Javaid Gadahi - Academia.edu (original) (raw)

Papers by Javaid Gadahi

Amplification efficiencies of target gene (API) and endogenous reference (β-Tubulin) gene were ve... more Amplification efficiencies of target gene (API) and endogenous reference (β-Tubulin) gene were verified to be similar by real time PCR. Figure S2. Amplification efficiencies of target genes (IL-2, IL-4, IL-10, Il-17, IFN-γ and TGF-β) and endogenous reference (β-Actin) gene were verified to be similar by real time PCR. (DOCX 57 kb)

Primers for amplification of API gene. Table S2. Primers for real time PCR. Table S3. Primers for... more Primers for amplification of API gene. Table S2. Primers for real time PCR. Table S3. Primers for quantification of cytokines transcription. (DOCX 18Â kb)

Cloning and expression of Hc-AK gene. The recombinant plasmid pMD19T-AK (a) and expression plasmi... more Cloning and expression of Hc-AK gene. The recombinant plasmid pMD19T-AK (a) and expression plasmid pET32a (+)-AK (b), were verified by restriction digestion with BamH I and EcoR I. Figure S2. Multiple sequence alignment of Hc-AK. Amino acid sequence of Hc-AK with that from other species, H. contortus (CDJ90032), C. briggsae (XP_002645008), C. brenneri (EGT52941), C. elegans (NP_509217), H. glycines (AAO49799), O. dentatum (KHJ89945), A. duodenale (KIH65495), D. viviparus (KJH41917), V. pacos (XP_006219889) and N. americanus (XP_013303820) using CLUSTAL W method and GeneDoc ( http://www.psc.edu/biomed/genedoc/ ). Figure S3. Phylogenetic analysis for Hc-AK gene. A phylogenetic tree was constructed by neighbour -joining method to verify relationships between the amino acid of Hc-AK to that of other nematode species, using MEGA ver. 6.1 programme. Figure S4. N-terminal signal peptide prediction. The amino acid sequences of Hc-AK (NCBI accession numbers: JX422018.1 was used to predict N-...

Parasites & Vectors, 2020

Background Methyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed... more Background Methyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals. MTFs proteins have been associated with various biological/cellular processes including transcriptional regulation, subcellular protein and RNA localization, signal transduction and DNA-damage repair. However, the role of MTFs in immune mechanism during host–parasite interaction has not been addressed yet. Results An open reading frame (764 bp) of methyltransferase-type 12 gene of H. contortus denoted as HcMTF-12, was successfully cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by prokaryotic expression in Escherichia coli BL21 (DE3 strain). The recombinant HcMTF-12 protein (rHcMTF-12) was about 47 kDa along with a fusion vector protein of 18 kDa. Immunoblot results identified the native protein MTF-12 with antibodies produced in rats against rHcMT-12, whereas rHcMTF-12 protein was recognized with sera...

Veterinaria, 2019

Babesia spp. are tick-transmitted apicomplexan parasites that infect erythrocytes of a wide range... more Babesia spp. are tick-transmitted apicomplexan parasites that infect erythrocytes of a wide range of vertebrates including domestic animals, and can be associated with a considerable economic loss due to introduction of prophylactic measures, decreased production and rates of morbidity and mortality. An efficient detection assay needs to be set up for accurate diagnosis and prevention of disease. The present work is aimed at verifying the occurrence of Babesia bigemina infection in water buffaloes in the southeastern region of Pakistan by using both traditional blood smear and molecular techniques. A total of 100 blood samples were collected from tick-free and tick-infested animals (50 animals each group). The overall prevalence was recorded as 20% and 17% by using thin blood smear and PCR methods, respectively. Moreover, the analysis of infection in tick-infested and tick-free animals showed the infection rates of 36% and 4% by using blood smear method, and 30% and 4% with that of ...

