Phage display based approach for identifying novel HDL particle binders: Recombinant HDL antibodies as novel diagnostic tool for risk assessment and monitoring of coronary artery disease

Cholesterol content of high density lipoprotein (HDL-C) has been a well established cardiac risk marker for coronary artery disease (CAD), a major cause of mortality worldwide. Various epidemiological and experimental animal studies have shown the inverse correlation between concentration of HDL-C and CAD. However, recent pharmacological studies and genetic studies have demonstrated lack of protection from CAD despite of elevated HDL-C. HDLs are a heterogenous group of particles with varying protein/lipid composition and anti-atherogenic (atheroprotective) function. Atheroprotective HDL particles could turn dysfunctional and thereby attenuate their atheroprotective properties under particular circumstances such as among subjects with metabolic syndrome. Therefore, it is hypothesised that risk assessment and clinical management of atherosclerotic CAD (ATCAD) should target these dysfunctional HDL particles rather than focusing on HDL-C. The aim of this thesis was to develop antibodies against HDL particles by using phage display based antibody library and implement them for development of immunoassay for diagnosis of CAD and its risk estimation in symptomatic and asymptomatic individuals. To that end in publication I, a large set of HDL antibodies were isolated against the HDL derived from CAD patients. These antibodies were characterized against different HDL preparations, most abundant proteins of HDL (i.e. apoA-I and apoA-II) and plasma. A variety of binders were identified. In publication II, three distinct pairs of HDL antibodies which were mainly recognizing the apoA-I were employed to develop three different phage based two-site apoA-I immunoassays identified as assay 022-454, assay 109-121 and assay 110-525. Assays 109-121 and 110-525 showed promise to further improve the diagnostic and predictive value for cardiac conditions. In publication III, simplified versions of two of the phage based two-site immunoassays (109-121 and 110-525) is presented and clinically assessed with a cohort of cardiac patients. Higher level of apoA-I measured with the assay 110-525 showed clear association with ATCAD especially in patients not using lipid lowering medication. In light of these observations, it can be said that that a large number of antibodies were obtained against the intact HDL molecules with phage display approach. The results from testing clinical specimens with the new combinations of HDL binders suggest a different and improved performance over existing test technologies in estimating risk of ATCAD However, further evaluation of these HDL binders with larger cohorts is clearly warranted. In addition, these antibodies could also serve as analytical tools for unravelling the etiology and the pathological process of atherosclerosis in relation to HDL.Uudenlaisten HDL-partikkelia tunnistavien sitojien kehittäminen faaginäyttötekniikan avulla: rekombinanttiset HDL-vasta-aineet uudenlaisina diagnostisina työkaluina sepelvaltimotaudin riskinarvioinnissa ja seurannassa Sepelvaltimotauti on merkittävä kuolinsyy maailmalaajuisesti. Suuritiheyksisen lipoproteiinin kolesterolipitoisuus (high density lipoprotein cholesterol, HDL-C) on laajalti tunnustettu sepelvaltimotaudin riskitekijä. Monet epidemiologiset sekä kokeelliset eläintutkimukset ovat osoittaneet, että HDL-C on käänteisesti yhteydessä sepelvaltimotautiin. Viimeaikaisissa farmakologisissa ja geneettisissä tutkimuksissa ei ole kuitenkaan pystytty osoittamaan, että koholla oleva HDL-C suojelisi sepelvaltimotaudilta. HDL-partikkelit ovat heterogeeninen ryhmä partikkeleja, joiden proteiini- ja lipidikoostumus sekä ateroskleroosilta suojelevat ominaisuudet vaihtelevat. Tietyissä yhteyksissä, kuten esimerkiksi metabolista oireyhtymää sairastavilla, ateroskleroosilta suojaavat HDL-partikkelit voivat muuntua toiminnaltaan, jolloin niiden ateroskleroosilta suojaavat ominaisuudet voivat heikentyä. Tästä syystä on esitetty, että ateroskleroosiin liittyvän sepelvaltimotaudin riskinarvio ja hoito pitäisi perustua toiminnaltaan heikentyneisiin HDL-partikkeleihin HDL-C:n sijaan. Tämän väitöskirjan tavoitteena oli löytää vasta-aineita HDL-partikkeleille faaginäyttötekniikkaan perustuvalla vasta-ainekirjastolla sekä kehittää niitä käyttäen immunomäärityksiä sepelvaltimotaudin diagnostiikkaan ja riskinarviointiin. Näiden tavoitteiden puitteissa julkaisussa I eristettiin suuri ryhmä HDL-vasta-aineita käyttämällä kohteena sepelvaltimotautipotilaiden HDL-partikkeleita. Vasta-aineet karakterisoitiin käyttämällä hyväksi erilaisia HDL-valmisteita, HDL-partikkelien yleisimpiä proteiineja (apoA-I ja apoA-II) ja plasmaa. Eristettyjen vasta-aineiden joukosta tunnistettiin erilaisia vasta-aineita. Julkaisussa II kehitettiin kolme apoA-I-proteiinia tunnistavaa kaksipuolista immunomääritystä (määritys 022-454, määritys 109-121 ja määritys 110-525), joissa hyödynnettiin kolmea keskenään erilaista, pääosin apoA-I -proteiinia tunnistavaa vastaaineparia. Määritykset 109-121 ja 110-525 näyttivät lupaavilta sen suhteen, että ne voisivat pystyä parantamaan sydänsairauksia diagnostiikkaa ja ennustettavuutta. Käsikirjoituksessa III esitetään yksinkertaistetut versiot kahdesta faagipohjaisesta kaksipuolisesta immunomäärityksestä (109-122 ja 110-525) ja arvioidaan niiden kliininen toiminta sydänpotilasryhmän kanssa. Määrityksellä 110-525 mitattu korkeampi apoA-I pitoisuus oli selvästi yhteydessä ateroskleroosiin liittyvään sepelvaltimotautiin erityisesti niillä potilailla, joilla ei ollut lipidejä laskevaa lääkitystä. Näiden havaintojen valossa voidaan todeta, että tutkimuksessa löydettiin faaginäyttötekniikalla useita HDL-molekyylejä tunnistavia vastaaineita. Kliinisillä näytteillä saatujen tulosten perusteella näiden uusien sitojien kombinaatiot näyttävät ennustavan ateroskleroosiin liittyvää sepelvaltimotautia eri tavalla ja paremmin kuin nykyään käytössä olevat testimenetelmät. Tulokset pitää kuitenkin vahvistaa laajemmilla potilasjoukoilla. Tutkimuksessa löydetyt vasta-aineet voisivat lisäksi olla hyödyllisiä analyyttisinä työkaluina tutkittaessa ateroskleroosin etiologiaa ja patologisia prosesseja suhteessa HDL-partikkeleihin

