ABSTRACT
Mechanisms by which electric (E) or magnetic (B) fields might be harnessed to affect tumor cell behavior remain poorly defined, presenting a barrier to translation. We hypothesized in early studies that the glycocalyx of lung cancer cells might play a role in mediating plasma membrane leak by low-frequency pulsed magnetic fields (Lf-PMF) generated on a low-energy solenoid platform. In testing glioblastoma and neuroblastoma cells known to overexpress glycoproteins rich in modifications by the anionic glycan sialic acid (Sia), exposure of brain tumor cells on the same platform to a pulse train that included a 5 min 50Hz Lf-PMF (dB/dt â¼ 2 T/s at 10 ms pulse widths) induced a very modest but significant protease leak above that of control nonexposed cells (with modest but significant reductions in long-term tumor cell viability after the 5 min exposure). Using a markedly higher dB/dt system (80 T/s pulses, 70 µs pulse-width at 5.9 cm from a MagVenture coil source) induced markedly greater leak by the same cells, and eliminating Sia by treating cells with AUS sialidase immediately preexposure abrogated the effect entirely in SH-SY5Y neuroblastoma cells, and partially in T98G glioblastoma cells. The system demonstrated significant leak (including inward leak of propidium iodide), with reduced leak at lower dB/dt in a variety of tumor cells. The ability to abrogate Lf-PMF protease leak by pretreatment with sialidase in SH-SY5Y brain tumor cells or with heparin lyase in A549 lung tumor cells indicated the importance of heavy Sia or heparan sulfate glycosaminoglycan glycocalyx modifications as dominant glycan species mediating Lf-PMF membrane leak in respective tumor cells. This "first-physical" Lf-PMF tumor glycocalyx event, with downstream cell stress, may represent a critical and "tunable" transduction mechanism that depends on characteristic anionic glycans overexpressed by distinct malignant tumors.
Subject(s)
Brain Neoplasms , Glioblastoma , Neuroblastoma , Humans , Glycocalyx/metabolism , Neuraminidase , Neuroblastoma/metabolism , Neuroblastoma/pathology , Magnetic Fields , Cell Line, Tumor , N-Acetylneuraminic Acid/metabolism , Peptide Hydrolases , PolysaccharidesABSTRACT
Extracellular vesicles (EVs) are nano-sized vehicles secreted by all live cells to establish communication with adjacent cells. In recent years, mammalian EVs (MEVs) have been widely investigated for their therapeutic implications in human disease conditions. As the understanding of MEV composition and nature is advancing, scientists are constantly exploring alternatives for EV production with similar therapeutic potential. Plant-derived exosome-like nanovesicles (PDEVs) may be a better substitute for MEVs because of their widespread sources, cost-effectiveness, and ease of access. Cissus quadrangularis (CQ), known as "bone setter or Hadjod", is a perennial plant utilized for its osteogenic potential. Its crude powder extract formulations are widely used as tablets and syrups. The present work elucidates the isolation of exosome-like nanovesicles (henceforth exosomes) from the culture supernatants of an in vitro cultured callus tissue derived from CQ. The physical and biological properties of the exosomes were successfully investigated using different characterization techniques. The therapeutic potential of the CQ exosomes was found to ameliorate the wound scratch injury and oxidative stress conditions in human-derived mesenchymal stem cells (hMSCs) and the pre-osteoblast (MC3T3) cell line. These exosomes also induced the proliferation and differentiation of hMSCs, as observed by alkaline phosphatase activity. These findings may serve as a proof of concept for further investigating the CQ exosomes as a nanocarrier for drug molecules in various therapeutic bone applications.
