) and 98.6% (95% CI, 97.4% to 99.3%), respectively. These results were mainly unchanged by any covariates tested and subgroup analysis. The diagnostic performance of PCR in respiratory samples was much better than that of UA. Compared to UA, PCR in respiratory samples (especially in sputum samples or swabs) revealed a significant advantage in sensitivity and an additional diagnosis of 18% to 30% of LD cases. The diagnostic performance of PCR in respiratory samples was excellent and preferable to that of the UA. Results were independent on the covariate tested. PCR in respiratory samples should be regarded as a valid tool for the diagnosis of LD.

The Clinical Significance of Early Imatinib Induced ABCB1 Overexpression in Chronic Phase CML Patients: A TIDEL II Sub-Study

▪Recent data from the TIDEL II trial indicate first-line imatinib (IM) treatment, with selective switching to nilotinib (NIL) for failure to meet specific molecular targets or for intolerance, results in excellent molecular response and overall survival in chronic phase Chronic Myeloid Leukaemia (CP-CML) patients. Drug transporters, particularly OCT-1 and ABCB1, have previously been demonstrated to impact IM response. Furthermore, ABCB1 overexpression has been implicated in IM and NIL resistance in vitro. In this TIDEL II sub-study the level of ABCB1 mRNA expression was retrospectively assessed by PCR at day 0 and day 22 post the start of IM therapy and where relevant, at cessation. A change in the level of expression of ABCB1 compared with baseline after 22 days exposure to IM was observed in all patients (Range: 0.6-12.3 fold; median: 2 fold), suggesting IM exposure resulted in altered ABCB1 expression that was variable between patients; this change was not related to the level of BCR-ABL1 expression (P =0.29 at day 0 and P =0.84 at 1 mo). Assessing the impact of >2 fold rise in ABCB1 expression after 22 days IM exposure on subsequent molecular response, and comparing this with patients where a <2 fold rise was observed revealed significant differences in outcome between the 2 groups (Table 1). Table 1% of patients achieving:Over the first 22 days of treatmentEarly Molecular ResponseMMR by 12 moEvent Free SurvivalMR4.5<2 fold rise in ABCB1 (n=22)86%64%77%64%>2 fold rise in ABCB1 (n=22)60%14%37%5%p -value0.0060.0010.018<0.001Importantly, change in ABCB1 mRNA over the first 22 days of treatment was predictive of MMR by 24 mo (Figure 1).These data support previous in vitro findings of ABCB1-mediated IM export and provide the first in vivo evidence for up regulation of ABCB1 in response to IM therapy. Because ABCB1-mediated export of NIL has also been demonstrated, we assessed the correlation between response to NIL therapy and changes in ABCB1 expression following 22 days IM exposure. Of the patients who demonstrated a >2 fold rise in ABCB1 compared with baseline, 16 of the remaining 20 patients (2/22 died at 3.2 mo (Cardiac event) and 3.7 mo (Blast Crisis)) switched to sequential NIL therapy because of sub-optimal response to IM (no prior MMR). Importantly, 0/16 patients receiving NIL subsequently achieved confirmed MMR, suggesting that NIL may provide a poor therapeutic option for patients with up-regulation of ABCB1. These data highlight the potential importance for monitoring increases in ABCB1 expression early in therapy in order to predict those patients likely to achieve poor molecular responses. We next determined the impact of ABCB1 expression on Event Free Survival (EFS) and demonstrated that the fold change in ABCB1 levels at day 22 was predictive for achieving EFS: 77% of patients with <2 rise in ABCB1 vs 37% of patients with >2 rise in ABCB1; p =0.018. Furthermore a sustained >2 fold rise in ABCB1 mRNA compared with levels at diagnosis was observed in 3/3 patients prior to progression to blast crisis and ABCB1 mRNA increased by >2 fold post therapy in 9/12 patients prior to development of KD mutations.Previous studies investigating the correlation between ABCB1 expression and response to therapy have focussed on absolute levels of ABCB1, usually at 12 mo post-therapy initiation when leukaemic cell burden would be low, and have observed no significant difference in ABCB1 expression in optimal vs sub-optimal responders. Conversely, our rapid PCR based assay performed pre and 22 days post the start of IM therapy in CP-CML patients provides a potent early predictor of subsequent IM response. The findings detailed here also suggest that NIL is likely a poor subsequent therapeutic option for patients with up-regulated ABCB1 expression in response to IM. Importantly, these data highlight the fact that better understanding of the effects of TKIs on factors with the potential to influence treatment outcome (such as drug transporter expression) can be used to personalise therapy. Overarchingly, these data exemplify the importance of drug transporters in the setting of TKI therapy and patient response, and, following validation in further studies, provide a new, effective and easily translatable prognostic biomarker based on ABCB1. [Display omitted] DisclosuresHughes:Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

