Genetics of asthma: current research paving the way for development of personalized drugs

Asthma is a complex genetic disorder involving the interplay between various environmental and genetic factors. In this review, efforts have been made to provide information on the recent advances in these areas and to discuss the future perspective of research in the area of developing personalized drugs using pharmacogenomic approach. Atopic asthma is found to be strongly familial, however the mode of inheritance is controversial. A large number of studies have been carried out and a number of candidate genes have been identified. In addition, a number of chromosomal regions have been identified using genome-wide scans, which might contain important unknown genes. It has been shown in studies carried out in different populations that the genetic predisposition varies with ethnicity. In other words, genes that are associated with asthma in one population may not be associated with asthma in another population. In addition to the involvement of multiple genes, gene-gene interactions play a significant role in asthma. The importance of environmental factors in asthma is beyond doubt. However, it remains controversial whether a cleaner environment or increased pollution is a trigger for asthma. Despite the increasing prevalence of the disorder, only a limited number of therapeutic modalities are available for the treatment. A number of novel therapeutic targets have been identified and drugs are being developed for better efficacy with less side-effects. With the rapid progress in the identification of genes involved in various ethnic populations combined with the availability in future of well-targeted drugs, it will be possible to have appropriate medicine as per the genetic make-up of an individual

New chemotherapy regimens and biomarkers for Chagas disease: The rationale and design of the TESEO study, an open-label, randomised, prospective, phase-2 clinical trial in the Plurinational State of Bolivia.

Introduction Chagas disease (CD) affects ∼7 million people worldwide. Benznidazole (BZN) and nifurtimox (NFX) are the only approved drugs for CD chemotherapy. Although both drugs are highly effective in acute and paediatric infections, their efficacy in adults with chronic CD (CCD) is lower and variable. Moreover, the high incidence of adverse events (AEs) with both drugs has hampered their widespread use. Trials in CCD adults showed that quantitative PCR (qPCR) assays remain negative for 12 months after standard-of-care (SoC) BZN treatment in ∼80% patients. BZN pharmacokinetic data and the nonsynchronous nature of the proliferative mammal-dwelling parasite stage suggested that a lower BZN/NFX dosing frequency, combined with standard or extended treatment duration, might have the same or better efficacy than either drug SoC, with fewer AEs. Methods and analysis New ThErapies and Biomarkers for ChagaS infEctiOn (TESEO) is an open-label, randomised, prospective, phase-2 clinical trial, with six treatment arms (75 patients/arm, 450 patients). Primary objectives are to compare the safety and efficacy of two new proposed chemotherapy regimens of BZN and NFX in adults with CCD with the current SoC for BZN and NFX, evaluated by qPCR and biomarkers for 36 months posttreatment and correlated with CD conventional serology. Recruitment of patients was initiated on 18 December 2019 and on 20 May 2021, 450 patients (study goal) were randomised among the six treatment arms. The treatment phase was finalised on 18 August 2021. Secondary objectives include evaluation of population pharmacokinetics of both drugs in all treatment arms, the incidence of AEs, and parasite genotyping. Ethics and dissemination The TESEO study was approved by the National Institutes of Health (NIH), U.S. Food and Drug Administration (FDA), federal regulatory agency of the Plurinational State of Bolivia and the Ethics Committees of the participating institutions. The results will be disseminated via publications in peer-reviewed journals, conferences and reports to the NIH, FDA and participating institutions. Trial registration number NCT03981523.We are very grateful to Marcelo Abril, Fundación Mundo Sano, Buenos Aires, Argentina, and Dr. Sergio Sosa-Estani, DNDi, Rio de Janeiro, Brazil, for their continuous support during the elaboration and implementation of this trial; Dr. Martin Springsklee (Medical Affairs Anti-Infectives), Dr. Ulrich-Dietmar Madeja (Head, Neglected Tropical Disease Programmes), and Dr. Maria-Luisa Rodriguez (Global Project Leader) at Bayer AG, Berlin, Germany, and this company for the kind donation of the nifurtimox to be used in this study; Dr. Pedro Albajar Viñas, WHO, for the support to the study through the kind advancement of nifurtimox from the WHO stockpile; Ernesto Palma (Business Development and External Markets Manager) and Luis Ferrero (former ELEA’s Especial Business Manager), at Laboratorio ELEA Phoenix S.A., Buenos Aires, Argentina, and this company for the generous donation of the benznidazole to be used in the TESEO study. We also thank Dr. Soyoung Jeon (currently at the New Mexico State University) and Dr. Xiaogang Su, Dept. of Mathematical Sciences, Border Biomedical Research Center (BBRC), University of Texas at El Paso, for the statistical analyses performed during the TESEO project evaluation by NIH. We are very thankful to all the medical, supporting (nurses, social workers, and laboratory staff) and administrative personnel of the three Chagas Platforms in Bolivia for their technical assistance and dedication in the recruitment, treatment, and follow-up of the CCD patients in this study. We would also like to thank all the staff (postdoctoral fellows, technicians, and administrative personnel) and graduate and undergraduate students of the participating institutions involved in this clinical trial and part of the TESEO Study Group

