Biology subjects

Kshetrapal, P.

Publications and source records attributed to Kshetrapal, P..

6 recordsLinked to original sources

bioRxiv2023

Abnormal trophoblast invasion in early onset preeclampsia: involvement of cystathionine β-synthase, specificity protein 1 and miR-22

IntroductionImpaired trophoblast invasion has been observed in early onset Preeclampsia patients (EOPE). Trophoblast cell invasion during human placentation is majorly regulated by the balance between MMPs 2, 9 and their inhibitors [tissue inhibitors of matrix metalloproteinases (TIMPs 1, 2)]. Exogenous NaHS (hydrogen sulphide donor) treatment was shown to significantly increase the expression levels of matrix metalloproteinases (MMPs 2, 9) in human bladder cancer EJ cells. Epigentically, the gene expression of hydrogen sulphide synthesising enzyme CBS (cystathionine {beta}-synthase) could be further regulated by various mi-RNAs via the transcription factors like Sp1. Specificity protein 1 (Sp1) has been identified as a target gene for miR-22 to regulate the invasion and metastasis of gastric cancer cells. However, the mechanism of MMPs regulation by either CBS, Sp1 and miRNA-22 in the pregnancies having EOPE is not known. Aims and ObjectivesTo determine and compare the expression of MMPs 2, 9, TIMPs 1, 2, CBS, Sp1 and miRNA-22 in EOPE patients and normotensive, non-proteinuric controls. Materials and methods100 pregnant women were enrolled from Department of Obstetrics and Gynaecology, AIIMS, New Delhi, India. EOPE women (n=50) after clinical diagnosis as per ACOG guidelines were enrolled as cases and normotensive, non-proteinuric pregnant women (n=50) were enrolled as controls. Protocol of the study was approved by Institute Ethics Committee, AIIMS, New Delhi. 5 ml of venous blood was collected from all the recruited women (2.5 ml in each EDTA and sera vial) followed by plasma and sera separation. Plasma samples were used subsequently to determine gene expression of MMPs 2, 9, TIMPs 1, 2, CBS, Sp1 and miRNA-22 by qRT-PCR and sera samples were used to estimate their protein levels by ELISA. Data were analyzed by STATA 14 and Graph Pad Prism 8. ResultsSignificantly down regulated expression of MMPs 2, 9, CBS and Sp1 whereas up regulated expression for that of TIMPs 1, 2 was observed in EOPE patients as compared to healthy pregnant women at both transcription and translation levels. Expression of miR-22 (pre miR-22 and miR-22-3p) was found to be significantly elevated in EOPE patients as compared to normotensive, non-proteinuric controls. ConclusionThis is the very first study of its kind which implicates that down regulated MMPs 2, 9, CBS, Sp1 levels and simultaneously upregulation of miR-22 expression in EOPE patients could have some association. In vitro experiments are needed to prove their association which if proven may provide a new, potential therapeutic target to treat early onset Preeclampsia.

developmental biology↗

bioRxiv2023

MicroRNA-22, Specificity protein-1 and Cystathionine β-synthase in early onset Preeclampsia: significance in trophoblast invasion