ABSTRACT
Developing advanced methods for effective bone reconstructive strategies in case of critical bone defects caused by tumor resection, trauma, and other implant-related complications remains a challenging problem in orthopedics. In the clinical management of bone diseases, there is a paradigm shift in using local drugs at the injury site; however, the dead space created during the surgical debridement of necrotic bone and soft tissues (periosteum and underlying muscle) leads to ineffective bone formation, thereby leading to secondary complications, and thus calls for better regenerative approaches. In this study, we have utilized an exosome-functionalized doxorubicin-loaded biodegradable nanocement (NC)-based carrier along with a Cissus quadrangularis (CQ) extract-laden antioxidant herbal membrane for simultaneously managing the periosteum as well as bone formation in the tumor resection model of osteosarcoma. We initially evaluated the efficacy of scaffolds for in vitro mineralization and bone formation. To examine the in vivo effectiveness, we developed a human osteosarcoma cell line (Saos-2)-induced tumor xenograft model with a critical-sized bone defect. The findings revealed that doxorubicin released from NC was successful in killing the tumor cells and was present even after 30 days of implantation. Additionally, the incorporation of exosomes aided the bone formation, resulting in around a 2.6-fold increase in the bone volume compared to the empty group as evaluated by micro-CT. The herbal membrane assisted in the development of periosteum and mineralizing bone callous as validated through histological and immunofluorescence analysis. Thus, our findings describe a one-step biomaterial-based cell-free approach to regenerate bone in osteosarcoma and prevent further fracture due to the complete development of periosteum and lost bone.
ABSTRACT
Cigarette smoking-induced oxidative stress has harmful effects on bone metabolism. Maqui berry extract (MBE) and ginseng extract (GE) are two naturally occurring antioxidants that have been shown to reduce oxidative stress. By using an osteoblast and osteoclast three-dimensional co-culture system, we investigated the effects of MBE and GE on bone cells exposed to cigarette smoke extract (CSE). The cell viability and function of the co-culture system were measured on day 14. Markers of bone cell differentiation and oxidative stress were evaluated at gene and protein levels on day 7. The results showed that exposure to CSE induced osteoporotic-like alterations in the co-culture system, while 1.5 µg/mL MBE and 50 µg/mL GE improved CSE-impaired osteoblast function and decreased CSE-induced osteoclast function. The molecular mechanism of MBE and GE in preventing CSE-induced bone cell damage is linked with the inhibition of the NF-κB signaling pathway and the activation of the Nrf2 signaling pathway. Therefore, MBE and GE can reduce CSE-induced detrimental effects on bone cells and, thus, prevent smoking-induced alterations in bone cell homeostasis. These two antioxidants are thus suitable supplements to support bone regeneration in smokers.
ABSTRACT
Survival from influenza A virus (IAV) infection largely depends on an intricate balance between pathogen clearance and immunomodulation in the lung. We demonstrate that genetic alteration of the glycan heparan sulfate (HS) in CD11c + cells via Ndst1f/f CD11cCre + mutation, which inhibits HS sulfation in a major antigen presenting cell population, reduces lung inflammation by A/Puerto Rico/8/1934(H1N1) influenza in mice. Mutation was also characterized by a reduction in lung infiltration by CD4+ regulatory T (Treg) cells in the late infection/effector phase, 9 days post inoculation (p.i.), without significant differences in lung CD8 + T cells, or Treg cells at an earlier point (day 5) following infection. Induction of under-sulfated HS via Ndst1 silencing in a model dendritic cell line (DC2.4) resulted in up-regulated basal expression of the antiviral cytokine interferon ß (IFN-ß) relative to control. Stimulating cells with the TLR9 ligand CpG resulted in greater nuclear factor-κB (NFκB) phosphorylation in Ndst1 silenced DC2.4 cells. While stimulating cells with CpG also modestly increased IFN-ß expression, this did not lead to significant increases in IFN-ß protein production. In further IFN-ß protein response studies using primary bone marrow DCs from Ndst1f/f CD11cCre + mutant and Cre- control mice, while trace IFN-ß protein was detected in response to CpG, stimulation with the TLR7 ligand R848 resulted in robust IFN-ß production, with significantly higher levels associated with DC Ndst1 mutation. In vivo, improved pathogen clearance in Ndst1f/f CD11cCre + mutant mice was suggested by reduced IAV AA5H nucleoprotein in lung examined in the late/effector phase. Earlier in the course of infection (day 5 p.i.), mean viral load, as measured by viral RNA, was not significantly different among genotypes. These findings point to novel regulatory roles for DC HS in innate and adaptive immunity during viral infection. This may have therapeutic potential and guide DC targeted HS engineering platforms in the setting of IAV or other respiratory viruses.