Research in Veterinary Science, 2021

Previously, it was found that several proteins of Haemonchus contortus were involved in the stimu... more Previously, it was found that several proteins of Haemonchus contortus were involved in the stimulation of the host immune system. However, the information about the selection of superlative antigens with immunogenic efficacies on host DCs is lacking. In the current study, the stimulatory effects of five recombinant proteins (elongation factor-1α, arginine kinase, ES-15, ES-24, and ADP-ribosylation factor 1) of H. contortus on the maturation of goat monocyte-derived dendritic cells (md-DCs) were reported. Recombinant proteins were purified separately in E. coli expression and incubated with isolated goat peripheral blood mononuclear cells (PBMC). Immunofluorescence assay (IFA) results confirmed the binding of these molecules to the md-DC's surface as compared to control groups. In the flow cytometry analysis, recombinant proteins induced md-DC stimulation via the up-regulation of the expression of the costimulatory molecule (CD80) and MHC-II. Quantitative RT-PCR data showed a significant increase in the expression of specific genes of the WNT and toll-like receptor (TLR) signaling pathways. The result of ELISA indicated the higher levels of cytokine (IL-10, IL-12, IFN-γ, and TNF-α) secretion in the md-DC compared to the negative (pET-32a His-Tag) and blank (PBS) control groups. The data gives valuable support in the selection of potential antigens for future studies on the immunomodulation of the host against the infection of H. contortus.

Vaccines, 2020

ADP-ribosylation factor 1 (HcARF1) is one of the Haemonchus contortus (H. contortus) excretory/se... more ADP-ribosylation factor 1 (HcARF1) is one of the Haemonchus contortus (H. contortus) excretory/secretory proteins involved in modulating the immune response of goat peripheral blood mononuclear cells (PBMC). Here, we evaluated the immunogenic potential of recombinant HcARF1 (rHcARF1) against H. contortus infection in Institute of Cancer Research (ICR) mice. Briefly, rHcARF1 was entrapped in poly (D, L-lactide-co-glycolide) (PLGA) and chitosan (CS) nanoparticles (NP) and injected into mice as a vaccine. Fifty-six ICR mice were assigned randomly into seven groups, with eight animals in each group, and they were vaccinated subcutaneously. At the end of the experiment (14th day), the blood and the spleen were collected from euthanized mice to detect lymphocyte proliferation, cytokine analysis, and the production of antigen-specific antibodies. Scanning electron microscope was used to determine the size, morphology, and zeta potential of nanoparticles. Flow cytometry was performed, which...

Background Probiotics and Medicinal plants are widely used as an alternative to treat different k... more Background Probiotics and Medicinal plants are widely used as an alternative to treat different kinds of the acute and chronic diseases in all over the world. Bidens pilosa is commonly used as foods and therapeutics against various pathogens including protozoan. In the present study probiotics and Bidens pilosa were used to explore the anticoccidial activity during experimental infection of Eimeria tenella ( E. tenella ) in chicken Methods One hundred and fifty one-day-old Chinese yellow breed chickens were divided into five equal groups. Group 1 was kept as control group (CG; un-treated, unchallenged). All other groups were inoculated with E. tenella sporulated oocysts on day 21 of age. Group 2 was kept as positive control (CPG; un-treated, challenged). Group 3 was treated with probiotics (PG) at the rate of 1g/kg of feed. Group 4 was treated with Bidens pilosa (BPG) at the dose of 0.5%/kg of feed. Group 5 was kept as probiotics + Bidens pilosa group (PG + BPG). All birds were weig...

Parasite Immunology, 2020

This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Pathogens, 2020

Small size excretory/secretory (ES) antigens of the Haemonchus contortus parasite have intense in... more Small size excretory/secretory (ES) antigens of the Haemonchus contortus parasite have intense interest among researchers for understanding the molecular basis of helminths immune regulation in term of control strategies. Immunomodulatory roles of H. contortus ES-15 kDa (HcES-15) on host immune cells during host–parasite interactions are unknown. In this study, the HcES-15 gene was cloned and expression of recombinant protein (rHcES-15) was induced by isopropyl-ß-d-thiogalactopyranoside (IPTG). Binding activity of rHcES-15 to goat peripheral blood mononuclear cells (PBMCs) was confirmed by immunofluorescence assay (IFA) and immunohistochemical analysis showed that H. contortus 15 kDa protein localized in the outer and inner structure of the adult worm, clearly indicated as the parasite’s ES antigen. The immunoregulatory role on cytokines production, cell proliferation, cell migration, nitric oxide (NO) production, apoptosis, and phagocytosis were observed by co-incubation of rHcES-1...