IntroductionTrophoblast cell invasion during human placentation is majorly regulated by the balance between MMPs 2, 9 and their inhibitors [tissue inhibitors of matrix metalloproteinases (TIMPs 1, 2)]. Exogenous NaHS (hydrogen sulphide donor) treatment was shown to significantly increase the expression levels of matrix metalloproteinases (MMPs 2, 9) in human bladder cancer EJ cells. Epigentically, the gene expression of hydrogen sulphide synthesising enzyme CBS (cystathionine {beta}-synthase) could be further regulated by various mi-RNAs via the transcription factors like Sp1. Specificity protein 1 (Sp1) has been identified as a target gene for miR-22 to regulate the invasion and metastasis of gastric cancer cells. However, the mechanism of MMPs regulation by either CBS, Sp1 and miRNA-22 in the pregnancies having early onset preeclampsia is not known. Aim of the studyTo determine and compare the expression of MMPs 2, 9, TIMPs 1, 2, CBS, Sp-1 and miR-22 in early onset preeclamptic patients and normotensive, non-proteinuric controls at both transcription and translation levels. Materials and methods30 pregnant women were enrolled from Department of Obstetrics and Gynaecology, AIIMS, New Delhi, India. EOPE women (n=15) after clinical diagnosis as per ACOG guidelines were enrolled as cases and normotensive, non-proteinuric pregnant women (n=15) were enrolled as controls. Protocol of the study was approved by Institute Ethics Committee, AIIMS, New Delhi. 30 caesarean delivered placentae (15 each of patients and controls) were collected to analyse the mRNA and protein expression/levels of MMPs 2, 9, TIMPs 1, 2, CBS, and Sp1. Protein activity of MMP-2 and 9 was determined. Gene expression of miR-22 was observed in placentae of the recruited patients and controls. Data were analysed by STATA 14 and Graph Pad Prism 8. ResultsSignificantly reduced mRNA and protein expression/levels of MMPs 2, 9, CBS and Sp1 whereas elevated for those of TIMP-1 and TIMP-2 was observed in EOPE patients as compared to controls. Gelatinolytic activity of MMP-2 and 9 was also found significantly reduced in placentae from EOPE patients whereas gene expression of miR-22 was found significantly up regulated in the early onset preeclamptic patients in comparison to controls. ConclusionThis is the first study of its kind which implicates that insufficient trophoblastic invasion may be because of down regulation of MMPs 2, 9, CBS, Sp1 and concomitant up regulation of TIMPs 1, 2 and miR-22 in the early onset preeclamptic patients as compared to controls.

developmental biology↗

bioRxiv2023

Role of MicroRNA-22 (miR-22)/Specificity protein 1 (Sp1)/Cystathionine beta-synthase (CBS) axis in regulating trophoblast invasion: an in vitro study

IntroductionPlacenta expresses many ubiquitous as well as specific miRNAs which regulate trophoblast cell differentiation, proliferation, apoptosis, invasion, migration, and angiogenesis. Aberrant miRNAs expression (miR-22) has been linked to pregnancy complications, such as preeclampsia. Specificity protein 1 (Sp1) was identified a direct target of miR-22 and an inverse linear correlation between expression of miR-22 and Sp1 mRNA in gastric tumour has been reported. Also, Sp1 has a critical and indispensable role in the regulation of cystathionine {beta}-synthase (H2S synthesizing enzyme). Exogenous H2S could promote cell proliferation and invasion by upregulating the expression of MMP-2 and MMP-9 in human bladder cancer EJ cells. Worldwide studies reported significant role of MMP-2 and MMP-9 in the regulation of trophoblast invasion. Aims and ObjectivesTo determine the role of MicroRNA-22 (miR-22)/Specificity protein 1 (Sp1)/Cystathionine {beta}-synthase (CBS) axis in regulating trophoblast invasion in the first-trimester trophoblast cells. Materials and methodsThe human first trimester trophoblast cell line (HTR-8/SVneo Cell line) was procured from American Type Culture Collection. Gain/Loss of function studies were conducted on HTR-8/SVneo cells and the expression of miR-22 (pre miR-22 and miR-22-3p), Sp1, CBS, MMP-2 and MMP-9 was observed. Invasion capacity of the cells was assessed by transwell invasion assay. Data were analyzed by STATA 14 and Graph Pad Prism 8. ResultsSignificantly down regulated regulated mRNA and protein expression of Sp1, CBS and and MMPs 2, 9 was observed when the cells were transfected with miR-22-3p mimic in comparison to miR-22-3p inhibitor transfected cells. Expression of CBS and MMPs 2, 9 was found to be significantly up regulated when the cells were transfected with Sp1 plasmid as compared to Sp1 inhibitor (2,4,5 trifluoroaniline) treated cells at both transcription and translation levels. MMP-2 and MMP-9 levels were found to be significantly elevated when the cells were subjected to NaHS [sodium hydrogen sulphide (H2S donor)] treatment as compared to AOAA (aminooxyacetic acid) treated cells. ConclusionThis is the first study which implies that miR-22 via targeting Sp1 must have influenced the mRNA and protein expression of CBS, MMP-2 and MMP-9 in order to regulate the trophoblast invasion.