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza A virus , Influenza, Human , Animals , Heparitin Sulfate , Humans , Inflammation/genetics , Mice , MutationABSTRACT
Early lung carcinoma development may be modulated by innate host cellular mechanisms that promote tumor growth and invasion. We recently identified how a loss-of-function mutation in the glycan sulfating enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1; involved in heparan sulfate biosynthesis) targeted to antigen presenting cells (APCs) may augment acquired anti-tumor T cell immune mechanisms. Crossing this mutation (Ndst1f/f CD11cCre+) onto a model of inducible spontaneous Kras mutant lung cancer [CCSP-rtTA; (tetO7) CMV-Kras-G12D] allowed us to examine how the APC mutation affects the formation and growth of early lung carcinoma. We examined early bronchocentric adenoma formation in the model, and the frequency of such events was significantly reduced on the mutant background. This was associated with significant reductions in tumor associated FOXP3+ cellular infiltration and CD163+ M2-type macrophage infiltration. The findings evolved prior to effector CD8+ T cell infiltration into tumors. The impact of this unique glycan under-sulfating mutation on inhibiting early Kras G12D mutant bronchocentric adenoma formation along with a cellular phenotype of inhibited tumor infiltration by cells involved in suppressive T-regulatory cell signaling (FOXP3+ cells) or tumor-permissive M2 macrophage functions (CD163+ cells) provides insight on how glycan targeting may modulate innate cellular mechanisms during early lung tumor development. The findings may also impact the future design of host-centered immunologic anti-tumor therapeutic strategies.
Subject(s)
Adenoma/pathology , CD11c Antigen/metabolism , Lung Neoplasms/pathology , Mutation , Myeloid Cells/immunology , Polysaccharides/chemistry , Proto-Oncogene Proteins p21(ras)/genetics , Adenoma/etiology , Adenoma/metabolism , Animals , CD8-Positive T-Lymphocytes , Heparitin Sulfate/chemistry , Humans , Lung Neoplasms/etiology , Lung Neoplasms/metabolism , Lymphocytes, Tumor-Infiltrating/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Cells/metabolism , Myeloid Cells/pathology , Sulfates/metabolism , Sulfotransferases/physiology , T-Lymphocytes, Regulatory/immunologyABSTRACT
In malaria, chemical genetics is a powerful method for assigning function to uncharacterized genes. MMV085203 and GNF-Pf-3600 are two structurally related napthoquinone phenotypic screening hits that kill both blood- and sexual-stage P. falciparum parasites in the low nanomolar to low micromolar range. In order to understand their mechanism of action, parasites from two different genetic backgrounds were exposed to sublethal concentrations of MMV085203 and GNF-Pf-3600 until resistance emerged. Whole genome sequencing revealed all 17 resistant clones acquired nonsynonymous mutations in the gene encoding the orphan apicomplexan transporter PF3D7_0312500 (pfmfr3) predicted to encode a member of the major facilitator superfamily (MFS). Disruption of pfmfr3 and testing against a panel of antimalarial compounds showed decreased sensitivity to MMV085203 and GNF-Pf-3600 as well as other compounds that have a mitochondrial mechanism of action. In contrast, mutations in pfmfr3 provided no protection against compounds that act in the food vacuole or the cytosol. A dihydroorotate dehydrogenase rescue assay using transgenic parasite lines, however, indicated a different mechanism of action for both MMV085203 and GNF-Pf-3600 than the direct inhibition of cytochrome bc1. Green fluorescent protein (GFP) tagging of PfMFR3 revealed that it localizes to the parasite mitochondrion. Our data are consistent with PfMFR3 playing roles in mitochondrial transport as well as drug resistance for clinically relevant antimalarials that target the mitochondria. Furthermore, given that pfmfr3 is naturally polymorphic, naturally occurring mutations may lead to differential sensitivity to clinically relevant compounds such as atovaquone.