Measles Virus Neutralizing Antibody Response, Cell-Mediated Immunity, and Immunoglobulin G Antibody Avidity Before and After Receipt of a Third Dose of Measles, Mumps, and Rubella Vaccine in Young Adults.

Two doses of measles, mumps, and rubella (MMR) vaccine are 97% effective against measles, but waning antibody immunity to measles and failure of the 2-dose vaccine occur. We administered a third MMR dose (MMR3) to young adults and assessed immunogenicity over 1 year. Measles virus (MeV) neutralizing antibody concentrations, cell-mediated immunity (CMI), and immunoglobulin G (IgG) antibody avidity were assessed at baseline and 1 month and 1 year after MMR3 receipt. Of 662 subjects at baseline, 1 (0.2%) was seronegative for MeV-neutralizing antibodies (level, <8 mIU/mL), and 23 (3.5%) had low antibody levels (8-120 mIU/mL). One month after MMR3 receipt, 1 subject (0.2%) was seronegative, and 6 (0.9%) had low neutralizing antibodies, with only 21 of 662 (3.2%) showing a ≥ 4-fold rise in neutralizing antibodies. One year after MMR3 receipt, no subject was seronegative, and 10 of 617 (1.6%) had low neutralizing antibody levels. CMI analyses showed low levels of spot-forming cells after stimulation, suggesting the presence of T-cell memory, but the response was minimal after MMR3 receipt. MeV IgG avidity did not correlate with findings of neutralization analyses. Most subjects were seropositive before MMR3 receipt, and very few had a secondary immune response after MMR3 receipt. Similarly, CMI and avidity analyses showed minimal qualitative improvements in immune response after MMR3 receipt. We did not find compelling data to support a routine third dose of MMR vaccine.

Effector and Central Memory Poly-Functional CD4+ and CD8+ T Cells are Boosted upon ZOSTAVAX® Vaccination

ZOSTAVAX® is a live attenuated varicella-zoster virus (VZV) vaccine that is licensed for the protection of individuals ≥50 years against shingles and its most common complication, postherpetic neuralgia. While IFNγ responses increase upon vaccination, the quality of the T cell response has not been elucidated. By using polychromatic flow cytometry, we characterized the breadth, magnitude, and quality of ex vivo CD4+ and CD8+ T cell responses induced 3–4 weeks after ZOSTAVAX vaccination of healthy adults. We show, for the first time that the highest frequencies of VZV-specific CD4+ T cells were poly-functional CD154+IFNγ+IL-2+TNFα+ cells, which were boosted upon vaccination. The CD4+ T cells were broadly reactive to several VZV proteins, with immediate early (IE) 63 ranking the highest among them in the fold rise of poly-functional cells, followed by IE62, gB, open reading frame (ORF) 9, and gE. We identified a novel poly-functional ORF9-specific CD8+ T cell population in 62% of the subjects, and these were boosted upon vaccination. Poly-functional CD4+ and CD8+ T cells produced significantly higher levels of IFNγ, IL-2, and TNFα compared to mono-functional cells. After vaccination, a boost in the expression of IFNγ by poly-functional IE63- and ORF9-specific CD4+ T cells and IFNγ, IL-2, and TNFα by ORF9-specific poly-functional CD8+ T cells was observed. Responding poly-functional T cells exhibited both effector (CCR7−CD45RA−CD45RO+), and central (CCR7+CD45RA−CD45RO+) memory phenotypes, which expressed comparable levels of cytokines. Altogether, our studies demonstrate that a boost in memory poly-functional CD4+ T cells and ORF9-specific CD8+ T cells may contribute toward ZOSTAVAX efficacy.