developmental biology↗

bioRxiv2023

Altered expression of MMP9 and TIMP1 leads to impaired placentation in early onset preeclamptic patients: a case control study

IntroductionMatrix metalloproteinases (MMPs) specifically MMP-9 is a key regulator of vascular and uterine spiral artery remodelling and its activity is controlled at multiple levels, including gene transcription, activation of its latent forms and endogenous inhibition by tissue inhibitors of metalloproteinases [TIMPs (specifically TIMP-1)]. Alteration in MMP-9 and TIMP-1 expression may contribute to uterine and vascular dysfunction leading to adverse pregnancy outcomes such as Preeclampsia (PE). Depending on time, PE is classified as early-onset preeclampsia, which is diagnosed before 34 weeks of gestation, or late-onset preeclampsia, which is diagnosed after 34 weeks. Early-onset preeclampsia (EOPE) is a severe obstetrics disease which threatens mother and foetus. Therefore, in the present study, we aimed to investigate the levels of MMP-9 and its inhibitor TIMP-1 in the placentae of EOPE patients and their maternal age matched normotensive, non-proteinuric controls at both mRNA and protein levels. MethodsA total of 30 caesarean delivered placentae (15 EOPE patients and 15 controls) were collected from Department of Obstetrics and Gynaecology, AIIMS, New Delhi after taking permission from Institute Ethics Committee. MMP-9 and TIMP-1 protein expression was observed by immunohistochemistry and immunofluorescence stainings. Gelatin Gel Zymography was done to evaluate MMP-9 activity and Western Blot was done for the quantification of MMP-9 and TIMP-1 proteins. mRNA levels of MMP-9 and TIMP-1 were determined by qRT-PCR ResultsImmunohistochemistry and Immunofluorescence staining discerned stronger expression of MMP-9 in normotensive placentae as compared to EOPE placentae whereas stronger expression of TIMP-1 was seen in EOPE placentae in comparison to those of normotensive placentae. Gelatin Gel Zymography reflected that MMP-9 activity was found elevated in maternal placental side in normotensive placentae as compared to EOPE placentae. Western Blot analysis revealed that protein expression of MMP-9 was elevated whereas for that of TIMP-1 was reduced in normotensive placentae in comparison to EOPE placentae. mRNA expression of MMP-9 was found up-regulated whereas for that of TIMP-1 was down-regulated in normotensive placentae as compared to EOPE placentae. ConclusionThe present study implies that aberrant functioning of MMP-9 and TIMP-1 in EOPE patients contribute to impaired placentation which might be relevant for possible future screening programs in order to predict and to design therapies for early onset preeclamptic patients.

developmental biology↗

bioRxiv2022

Ancestral SARS-CoV-2 driven antibody repertoire diversity in an unvaccinated individual correlates with expanded neutralization breadth