Subject(s)
Antimalarials , Malaria , Antimalarials/pharmacology , Drug Resistance , Humans , Malaria/drug therapy , Mutation , Plasmodium falciparum/geneticsABSTRACT
Acute respiratory distress syndrome (ARDS) is a common cause of mortality in patients with acute kidney injury (AKI). Inflammatory crosstalk from the kidney to the lung has been shown to contribute to lung injury after AKI, but anti-inflammatory therapies have not been proven beneficial in human studies. Recently, AKI was shown to alter mitochondria and related metabolic pathways in the heart, but the impact of AKI on lung metabolism has not been investigated to our knowledge. In this study, we evaluated the metabolomic profile of the lung following renal ischemia and reperfusion to identify novel pathways that may be modifiable. We randomized C57BL/6 mice to 20 minutes of bilateral renal arterial clamping or sham operation under ketamine/xylazine anesthesia. At 4 hours after reperfusion, we found a significant increase in markers of lung injury, as well as significant metabolomic changes across lung, kidney, plasma and bronchoalveolar lavage fluid (BALF) compared to shams. Comparative analyses revealed that the fatty acid oxidation pathway was the most significantly altered metabolic pathway, a finding which is consistent with mitochondrial dysfunction systemically and in the lung. These metabolomic changes correlated with the extracellular accumulation of the mitochondrial damage associated molecular patterns (mtDAMPs), mitochondrial DNA (mtDNA) and transcription factor A, mitochondria (TFAM). Finally, we found that intraperitoneal injection of renal mtDAMPs caused metabolomic changes consistent with mitochondrial dysfunction in the lung in vivo. Mitochondrial function and mtDAMPs warrant further investigation as potential therapeutic targets in preventing lung injury because of AKI.
Subject(s)
Acute Kidney Injury/complications , Alarmins/metabolism , Lung Injury/pathology , Metabolome , Mitochondria/pathology , Animals , Lung Injury/etiology , Lung Injury/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolismABSTRACT
While recent research points to the importance of glycans in cancer immunity, knowledge on functional mechanisms is lacking. In lung carcinoma among other tumors, anti-tumor immunity is suppressed; and while some recent therapies boost T-cell mediated immunity by targeting immune-checkpoint pathways, robust responses are uncommon. Augmenting tumor antigen-specific immune responses by endogenous dendritic cells (DCs) is appealing from a specificity standpoint, but challenging. Here, we show that restricting a heparan sulfate (HS) loss-of-function mutation in the HS sulfating enzyme Ndst1 to predominantly conventional DCs (Ndst1f/f CD11cCre+ mutation) results in marked inhibition of Lewis lung carcinoma growth along with increased tumor-associated CD8+ T cells. In mice deficient in a major DC HS proteoglycan (syndecan-4), splenic CD8+ T cells showed increased anti-tumor cytotoxic responses relative to controls. Studies examining Ndst1f/f CD11cCreâ¯+ mutants revealed that mutation was associated with an increase in anti-tumor cytolysis using either splenic CD8+ T cells or tumor-infiltrating (TIL) CD8+ T cells purified ex-vivo, and tested in pooled effector-to-target cytolytic assays against tumor cells from respective animals. On glycan compositional analysis, HS purified from Ndst1f/f CD11cCreâ¯+ mutant DCs had reduced overall sulfation, including reduced sulfation of a tri-sulfated disaccharide species that was intriguingly abundant on wildtype DC HS. Interestingly, antigen presentation in the context of major histocompatibility complex class-I (MHC-I) was enhanced in mutant DCs, with more striking effects in the setting of HS under-sulfation, pointing to a likely regulatory role by sulfated glycans at the antigen/MHC-I - T-cell interface; and possibly future opportunities to improve antigen-specific T cell responses by immunologic targeting of HS proteoglycans in cancer.