A descriptive analysis of caesarean section rate in a tertiary care hospital according to Robson’s criteria

Background: There has been a rapid increase in rates of caesarean section (CS) in the past decade, leading to increased complications not only in the current pregnancy but also in subsequent pregnancies. Hence Robson’s ten group classification has been used to analyse the indications, scrutinize the rate of CS so that only obstetrically indicated CS are carried out.Methods: The cross-sectional study was carried out for a period of 6 months from 1/7/2019 to 31/12/2019 at Silchar medical college and hospital. All the women ≥28 weeks who delivered during the said period were taken into account. The data was then regrouped according to Robson’s classification.Results: The overall CS rate in our study was 38.09%. Most of the women who underwent CS belonged to age group 20-24 years (49%). 43.26% were nullipara and 91.86% females were term. On analysis of CS according to Robson’s classification, it was observed that group 1 (35.14%) constituted the largest number of women. However, group CS rate was highest in group 9 (100%). Group 5 made the greatest relative (38.83%) as well as absolute contribution (14.79%). Fetal compromise seems to be the leading cause for primary CS.Conclusions: The CS rates in our study have been found to be comparatively higher as the hospital is a tertiary referral centre. Since the maximum contribution has been made by the previous CS group, the rate of primary CS should be reduced and more vaginal birth after CS may be encouraged