Understanding the quality of immune repertoire triggered during natural infection can provide vital clues that form the basis for development of humoral immune response in some individuals capable of broadly neutralizing pan SARS-CoV-2 variants. We assessed the diversity of neutralizing antibody responses developed in an unvaccinated individual infected with ancestral SARS-CoV-2 by examining the ability of the distinct B cell germline-derived monoclonal antibodies (mAbs) in neutralizing known and currently circulating Omicron variants by pseudovirus and authentic virus neutralization assays. The ability of the antibodies developed post vaccination in neutralizing Omicron variants was compared to that obtained at baseline of the same individual and to those obtained from Omicron breakthrough infected individuals by pseudovirus neutralization assay. Broadly SARS-CoV-2 neutralizing mAbs representing unique B cell lineages with non-overlapping epitope specificities isolated from a single donor varied in their ability to neutralize Omicron variants. Plasma antibodies developed post vaccination from this individual demonstrated neutralization of Omicron BA.1, BA.2 and BA.4 with increased magnitude and found to be comparable with those obtained from other vaccinated individuals who were infected with ancestral SARS-CoV-2. Development of B cell repertoire capable of producing antibodies with distinct affinity and specificities for the antigen immediately after infection capable of eliciting broadly neutralizing antibodies offers highest probability in protecting against evolving SARS-CoV-2 variants. ImportanceDevelopment of robust neutralizing antibodies in SARS-CoV-2 convalescent individuals is known, however varies at population level. We isolated monoclonal antibodies from an individual infected with ancestral SARS-CoV-2 in early 2020 that not only varied in their B cell lineage origin but also varied in their capability and potency to neutralize all the known VOC and currently circulating Omicron variants. This indicated establishment of unique lineages that contributed in forming B cell repertoire in this particular individual immediately following infection giving rise to diverse antibody responses that could compensate each other in providing broadly neutralizing polyclonal antibody response. Individuals who were able to produce such potent polyclonal antibody responses after infection have a higher chance of being protected from evolving SARS-CoV-2 variants.

immunology↗

bioRxiv2021

A combination of potently neutralizing monoclonal antibodies isolated from an Indian convalescent donor protects against the SARS-CoV-2 delta variant

Although efficacious vaccines have significantly reduced the morbidity and mortality due to COVID-19, there remains an unmet medical need for treatment options, which monoclonal antibodies (mAbs) can potentially fill. This unmet need is exacerbated by the emergence and spread of SARS-CoV-2 variants of concern (VOCs) that have shown some resistance to vaccine responses. Here we report the isolation of two highly potently neutralizing mAbs (THSC20.HVTR04 and THSC20.HVTR26) from an Indian convalescent donor, that neutralize SARS-CoV-2 VOCs at picomolar concentrations including the delta variant (B.1.617.2). These two mAbs target non-overlapping epitopes on the receptor-binding domain (RBD) of the spike protein thereby preventing the virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Furthermore, the mAb cocktail demonstrated protection against the Delta variant at low antibody doses when passively administered in the K18 hACE2 transgenic mice model, highlighting their potential as cocktail for prophylactic and therapeutic applications. Developing the capacity to rapidly discover and develop mAbs effective against highly transmissible pathogens like coronaviruses at a local level, especially in a low- and middle-income country (LMIC) such as India, will enable prompt responses to future pandemics as an important component of global pandemic preparedness. HighlightsO_LIIdentification of an Indian convalescent donor prior to emergence of SARS-CoV-2 Delta variant whose plasma demonstrated neutralization breadth across SARS-CoV-2 variants of concern (VOCs). C_LIO_LITwo (THSC20.HVTR04 and THSC20.HVTR26) monoclonal antibodies isolated from peripheral memory B cells potently neutralize SARS-CoV-2 VOCs: Alpha, Beta, Gamma, Delta and VOIs: Kappa and Delta Plus. C_LIO_LITHSC20.HVTR04 and THSC20.HVTR26 target non-competing epitopes on the receptor binding domain (RBD) and represent distinct germline lineages. C_LIO_LIPassive transfer of THSC20.HVTR04 and THSC20.HVTR26 mAbs demonstrated protection against Delta virus challenge in K18-hACE2 mice at low antibody doses. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/474152v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1f1b55corg.highwire.dtl.DTLVardef@1b9b438org.highwire.dtl.DTLVardef@e6d2a6org.highwire.dtl.DTLVardef@f92cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