Subject(s)
Carcinoma, Lewis Lung/drug therapy , Major Histocompatibility Complex/genetics , Polysaccharides/genetics , Proteoglycans/genetics , Sulfotransferases/genetics , Animals , CD11c Antigen/genetics , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Lewis Lung/genetics , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Dendritic Cells/immunology , Dendritic Cells/pathology , Heparitin Sulfate/pharmacology , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Immunity, Cellular/genetics , Immunity, Cellular/immunology , Loss of Function Mutation/genetics , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Major Histocompatibility Complex/immunology , Mice , Polysaccharides/antagonists & inhibitors , Proteoglycans/antagonists & inhibitors , Proteoglycans/immunology , T-Lymphocytes/immunology , T-Lymphocytes/pathologyABSTRACT
Although many new anti-infectives have been discovered and developed solely using phenotypic cellular screening and assay optimization, most researchers recognize that structure-guided drug design is more practical and less costly. In addition, a greater chemical space can be interrogated with structure-guided drug design. The practicality of structure-guided drug design has launched a search for the targets of compounds discovered in phenotypic screens. One method that has been used extensively in malaria parasites for target discovery and chemical validation is in vitro evolution and whole genome analysis (IVIEWGA). Here, small molecules from phenotypic screens with demonstrated antiparasitic activity are used in genome-based target discovery methods. In this Review, we discuss the newest, most promising druggable targets discovered or further validated by evolution-based methods, as well as some exceptions.
Subject(s)
Antimalarials/isolation & purification , DNA Mutational Analysis , Drug Discovery/methods , Plasmodium/drug effects , Whole Genome Sequencing , Drug Resistance , Plasmodium/genetics , Selection, GeneticABSTRACT
Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target-inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.
Subject(s)
Antimalarials/pharmacology , Drug Resistance/genetics , Genome, Protozoan , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Activation, Metabolic , Alleles , DNA Copy Number Variations , Directed Molecular Evolution , Drug Resistance, Multiple/genetics , Genes, Protozoan , Metabolomics , Mutation , Plasmodium falciparum/growth & development , Selection, Genetic , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The malaria infection is the interplay of several complex factors, among which drug resistance and gametocyte biology are the crucial ones. Measurements of the drug sensitivity assays and prevalence of gametocyte genes play a major role in malaria eradication programmes. The purpose of this study was to assess the current drug sensitivity profile in the field isolates and type the Pfg377 gene. In vitro drug sensitivity assay was used to assess the susceptibility of Plasmodium falciparum field isolates collected from Mewat (Haryana), located in Northern region of India to four anti-malarial drugs. The inhibitory concentrations (IC50) for the four drugs viz Chloroquine (CQ), Artesunate (AS), Sulphadoxine (SD) and Pyrimethamine (PYR) were in the range of 10.11-113.2, 2.26-4.08, 13.31-43.91 and 0.76-4.91 nM respectively in the evaluated 25 field isolates. The Pfg377 allele typing in the 26 field isolates revealed different types of haplotypes (A, B, C and D) varying by the size and number of repeats and deletions. The Pfg377 allelic types in the cryopreserved and adapted isolates of P. falciparum were compared to determine the parasite lines in them. The allele types or presence of different clones in the same sample could not be correlated significantly with resistance to any of the four drugs tested in the study. Our study revealed different drug sensitivity profiles of field isolates from Mewat region and allele typing of Pfg377 gene revealed different haplotypes of field isolates were circulating in parasite population.