Optimal Cutoff Titers for Indirect Immunofluorescence Assay for Diagnosis of Scrub Typhus.

We determined the optimal cutoff titers in admission and convalescent-phase samples for scrub typhus indirect immunofluorescence assay using Bayesian latent class models. Cutoff titers of ≥1:3,200 in an admission sample or of a ≥4-fold rise to ≥1:3,200 in a convalescent-phase sample provided the highest accuracy (sensitivity, 81.6%; specificity, 100%).

Pharmacological Characterization of ZYAN1, a Novel Prolyl Hydroxylase Inhibitor for the Treatment of Anemia.

Prolyl hydroxylase (PHD) inhibitors stabilize hypoxia inducible factor (HIF), and exert antianemic effect by potentiating erythropoietin (EPO) expression and down-regulation of hepcidin. ZYAN1 is a novel PHD inhibitor under clinical development for the treatment of anemia. The pharmacodynamic effects of acute and chronic dosing of ZYAN1 were assessed in normal and 5/6 nephrectomized Wistar rats. The effect of ZYAN1 was also investigated in cisplatin-induced anemia using C57 mice. Acute treatment with ZYAN1 increased circulating EPO levels (10.3 ± 3.7 and 40.0 ± 8.5 fold rise at 15 and 30 mg/kg, respectively), reticulocyte count (4.2 ± 0.5 and 6.0 ± 0.2 fold rise at 15 and 30 mg/kg, respectively) and stabilized HIF (28% increase at 45 mg/kg) in normal rats. Nephrectomized rats showed similar dose-related pharmacodynamic effects. In a 28-day study in nephrectomized rats, ZYAN1 administered every alternate day, caused increase in hemoglobin (1.9 ± 0.3 and 2.5 ± 0.4 g/dL) and RBC count (10.7 ± 4.0 and 14.0 ± 4.1%) at 15 and 30 mg/kg respectively. In cisplatin-treated mice also an increase in hemoglobin (3.4 ± 0.2 and 5.9 ± 0.2 g/dL) and RBC count (22.5 ± 2.2 and 37.3 ± 1.7%) at 15 and 30 mg/kg respectively was observed. ZYAN1's effects on hemoglobin and RBC count were distinct from darbepoietin. ZYAN1 demonstrated hematinic potential by combined effects on EPO release and efficient iron utilization. The efficacy of ZYAN1 in disease models of different etiologies suggests that it will be useful in treating wide spectrum of anemia patients.

Interference of Monovalent, Bivalent, and Trivalent Oral Poliovirus Vaccines on Monovalent Rotavirus Vaccine Immunogenicity in Rural Bangladesh.

Trivalent oral poliovirus vaccine (OPV) is known to interfere with monovalent rotavirus vaccine (RV1) immunogenicity. The interference caused by bivalent and monovalent OPV formulations, which will be increasingly used globally in coming years, has not been examined. We conducted a post hoc analysis to assess the effect of coadministration of different OPV formulations on RV1 immunogenicity. Healthy infants in Matlab, Bangladesh, were randomized to receive 3 doses of monovalent OPV type 1 or bivalent OPV types 1 and 3 at either 6, 8, and 10 or 6, 10, and 14 weeks of age or trivalent OPV at 6, 10, and 14 weeks of age. All infants received 2 doses of RV1 at about 6 and 10 weeks of age. Concomitant administration was defined as RV1 and OPV given on the same day; staggered administration as RV1 and OPV given ≥1 day apart. Rotavirus seroconversion was defined as a 4-fold rise in immunoglobulin A titer from before the first RV1 dose to ≥3 weeks after the second RV1 dose. There were no significant differences in baseline RV1 immunogenicity among the 409 infants included in the final analysis. Infants who received RV1 and OPV concomitantly, regardless of OPV formulation, were less likely to seroconvert (47%; 95% confidence interval, 39%-54%) than those who received both vaccines staggered ≥1 day (63%; 57%-70%; P < .001). For staggered administration, we found no evidence that the interval between RV1 and OPV administration affected RV1 immunogenicity. Coadministration of monovalent, bivalent, or trivalent OPV seems to lower RV1 immunogenicity. NCT01633216.