Effect of melatonin addition in ovulation induction protocols with clomiphene citrate in management of infertility

Background: About 10-12% of couples are unable to conceive by natural means despite concerted efforts and these figures have risen in past few decades. In recent years, effect of oxidative stress on the fertility has been widely recognised. Oxidative stress has been known to affect both male and female fertility reducing sperm count and motility in men, and affecting ovum reserve and quality in women. Melatonin is the secretion of the pineal gland responsible for circadian sleep rhythm has also shown to be good antioxidant. Aim and objective of current study was to study effect of melatonin addition to clomiphene citrate for induction of ovulation with a view to improve conception rates.Methods: This was a retrospective analysis of 52 women with infertility who were given clomiphene citrate with melatonin. Ovulation, conception rates and pregnancy outcomes were noted.Results: We observed 77.8%ovulation rates, and a significantly better conception and live birth rates.Conclusions: Melatonin, as an anti-oxidant may improve, conception and live birth rates when added to clomiphene citrate induction protocols

Malnutrition among under Five Children in Uttarakhand

BACKGROUND: Globally more than one third of child deaths are attributable to under nutrition. Eighty percent of the world’s undernourished children live in 20 countries, with India being home to nearly 60 million children who are underweight. OBJECTIVE: This study aimed to assess malnutrition among under five children in a selected hospital of Dehradun, Uttarakhand. METHODOLOGY: A quantitative research approach was used with descriptive cross-sectional research design. Non probability convenient sampling technique was used to select 70 under five children who fulfilled the inclusion criteria. The data collection tools included Socio -demographic profile and Anthropometric assessment of the children. The data was analyzed based on objectives by using descriptive and inferential statistics. Data was analyzed using WHO anthroplus software and SPSS 21.0. RESULT: The result showed that out of 70 children 17.14% showed mild wasting, 18.57% showed moderate wasting and (21.43%) showed severe wasting. 11.43% children showed mild stunting, moderate stunting was present in 15.71% children and severe stunting was present in 27.14% children. 31.43% children were moderately underweight and 30% were severely underweight. CONCLUSION: The study concluded that majority of the children were malnourished

Management practices for west syndrome in south Asia: A survey study and meta-analysis

Objectives: Considering the dearth of literature on West syndrome (WS) from South Asian countries, this study aimed to evaluate the management practices in South Asia by an online survey and meta-analysis.Methods: An online questionnaire was sent to 223 pediatric neurologists/pediatricians in India, Pakistan, Myanmar, Sri Lanka, Bhutan, Nepal, and Bangladesh. Their responses were evaluated and supplemented by a meta-analysis.Results: Of 125 responses received (response rate: 56%), around 60% of responders observed male preponderance and an approximate lead-time-to-treatment (LTTT) of 4-12 weeks. The commonest etiology observed was a static structural insult (88.6% of responders). Most commonly used first-line drug (country-wise) was as follows: India-adrenocorticotropin hormone (ACTH, 50%); Pakistan-oral steroids (45.5%); Myanmar, Sri Lanka, and Nepal-oral steroids (94.4%); Bangladesh-ACTH (2/2); Bhutan-vigabatrin (3/5). ACTH and vigabatrin are not available in Myanmar and Nepal. The most commonly used regime for ACTH was maximal-dose-at-initiation-regime in India, Sri Lanka, and Bangladesh and gradually escalating-regime in Pakistan. Maximum dose of prednisolone was variable-most common response from India: 3-4 mg/kg/d; Pakistan, Bhutan, and Bangladesh: 2 mg/kg/d; Sri Lanka, Nepal, and Myanmar: 5-8 mg/kg/d or 60 mg/d. The total duration of hormonal therapy (including tapering) ranged from 4 to 12 weeks (67/91). Most responders considered cessation of spasms for four weeks as complete response (54/111) and advised electroencephalography (EEG; 104/123) to check for hypsarrhythmia resolution. Difficult access to pediatric EEG in Bhutan and Nepal is concerning. More than 95% of responders felt a need for more awareness. The meta-analysis supported the preponderance of male gender (68%; confidence interval [CI]: 64%-73%), structural etiology(80%; CI 73%-86%), longer LTTT (2.4 months; CI 2.1-2.6 months), and low response rate to hormonal therapy(18% and 28% for ACTH and oral steroids respectively) in WS in South Asia.Significance: This study highlights the practices and challenges in the management of WS in South Asia. These include a preponderance of male gender and structural etiology, a longer LTTT, difficult access to pediatric EEG, nonavailability of ACTH and vigabatrin in some countries, and low effectiveness of hormonal therapy in this region