Subject(s)
Antimalarials/pharmacology , Genes, Protozoan , Genetic Variation , Malaria, Falciparum/parasitology , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Child , Female , Genotype , Genotyping Techniques , Humans , India , Inhibitory Concentration 50 , Male , Middle Aged , Parasitic Sensitivity Tests , Plasmodium falciparum/isolation & purification , Young AdultABSTRACT
Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite's acidic digestive vacuole as the primary mode of both the bis-aminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates.IMPORTANCE The global agenda to eliminate malaria depends on the continued success of artemisinin-based combination therapies (ACTs), which target the asexual blood stages of the intracellular parasite Plasmodium Partial resistance to artemisinin, however, is now established in Southeast Asia, exposing the partner drugs to increased selective pressure. Plasmodium falciparum resistance to the first-line partner piperaquine (PPQ) is now spreading rapidly in Cambodia, resulting in clinical treatment failures. Here, we report that a variant form of the Plasmodium falciparum chloroquine resistance transporter, harboring a C101F mutation edited into the chloroquine (CQ)-resistant Dd2 isoform prevalent in Asia, can confer PPQ resistance in cultured parasites. This was accompanied by a loss of CQ resistance. Biochemical assays showed that PPQ, like CQ, inhibits the detoxification of reactive heme that is formed by parasite-mediated catabolism of host hemoglobin. We propose that novel PfCRT variants emerging in the field could contribute to a multigenic basis of PPQ resistance.
Subject(s)
Antimalarials/pharmacology , Drug Resistance/genetics , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Plasmodium falciparum/drug effects , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Quinolines/pharmacology , Antimalarials/chemistry , Artemisinins/therapeutic use , Cambodia , Gene Editing , Humans , Lethal Dose 50 , Malaria, Falciparum/epidemiology , Malaria, Falciparum/parasitology , Membrane Transport Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Mutation/drug effects , Plasmodium falciparum/genetics , Protein Isoforms , Protozoan Proteins/metabolism , Quinolines/chemistryABSTRACT
BACKGROUND: Plasmodium vivax once considered benign is now being increasingly associated with complicated malaria where the spectrum of complications is vast and like Plasmodium falciparum. The clinical data is important with respect to the immunopathological status of the patient. Several genes like the vir genes and pvcrt-o are speculated to be attributing to the severity of P. vivax malaria. METHODS: In the present study we carried out the transcription analysis of five vir genes (vir 14-related, vir 12, vir 17-like, putative vir 14 and vir 10-related) and pvcrt-o gene in severe (n=12) and non-severe (n=7) P. vivax clinical infections and studied the correlation of these genes with clinical disease severity. RESULTS: This study revealed multiorgan involvement in severe vivax cases with severe thrombocytopenia and anemia, the predominantly occurring symptoms. Four out of five vir genes and pvcrt-o showed a significant increase in expression levels of severe infections compared to the non-severe infections indicating their possible role in the changing pathogenesis of P. vivax. CONCLUSIONS: The increased virulence in vivax malaria seems to be the result of multifactorial parameters changing it phenotypically as well as genotypically. However more studies are needed to understand the still nascent severity of P. vivax malaria.
Subject(s)
Malaria, Vivax/diagnosis , Malaria, Vivax/parasitology , Plasmodium vivax/physiology , Adolescent , Child , Child, Preschool , Erythrocytes/parasitology , Female , Gene Expression , Humans , India/epidemiology , Infant , Malaria, Vivax/epidemiology , Male , Protozoan Proteins/geneticsABSTRACT
Genotyping the sulfadoxine-pyrimethamine (SP) genes will help in identifying the genes under drug selection and the emergence of resistance in dhfr and dhps genes. India is an important hotspot for studying malaria due to the immense climatic diversity prevalent in the country. The central and eastern parts of the country are most vulnerable sites where malaria cases are reported throughout the year. From different regions of the country 173 field isolates were genotyped at various loci in dhfr and dhps genes collected between 1994 and 2013. This encompasses the period before antimalarial resistance emerged and the period after the use of combination therapy was made mandatory in the country. We observed the rise of resistant SP alleles from very low frequencies (in the year 1994) to steadily rising (in the year 2000) and maintaining this increasing trend subsequently (in the year 2013) as shown by the sequence analysis of dhfr and dhps genes. This study assessed the prevalence of mutations in dhfr and dhps genes associated with SP resistance in samples indicative of increase in resistance levels of Plasmodium falciparum to SP even after the change in malaria treatment policy in the country.