Functional immune responses to twelve serotypes after immunization with a 23-valent pneumococcal polysaccharide vaccine in older adults

BackgroundThe 23-valent pneumococcal polysaccharide vaccine (PPSV23) was introduced as part of the national immunization program for the elderly (≥65 years of age) in Korea on 2013. To evaluate immune responses in this population, serotype-specific anti-pneumococcal antibodies were studied with opsonophagocytic assay (OPA). MethodsPneumococcal vaccine-naïve participants ≥65 years of age were enrolled. They were divided into two groups according to their age: 30 in (65–74 years) and 32 in group (≥75 years). The functional antibody response was determined by multiplexed OPA (MOPA) for 12 serotypes (1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) before and 4 weeks after vaccination with PPSV23. ResultsGeometric mean titers (GMTs) to all tested serotypes significantly increased in both groups after vaccination compared to those before vaccination. There were no significant differences in either the fold rise (post-vaccination to pre-vaccination) or the percentage of participants with a ≥4-fold increase in OPA titers between two groups for any of the 12 serotypes. Following vaccination, GMT for serotype 9V was higher in group 1 than in group 2 (P=0.011). ConclusionsPPSV23 induces functional immune response for 12 vaccine serotypes in both age groups. Further analysis is needed for the remaining 11 serotypes in the PPSV23, in order to develop a better understanding of the immune responses induced by PPV23 in older adults.

Peru-15 (Choleragarde®), a live attenuated oral cholera vaccine, is safe and immunogenic in human immunodeficiency virus (HIV)-seropositive adults in Thailand

BackgroundMany areas with endemic and epidemic cholera report significant levels of HIV transmission. According to the World Health Organization (WHO), over 95% of reported cholera cases occur in Africa, which also accounts for nearly 70% of people living with HIV/AIDS globally. Peru-15, a promising single dose live attenuated oral cholera vaccine (LA-OCV), was previously found to be safe and immunogenic in cholera endemic areas. However, no data on the vaccine's safety among HIV-seropositive adults had been collected. MethodsThis study was a double-blinded, individually randomized, placebo-controlled trial enrolling HIV-seropositive adults, 18–45 years of age, conducted in Bangkok, Thailand, to assess the safety of Peru-15 in a HIV-seropositive cohort. Results32 HIV infected subjects were randomized to receive either a single oral dose of the Peru-15 vaccine with a buffer or a placebo (buffer only). No serious adverse events were reported during the follow-up period in either group. The geometric mean fold (GMF) rise in V. cholerae O1 El Tor specific antibody titers between baseline and 7 days after dosing was 32.0 (p<0.001) in the vaccine group compared to 1.6 (p<0.14) in the placebo group. Among the 16 vaccinees,14 vaccinees (87.5%) had seroconversion compared to 1 of 16 placebo recipients (6.3%). V. cholerae was isolated from the stool of one vaccinee, and found to be genetically identical to the Peru-15 vaccine strain. There were no significant changes in HIV viral load or CD4 T-cell counts between vaccine and placebo groups. ConclusionPeru-15 was shown to be safe and immunogenic in HIV-seropositive Thai adults.

Circulating microRNAs - a new horizon in molecular diagnosis of breast cancer.