Analysis of Throughput in Infrastructure based Multi-Radio Network

This paper deals with the design of an infrastructure based network consisting of Multi-radio Hybrid mobile nodes that perform traffic splitting over the network. The multi- radio mobile nodes have a WiMAX and a WiFi Radio that are used in transmitting data traffic over two different radio channels. Data traffic is split statically over a node and transmitted over the two radio channels. We analyse the Throughput and End-to-End delay for data transmission in the network

Three two-site apoA-I immunoassays using phage expressed detector antibodies - Preliminary clinical evaluation with cardiac patients

High density lipoproteins (HDL) are a heterogenous group of subpopulations differing in protein/lipid composition and in their anti-atherogenic function. There is a lack of specific and robust assays which can target the functionality of HDL with respect to atherosclerosis. With recently generated CAD HDL targeted, single chain recombinant antibodies (scFvs) we set out to design and optimize apo A-I tests to compare it with conventional HDL-C and apo A-I analyses for diagnosis and risk assessment of coronary artery disease (CAD) and its outcome. Three highly sensitive two-site apo A-I assays: 022-454, 109-121 and 110-525 were optimized. A preliminary clinical evaluation of these assays, after proper sample dilution procedure, was performed using samples derived from 195 chest pain patients (myocardial infarction (MI), n = 86 and non-MI, n = 109), collected at the time of admission and at discharge from hospital (hospital stay <= 24 h). The clinical performance of the assays was compared with apo A-I measured with polyclonal anti-apo A-I antibody using conventional ELISA. Apo A-I data was in addition compared with HDL-C concentration of the samples. The concentration of apo A-I was significantly lower in MI patients than in non-MI individuals with assay 022-454 (admission and discharge samples, P < 0.0001 and = 0.004); assay 109-121 (admission and discharge samples, P = 0.04 and 0.0009), and, ELISA based apo A-I test (admission and discharge samples, P = 0.008 and < 0.0001). HDL-C (admission and discharge samples, P = 0.002 and P = 0.01) was also significantly lower in MI patients. In Kaplan- Meier analysis, two-site assay 109-121 assay predicted mortality from admission samples at 1.5 yrs (whole cohort, P = 0.01 and in MI patients, P = 0.05) and at 6 months (whole cohort, P = 0.04). Assay 110-525 predicted mortality at 1.5 yrs from admission samples of non-MI patients (P = 0.01) and at 6 months from whole discharge sample cohort (P = 0.04). Polyclonal anti-apo A-I based conventional assay predicted mortality at 1.5 yrs from admission samples of whole cohort (P = 0.03). Two-site apo A-I assay 022-454 and HDL-C provided no capability of predicting mortality in the whole cohort or any sub-group. In conclusion, two of the tested recombinant apo A-I antibody combinations (sc 109-121 and sc 110-525) display promising outcome to improve diagnosis and prediction of future cardiac events in cardiac patients over polyclonal apo A-I ELISA and HDL-C assays. The noted differences, while interesting, are preliminary and need however to be verified in extensive cohorts of pathological cardiac conditions and healthy controls. (C) 2020 The Authors. Published by Elsevier B.V

Time-resolved fluorescence based direct two-site apoA-I immunoassays and their clinical application in patients with suspected obstructive coronary artery disease