HDP—A Novel Heme Detoxification Protein from the Malaria Parasite

When malaria parasites infect host red blood cells (RBC) and proteolyze hemoglobin, a unique, albeit poorly understood parasite-specific mechanism, detoxifies released heme into hemozoin (Hz). Here, we report the identification and characterization of a novel Plasmodium Heme Detoxification Protein (HDP) that is extremely potent in converting heme into Hz. HDP is functionally conserved across Plasmodium genus and its gene locus could not be disrupted. Once expressed, the parasite utilizes a circuitous “Outbound–Inbound” trafficking route by initially secreting HDP into the cytosol of infected RBC. A subsequent endocytosis of host cytosol (and hemoglobin) delivers HDP to the food vacuole (FV), the site of Hz formation. As Hz formation is critical for survival, involvement of HDP in this process suggests that it could be a malaria drug target

Identification, Cloning, Expression, and Characterization of the Gene for Plasmodium knowlesi Surface Protein Containing an Altered Thrombospondin Repeat Domain

Proteins present on the surface of malaria parasites that participate in the process of invasion and adhesion to host cells are considered attractive vaccine targets. Aided by the availability of the partially completed genome sequence of the simian malaria parasite Plasmodium knowlesi, we have identified a 786-bp DNA sequence that encodes a 262-amino-acid-long protein, containing an altered version of the thrombospondin type I repeat domain (SPATR). Thrombospondin type 1 repeat domains participate in biologically diverse functions, such as cell attachment, mobility, proliferation, and extracellular protease activities. The SPATR from P. knowlesi (PkSPATR) shares 61% and 58% sequence identity with its Plasmodium falciparum and Plasmodium yoelii orthologs, respectively. By immunofluorescence analysis, we determined that PkSPATR is a multistage antigen that is expressed on the surface of P. knowlesi sporozoite and erythrocytic stage parasites. Recombinant PkSPATR produced in Escherichia coli binds to a human hepatoma cell line, HepG2, suggesting that PkSPATR is a parasite ligand that could be involved in sporozoite invasion of liver cells. Furthermore, recombinant PkSPATR reacted with pooled sera from P. knowlesi-infected rhesus monkeys, indicating that native PkSPATR is immunogenic during infection. Further efficacy evaluation studies in the P. knowlesi-rhesus monkey sporozoite challenge model will help to decide whether the SPATR molecule should be developed as a vaccine against human malarias

Aptamer-based detection of disease biomarkers in mouse models for chagas drug discovery.

Drug discovery initiatives, aimed at Chagas treatment, have been hampered by the lack of standardized drug screening protocols and the absence of simple pre-clinical assays to evaluate treatment efficacy in animal models. In this study, we used a simple Enzyme Linked Aptamer (ELA) assay to detect T. cruzi biomarker in blood and validate murine drug discovery models of Chagas disease. In two mice models, Apt-29 ELA assay demonstrated that biomarker levels were significantly higher in the infected group compared to the control group, and upon Benznidazole treatment, their levels reduced. However, biomarker levels in the infected treated group did not reduce to those seen in the non-infected treated group, with 100% of the mice above the assay cutoff, suggesting that parasitemia was reduced but cure was not achieved. The ELA assay was capable of detecting circulating biomarkers in mice infected with various strains of T. cruzi parasites. Our results showed that the ELA assay could detect residual parasitemia in treated mice by providing an overall picture of the infection in the host. They suggest that the ELA assay can be used in drug discovery applications to assess treatment efficacy in-vivo