The potential use of microRNAs (miRNAs) as ideal tumor markers has been the focus of recent research. Our hypothesis was that circulating miRNAs are differentially expressed in pretherapeutic sera of breast cancer patients compared to controls. Using real-time quantitative polymerase chain reaction (qPCR) analysis, levels of 5 candidate miRNAs (miR10b, miR34a, miR155, miR195 and miR16) were quantified in sera of breast cancer patients and control individuals. Levels of preoperative sera showed significant upregulation of 3.36 fold rise in miR10b (p<0.001), a 2.07 fold rise in miR155 (p =0.005) and remarkable over expression of 11.9 fold rise in miR195 (p<0.001) of cases than controls. There was significant down regulation of miR34a (0.032, p<0.001). The comparison with the clinicopathological data of the breast cancer patients revealed significant high serum level of miR155 (p =0.004) and miR195 (p =0.002) in patients with lymph node metastasis and higher levels of miR10b (p =0.001) and miR155 (p <0.001) with distant metastasis (M1) than without metastasis (M0), in addition to significant decrease in miR34a (p <0.001) level in M1 than M0 cases. These findings suggest that systemic circulating miRNAs have potential use as novel biomarkers for breast cancer.

Abstract 535: Lipid Phosphate Phosphatase 3: A Novel Mediator of Heart Failure Development and Progression

Lysophosphatidic acid (LPA) is a bioactive lipid mediator that is found in abundance in atherosclerotic plaques and whose production is accelerated by activated platelets. Two fold rise in serum LPA concentration in acute myocardial infarct patients (6.4±1.3 micro mol/L vs 2.5 ± 1.1 micro mol/L, P = 0.0001) and its effect to cause cardiomyocyte hypertrophy suggests, a critical role for LPA regulation in the myocardium. The actions of local and circulating LPA may be terminated by enzymatic dephosphorylation of the lipid by a family of hexahelical membrane spanning proteins, termed lipid phosphate phosphatases (LPP). Of the three LPP enzymes with preference for LPA, LPP3 appears the most likely to play a biologic role in regulating LPA levels, and deficiency of LPP3 results in embyronic lethality in mice due to vascular and neural tube defects. We have generated mice that specifically lack LPP3 in cardiomyocytes. These mice are viable and fertile but showed increased mortality ~8 months (Fig1). Blood pressure was similar in Ppap2b fl/fl (96 ± 9 mmHg; n = 19) and Myh6-Ppap2b Δ mice (92 ± 7 mmHg; n = 19), although heart rates were significantly higher in Myh6-Ppap2b Δ 3 month old mice (642 ± 21 bpm, compared to Ppap2b fl/fl with 600± 17 bpm; P&lt;0.001). Knockdown of LPP3 enhanced cardiomyocyte hypertrophy induced by LPA based on analysis of sarcomere organization, cell surface area, levels of fetal genes ANP and BNP, and ANF release from nuclei, which are hallmarks of cardiomyocyte hypertrophy, indicating that LPP3 negatively regulates cardiomyocyte hypertrophy induced by LPA.

(345) - Prior Coronary Artery Bypass Surgery- Is It Still a Contraindication for Lung Transplantation?