Biomolecules, 2020

Galectins are glycan-binding proteins that are widely expressed and distributed in mammalian tiss... more Galectins are glycan-binding proteins that are widely expressed and distributed in mammalian tissues as well as cells of innate and adaptive immune responses. CD4+ T-helper cells differentiate into effector subsets in response to cytokines. T helper 9 cells are one of the recently described subsets of effector T cells that are relatively new and less studied. In this study, galectin domain containing protein from Haemonchus contortus (Hc-GDC) was cloned, expressed in pET32a, and immunoblotting was performed. Localization of recombinant (r)Hc-GDC on outer and inner surface of H. contortus worm and binding with goat Peripheral Blood Mononuclear cells (PBMCs) were performed using immunofluorescence assay. Moreover, effects of rHc-GDC on proliferation, apoptosis, cell migration, and the nitric oxide production in goat PBMCs were evaluated. Furthermore, modulatory effects of rHc-GDC on production of Th1, Th2, and Th9 cells were evaluated by flowcytometry and on interferon gamma, interleu...

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Frontiers in immunology, 2018

Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/sec... more Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/secretory proteins, are assumed to be released during the host-parasite interactions. However, knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host peripheral blood mononuclear cells (PBMCs) is scant. In this study, an open reading frame of STP from designated as HcSTP-1 was amplified and cloned using reverse-transcription-polymerase chain reaction (RT-PCR) method. The 951-bp nucleotides sequence was encoded to a protein of 316 amino acid residues, conserved in characteristics motifs GDXHG, GDYVDRG, GNHE, HGG, RG, and H. The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunohistochemically, HcSTP-1 was found to be localized in both male and female adult worm sections. Using immunofluorescence ass...

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Oncotarget, Jan 22, 2017

ADP-ribosylation factors (ARFs) are members of the Ras-related small GTPase family involved in th... more ADP-ribosylation factors (ARFs) are members of the Ras-related small GTPase family involved in the vesicular trafficking regulation. Immunomodulatory effects of these proteinson host cell arenot being addressed yet.small GTPase ADP-ribosylation 1 gene (HcARF1) was cloned and recombinant protein of HcARF1 (rHcARF1) was successfully expressed in. Binding activity of rHcARF1 to goat PBMCs was confirmed by immunofluorescence assay (IFA) and its immunomudulatory effects on cytokine secretion, cell proliferation, cell migration and nitric oxide production (NO) were observed by co-incubation of rHcARF1. IFA results revealed that rHcARF1 could bind to the PBMCs. The interaction of rHcARF1 modulated the cytokine production, the production of IL-4, IL-10 and IL-17 was increased in a dose dependent manner, however, the IFN-γ production was significantly decreased. Cell migration and NO production were significantly increased by rHcARF1, whereas, rHcARF1 treatment significantly suppressed the p...

Oncotarget, 2017

Excretory/secretory antigens (ESAs) produced by Toxoplasma gondii enable this parasite to invade ... more Excretory/secretory antigens (ESAs) produced by Toxoplasma gondii enable this parasite to invade and survive within the host cells through immunomodulation. In this study, the modulating effects of T. gondii excretory/secretory antigens (TgESAs) on the Ana-1 murine macrophage cell line were evaluated. Ana-1 cells were incubated with several concentrations of TgESAs, and the resulting effects on cellular viability, phagocytotic ability, and apoptosis induction were determined. Pro-inflammatory and anti-inflammatory cytokine secretion, nitric oxide production, toll-like receptor expression, and nuclear translocation of NF-κB were all measured after incubation with TgESAs. After TgESAs treatment, the proliferation and phagocytosis ability of Ana-1 cells decreased, and apoptosis was induced in a dose dependent manner. Analysis of Ana-1 cell culture supernatants showed that TgESAs not only upregulated secretion of anti-inflammatory cytokines (interleukin-10 and transforming growth factor-β1), they also inhibited secretion of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1β). Additionally, TgESAs inhibited nitric oxide production, toll-like receptor (TLR) 2 and 4 activation, and the nuclear translocation of NF-κB in lipopolysaccharide-stimulated Ana-1 macrophages. These results suggest TgESAs inhibit the functional activity of Ana-1 murine macrophages by inhibiting TLRinduced NF-κB activation, which suppresses pro-inflammatory cytokine secretion.

Parasites & vectors, Jan 26, 2017

Arginine kinase (AK), an important member of phosphagen kinase family has been extensively studie... more Arginine kinase (AK), an important member of phosphagen kinase family has been extensively studied in various vertebrates and invertebrates. Immunologically, AKs are important constituents of different body parts, involved in various biological and cellular functions, and considered as immune-modulator and effector for pro-inflammatory cytokines. However, immunoregulatory changes of host cells triggered by AK protein of Haemonchus contortus, a parasitic nematode of ruminants, are still unknown. The current study was focused on cloning and characterisation of Hc-AK, and its regulatory effects on cytokines level, cell migration, cell proliferation, nitric oxide production and apoptosis of goat peripheral blood mononuclear cells (PBMCs) were observed. The full-length sequence of the Hc-AK gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sub-cloned into the prokaryotic expression vector pET-32a. The biochemical characteristics of recombinant protein Hc-...