Signal transducer and activator of transcription 6 haplotypes and asthma in the Indian population

In this paper, we report for the first time the results of an investigation on the association of signal transducer and activator of transcription 6 (STAT6) with asthma in the Indian population. A novel polymorphic CA-repeat in the proximal promoter region [R1] and a previously identified CA-repeat in the 5'-untranslated region [R3] were genotyped, and haplotypes [R1_R3] were generated using PHASE software. The 16 repeat allele at the R1 locus was positively associated (P = 0.01) with asthma. The 15 and 16 repeat alleles at the R3 locus were positively (P < 10−4) and negatively (P < 10−5) associated with asthma, respectively. Further, the 17_15 (P = 0.0031) and 16_15 (P = 0.001) haplotypes were found to be positively associated with asthma, whereas 17_14, 24_16, and 23_16 were negatively associated (P < 10−5). It appears that the R3 and R1 loci together play a bigger role in asthma than either of them alone, and the R3 locus has a larger effect than the R1 locus. Although alleles at the R1 locus appeared to be associated with total serum immunoglobulin E level, the genotypes showed no association, and the R3 locus showed no effect. As no exonic variants of STAT6 are known as yet, repeat polymorphisms in the regulatory regions and their haplotypes could be important in deciphering the genetic role of STAT6 in asthma and atopy

Aptamer Based, Non-PCR, Non-Serological Detection of Chagas Disease Biomarkers in <i>Trypanosoma cruzi</i> Infected Mice

<div><p>Chagas disease affects about 5 million people across the world. The etiological agent, the intracellular parasite <i>Trypanosoma cruzi (T. cruzi)</i>, can be diagnosed using microscopy, serology or PCR based assays. However, each of these methods has their limitations regarding sensitivity and specificity, and thus to complement these existing diagnostic methods, alternate assays need to be developed. It is well documented that several parasite proteins called <i>T. cruzi</i> Excreted Secreted Antigens (TESA), are released into the blood of an infected host. These circulating parasite antigens could thus be used as highly specific biomarkers of <i>T. cruzi</i> infection. In this study, we have demonstrated that, using a SELEx based approach, parasite specific ligands called aptamers, can be used to detect TESA in the plasma of <i>T. cruzi</i> infected mice. An Enzyme Linked Aptamer (ELA) assay, similar to ELISA, was developed using biotinylated aptamers to demonstrate that these RNA ligands could interact with parasite targets. Aptamer L44 (Apt-L44) showed significant and specific binding to TESA as well as <i>T. cruzi</i> trypomastigote extract and not to host proteins or proteins of <i>Leishmania donovani</i>, a related trypanosomatid parasite. Our result also demonstrated that the target of Apt-L44 is conserved in three different strains of <i>T. cruzi</i>. In mice infected with <i>T. cruzi</i>, Apt-L44 demonstrated a significantly higher level of binding compared to non-infected mice suggesting that it could detect a biomarker of <i>T. cruzi</i> infection. Additionally, Apt-L44 could detect these circulating biomarkers in both the acute phase, from 7 to 28 days post infection, and in the chronic phase, from 55 to 230 days post infection. Our results show that Apt-L44 could thus be used in a qualitative ELA assay to detect biomarkers of Chagas disease.</p></div

Apt-L44 ELA assay can detect mice infected with <i>T. cruzi</i>.

<p>Plasma, at 1∶200 dilution, from groups of mice infected either, the <i>T. cruzi</i> Colombiana (Col., n = 4) or, the 0704 (n = 4) strain, was coated on a polystyrene 96 well plate and ELA assay was performed using biotinylated Apt-L44. Signal generated, relative fluorescence units (RFU), are plotted on the Y-axis and each point represents the mean of duplicate values from individual mice. Group means and standard deviations are shown and statistical significance, compared with the non-infected group (NI, n = 4), determined using the unpaired t-test with Welch correction with a 95% confidence intravel. Biotinylated Apt-L44 showed significant binding to plasma from mice infected with either the Colombiana or the 0704 strains of <i>T. cruzi</i> and showed no binding with plasma from <i>L. donovani</i> infected mice (L.d 1 s, n = 4).</p