Purpose: Historically, coronary artery bypass grafting (CABG) has been a contraindication to lung transplantation (LTx) due to the severity of coronary artery disease and technical considerations of injuring a left internal mammary artery during LTx. However, an increasing number of patients with previous CABG are being referred for and receiving LTx. Whether prior CABG should remain a contraindication to LTx is unknown. We, therefore, used the UNOS data set to define the prevalence of LTx following CABG and determine its effect on short and long-term outcomes. Methods: The UNOS STAR dataset was queried to define the prevalence of patients undergoing LTx following CABG through end of 2013. Only adult LTx patients who underwent primary left, right or bilateral LTx were included. The primary end points were 30-day and 1-year survival. Results: During the study period, 24,655 primary lung transplants were performed. CABG became a mandatory reporting field in UNOS in 2004 so our study cohort included 14,791 patients transplanted between 2004 and 2013 (single left (SL) n= 2,787, single right (SR) n= 2,447, bilateral (B) n= 9,557). During that time period, a total of 292 transplants were performed in patients who had previously undergone CABG (SL n= 68, SR n= 181, B n= 43) representing 2% of all transplants. Over the past 10 years, we observed a 4-fold rise in LTx following CABG which increased from 0.5% to 2.5%. SR LTx was the most common type of LTx in CABG patients representing 62% (181/292). Thirty-day survival 95% for the entire cohort (n= 292) with no difference observed between the CABG-LTx and LTx groups. In contrast, 1-year survival was 80% for CABG-LTx cohort compared to 85% for the non-CABG cohort (P= 0.004). CABG persisted as a predictor of 1-year mortality on multivariable analysis (P= 0.03; HR 1.34; CI 1.03, 1.74). Conclusion: LTx following CABG is uncommon but being performed more frequently in the current era. Single right LTx is the most common procedure performed in patients with prior CABG. Despite a higher risk cohort, CABG LTx patients had equivalent short-term and only slightly inferior 1-year survival. This suggests CABG should not be a contraindication to LTx in well-selected patients.

Mechanistic insight into ultrasound induced enhancement of simultaneous saccharification and fermentation of Parthenium hysterophorus for ethanol production

This paper presents investigations into mechanism of ultrasound assisted bioethanol synthesis using Parthenium hysterophorus biomass through simultaneous saccharification and fermentation (SSF) mode. Approach of coupling experimental results to mathematical model for SSF using Genetic Algorithm based optimization has been adopted. Comparison of model parameters for experiments with mechanical shaking and sonication (10% duty cycle) give an interesting mechanistic account of influence of ultrasound on SSF system. A 4-fold rise in ethanol and cell mass productivity is seen with ultrasound. The analysis reveals following facets of influence of ultrasound on SSF: increase in Monod constant for glucose for cell growth, maximal specific growth rate and inhibition constant of cell growth by glucose and reduction in specific cell death rate. Values of inhibition constant of cell growth by ethanol (K3E), and constants for growth associated (a) and non-growth associated (b) ethanol production remained unaltered with sonication. Beneficial effects of ultrasound are attributed to enhanced cellulose hydrolysis, enhanced trans-membrane transport of substrate and products as well as dilution of the toxic substances due to micro-convection induced by ultrasound. Intrinsic physiological functioning of cells remained unaffected by ultrasound as indicated by unaltered values of K3E, a and b.

Safety and immunogenicity of single dose live attenuated varicella vaccine (VR 795 Oka strain) in healthy Indian children: A randomized controlled study

Varicella, an acute viral systemic infection that may cause lifelong latent infection with the potential for causing clinical reactivation, may be prevented by immunization. The present study was an open label, randomized, controlled, phase III, multicentre trial, conducted to evaluate and compare the safety, tolerability and immunogenicity of a freeze dried live attenuated Oka strain Varicella Vaccine (VR 795 Oka strain) with Varilrix (Oka-RIT strain) in children. A total of 268 healthy Indian children aged 12 months to 12 y with baseline VZV IgG antibody (<100 mIU/ mL) were enrolled, and 256 children completed the study. The extent of rise of VZV IgG antibody titer assessed as 3-fold and 4-fold rise from baseline was found to be significantly higher (89.1% and 85.2%) in the test group as compared to control group (73.4% and 61.7%). The post-vaccination GMT of the test group was significantly higher (112.5 mIU/mL) as compared with the control group (67.8 mIU/mL) (P < 0.001). The seroconversion rate considering the 5 gp ELISA units/ml equivalent to 10mIU/ml were similar in the control (96.5%) and the test (98.3%) groups. The adverse events were not different in the control and test groups (P > 0.05). The test live attenuated vaccine was found to be highly immunogenic, safe and comparable to Varilrix used in control arm.