Frontiers in microbiology, 2017

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite which can infect almost all w... more Toxoplasma gondii (T. gondii) is an obligate intracellular parasite which can infect almost all warm-blood animals, leading to toxoplasmosis. Screening and discovery of an effective vaccine candidate or new drug target is crucial for the control of this disease. In this study, the recombinant T. gondii elongation factor 1-alpha (rTgEF-1α) was successfully expressed in in Escherichia coli. Passive immunization of mice with anti-rTgEF-1α polyclonal antibody following challenge with a lethal dose of tachyzoites significantly increased the survival time compared with PBS control group. The survival time of mice challenged with tachyzoites pretreated with anti-rTgEF-1α PcAb also was significantly increased. Invasion of tachyzoites into mouse macrophages was significantly inhibited in the anti-rTgEF-1α PcAb pretreated group. Mice vaccinated with rTgEF-1α induced a high level of specific anti-T. gondii antibodies and production of IFN-gamma, interleukin-4. The expression levels of MHC-I an...

Parasites & vectors, Jan 19, 2017

Aspartyl protease inhibitor (API) was thought to protect intestinal parasitic nematodes from thei... more Aspartyl protease inhibitor (API) was thought to protect intestinal parasitic nematodes from their hostile proteolytic environment. Studies on Ostertagia ostertagi, Ascaris suum and Brugia malayi indicated that aspins might play roles in nematode infection. In a recent study, proteins differentially expressed between free-living third-stage larvae (L3) and activated L3 (xL3) of Haemonchus contortus were identified by 2D-DIGE. API was found downregulated in xL3 when compared with L3. However, there was no report about the functions of H. contortus API in the parasite-host interaction. In this study, the gene encoding API from H. contortus was cloned, expressed, and part of its biological characteristics were studied. A DNA fragment of 681 bp was amplified by RT-PCR. Ninety one percent of the amino acid sequence was similar with that for aspin from O. ostertagi. The recombinant API protein was fusion-expressed with a molecular weight of 48 × 10(3). Results of Western blot showed that ...

Oncotarget, Jan 21, 2016

A 24 kDa protein is one of the important components in Haemonchus contortus (barber pole worm) ex... more A 24 kDa protein is one of the important components in Haemonchus contortus (barber pole worm) excretory/secretory products (HcESPs), which was shown to have important antigenic function. However, little is known about the immunomodulatory effects of this proteinon host cell. In the present study gene encoding 24kDa excretory/secretory protein (HcES-24) was cloned. The recombinant protein of HcES-24 (rHcES-24) was expressed in a histidine-tagged fusion protein soluble form in Escherichia coli. Binding activity of rHcES-24 to goat PBMCs was confirmed by immunofluorescence assay (IFA) and its immunomudulatory effect on cytokine secretion, cell proliferation, cell migration and nitric oxide production were observed by co-incubation of rHcES-24. IFA results revealed that rHcES-24 could bind to the PBMCs. The interaction of rHcES-24 increased the production of IL4, IL10, IL17 and cell migration in dose dependent manner. However, rHcES-24 treatment significantly suppressed the production ...

Experimental parasitology, Jan 14, 2016

14-3-3 proteins have been found to be an excreted/secreted antigen and assumed to be released int... more 14-3-3 proteins have been found to be an excreted/secreted antigen and assumed to be released into the host-parasite interface and described in several unicellular and multicellular parasites. However, little is known about the immunomodulatory effects of H. controtus 14-3-3 protein on host cell. In present study, 14-3-3 isoform 2 gene, designated as Hcftt-2, was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from the adult H. contortus cDNA and cloned into expression plasmid pET32a (+) and expression of the recombinant protein (rHcftt-2) was induced by IPTG. Binding activity of rHcftt-2 to goat peripheral blood mononuclear cells (PBMCs) was confirmed by immunofluorescence assay (IFA) and modulatory effects on cytokine production, cell proliferation, cell migration and nitric oxide (NO) production were observed by co-incubation of rHcftt-2 with goat PBMCs. Sequence analysis showed that it had significant homology with the known 14-3-3 protein isoform 2. Result...