Objective: High-density lipoprotein (HDL) is a heterogeneous group of subpopulations differing in protein/lipid composition and in their anti-atherogenic function. There is a lack of assays that can target the functionality of HDL particles related to atherosclerosis. The objective of this study was to construct two-site apolipoprotein A-I (apoA-I) assays and to evaluate their clinical performance in patients with suspected obstructive coronary artery disease (CAD).Approach and results: Direct two-site apoA-I assays (named 109–121 and 110–525) were developed to identify the presence of apoA-I in the HDL of patients with CAD using apoA-I antibodies as a single-chain variable fragment fused with alkaline phosphatase. ApoA-I109−121 and apoA-I110−525 were measured in 197 patients undergoing coronary computed tomography angiography (CTA) and myocardial positron emission tomography perfusion imaging due to suspected obstructive CAD. Among patients not using lipid-lowering medication (LLM, n = 125), the level of apoA-I110−525 was higher in the presence than in the absence of coronary atherosclerosis [21.88 (15.89–27.44) mg/dl vs. 17.66 (13.38–24.48) mg/dl, P = 0.01)], whereas there was no difference in apoA-I109−121, HDL cholesterol, and apoA-I determined using a polyclonal apoA-I antibody. The levels of apoA-I109−121 and apoA-I110−525 were similar in the presence or absence of obstructive CAD. Among patients not using LLM, apoA-I110−525 adjusted for age and sex identified individuals with coronary atherosclerosis with a similar accuracy to traditional risk factors [area under the curve [AUC] (95% CI): 0.75(0.66–0.84) 0.71 (0.62–0.81)]. However, a combination of apoA-I110−525 with risk factors did not improve the accuracy [AUC (95% CI): 0.73 (0.64–0.82)].Conclusion: Direct two-site apoA-I assays recognizing heterogeneity in reactivity with apoA-I could provide a potential approach to identify individuals at a risk of coronary atherosclerosis. However, their clinical value remains to be studied in larger cohorts.</p

Gaia GraL: Gaia DR2 Gravitational Lens Systems. VII. XMM-Newton Observations of Lensed Quasars

© 2022. The Author(s). Published by the American Astronomical Society. This is the accepted manuscript version of an article which has been published in final form at https://doi.org/10.3847/1538-4357/ac4476We present XMM-Newton X-ray observations of nine confirmed lensed quasars at 1 ≲ z ≲ 3 identified by the Gaia Gravitational Lens program. Eight systems are strongly detected, with 0.3-8.0 keV fluxes F 0.3-8.0 ≳ 5 ×10-14 erg cm-2 s-1. Modeling the X-ray spectra with an absorbed power law, we derive power-law photon indices and 2-10 keV luminosities for the eight detected quasars. In addition to presenting sample properties for larger quasar population studies and for use in planning for future caustic-crossing events, we also identify three quasars of interest: a quasar that shows evidence of flux variability from previous ROSAT observations, the most closely separated individual lensed sources resolved by XMM-Newton, and one of the X-ray brightest quasars known at z > 3. These sources represent the tip of the discoveries that will be enabled by SRG/eROSITA.Peer reviewe

Gaia GraL: Gaia DR2 gravitational lens systems – VIII. A radio census of lensed systems

© 2024 The Author(s). Published by Oxford University Press on behalf of Royal Astronomical Society. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/We present radio observations of 24 confirmed and candidate strongly lensed quasars identified by the Gaia Gravitational Lenses working group. We detect radio emission from eight systems in 5.5 and 9 GHz observations with the Australia Telescope Compact Array (ATCA), and 12 systems in 6 GHz observations with the Karl G. Jansky Very Large Array (VLA). The resolution of our ATCA observations is insufficient to resolve the radio emission into multiple lensed images, but we do detect multiple images from 11 VLA targets. We have analysed these systems using our observations in conjunction with existing optical measurements, including measuring offsets between the radio and optical positions for each image and building updated lens models. These observations significantly expand the existing sample of lensed radio quasars, suggest that most lensed systems are detectable at radio wavelengths with targeted observations, and demonstrate the feasibility of population studies with high-resolution radio imaging.Peer reviewe