Time to Complete Remission As a Function of Kinetics of White Blood Cell Elimination and Recovery in Acute Myeloid Leukemia Patients Undergoing Remission Induction Chemotherapy

Background: While achieving complete remission (CR) is a major treatment milestone in acute myeloid leukemia (AML) patients (pts) undergoing induction chemotherapy (IC), it is the time to achieve CR (Tc) that has greater prognostic value in predicting subsequent survival. We are particularly interested in seeing whether trends in white blood cell (WBC) count elimination and recovery during induction phase can aid in prediction of Tc, thus enabling real-time prognostication.Methods: Adult pts diagnosed with AML (excluding acute promyelocytic leukemia) at the Cleveland Clinic from 10/08 - 11/12 who underwent IC with 7+3 (cytarabine and anthracycline) and achieved CR were included. Pretreatment variables including age, gender, race, smoking status, body mass index at dx, peripheral blood counts at diagnosis (dx), peripheral and marrow blasts at dx, disease classification and metaphase cytogenetics (per CALGB/Alliance 8461 criteria) were assessed. For mapping WBC elimination and recovery kinetics, we collected data on total WBC, absolute neutrophil count (ANC) and absolute lymphocyte count (ALC) at several different time points after IC – day 1, day 7, day 14 (range, 13-17), day 21, day 35 (range 30-45) and the day CR was achieved. Time intervals selected for analysis included time taken for clearance of peripheral circulating blasts; time to reach nadirs for WBC, ANC and ALC; and time to ANC recovery (from ANC nadir to > 500/µL). Tc was assessed as a function of WBC, ANC and ALC measurements (at the above predefined time points); time intervals; and magnitude of drop and rise in WBC, ANC and ALC (log reductions and improvements in counts) using multivariable logistic and Cox proportional hazards models and stepwise regression using Akaike’s Information Criterion (AIC) for model selection. Due to multiple testing, parameters are reported as significant if p<0.01. Cell counts and time interval metrics were also analyzed as a function of pretreatment covariates using standard linear regression.Results: Of 80 pts, 58% (n=46) were female, 79% (n=63) were Caucasian, 68% (n=54) were younger than 60 years, the median age at treatment was 53.5 years (range, 19 to 77) & 18% (n=14) received 2 cycles of induction therapy. Twenty one pts (26%) had secondary AML. Disease characteristics (per WHO classification) were – AML with recurrent cytogenetic abnormalities 31% (n=25), secondary AML 27% (n=22), therapy-related AML 6% (n=5), AML not otherwise specified 34% (n=26), myeloid sarcoma 1% (n=1) and unknown 1% (n=1). Cytogenetic risk groups per CALGB 8461 were – favorable 19% (n=15), intermediate 53% (n=42), unfavorable 23% (n=18), & unknown cytogenetics (n=5). In the final model of a multivariable stepwise regression analysis, none of the cell counts at the defined time points of IC predicted for Tc. Of pretreatment covariates, only male sex correlated slightly with longer Tc (p=.04). Longer times to reach ANC (p=.01) and ALC (p=.03) nadirs had borderline correlations with longer Tc. In contrast, longer times to reach ANC > 500/µL was significantly associated with delayed Tc (p<<.001). Greater fold change drop and rise in ANC counts correlated with Tc but was not significant at the p<.01 level.Conclusions: In this study the only WBC cell kinetic parameter post IC that independently predicted Tc was time to ANC recovery, which seems intuitive. Biologically, this could represent resiliency of the underlying hematopoietic stem cell reserve which is a function of age, prior therapies or antecedent hematologic disorder. Interestingly, in our series, WBC kinetics did not correlate with the number of induction cycles required (1 vs. 2), age (using age cut-offs of 60 and 70 years) or body mass index. Although the concept of using WBC kinetics during IC as a clinical surrogate for chemotherapy responsiveness or toxicity (overdosing vs. underdosing) is appealing, it is not supported by our data. DisclosuresSekeres:Celgene: Membership on an entity’s Board of Directors or advisory committees; Amgen Corp: Membership on an entity’s Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity’s Board of Directors or advisory committees. Mukherjee:GlaxoSmithKline: Research Funding.