Subject(s)
Antimalarials/pharmacology , Drug Resistance/genetics , Malaria, Falciparum/parasitology , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Pyrimethamine/pharmacology , Sulfadoxine/pharmacology , Dihydropteroate Synthase/genetics , Drug Combinations , Humans , India , Malaria, Falciparum/epidemiology , Polymorphism, Single Nucleotide , Prevalence , Protozoan Proteins/genetics , Tetrahydrofolate Dehydrogenase/geneticsABSTRACT
About 50% of malaria infections in India are attributed to Plasmodium falciparum but relatively little is known about the genetic structure of the parasite populations. The molecular genotyping of the parasite populations by merozoite surface protein (msp1 and msp2) and glutamate-rich protein (glurp) genes identifies the existing parasite population in the regions which help in understanding the molecular mechanisms involved in the parasite's drive for survival. This study reveals the genetic profile of the parasite population in selected regions across the country with varying degree of endemicity among them. We also report the prevalence of Pfcrt mutations in this parasite population to evaluate the pattern of drug resistance development in them.
Subject(s)
Epitopes/genetics , Gene Expression Profiling/methods , Genetic Markers/genetics , Plasmodium falciparum/genetics , Humans , India/epidemiology , Malaria, Falciparum/diagnosis , Malaria, Falciparum/epidemiology , Malaria, Falciparum/genetics , Plasmodium falciparum/isolation & purificationABSTRACT
The vir genes are antigenic genes and are considered to be possible vaccine targets. Since India is highly endemic to Plasmodium vivax, we sequenced 5 different vir genes and investigated DNA sequence variations in 93 single-clonal P. vivax isolates. High variability was observed in all the 5 vir genes; the vir 1/9 gene was highly diverged across Indian populations. The patterns of genetic diversity do not follow geographical locations, as geographically distant populations were found to be genetically similar. The results in general present complex genetic diversity patterns in India, requiring further in-depth population genetic and functional studies.
Subject(s)
Antigens, Protozoan/genetics , Plasmodium vivax/genetics , Polymorphism, Genetic , Protozoan Proteins/metabolism , Humans , India/epidemiology , Malaria, Vivax/epidemiology , Malaria, Vivax/parasitology , Phylogeny , Protozoan Proteins/geneticsABSTRACT
Malaria is an infectious disease that is widespread in tropical and subtropical regions. The malaria parasite is able to skip the host immunity and thus maintains not only persistent but also repeated infections. There are a number of multigene families in Plasmodium that code for the variant antigens and are targets for protective immunity. In this article, we summarize the virulence genes of P. falciparum (var genes) and P. vivax (vir genes) which play key roles in disease pathogenesis by evading elimination by the host immune system. These genes occurring within the parasite population are mostly present in the subtelomeric regions of the chromosome.
Subject(s)
Genetic Variation , Immune Evasion/genetics , Multigene Family/genetics , Plasmodium falciparum/genetics , Plasmodium vivax/genetics , Protozoan Proteins/genetics , Virulence/genetics , Plasmodium falciparum/pathogenicity , Plasmodium vivax/pathogenicity , Species SpecificityABSTRACT
Control and eradication of malaria is hindered by the acquisition of drug resistance by Plasmodium species. This has necessitated a persistent search for novel drugs and more efficient targets. Plasmodium species specific lactate dehydrogenase is one of the potential therapeutic and diagnostic targets, because of its indispensable role in endoerythrocytic stage of the parasite. A target molecule that is highly conserved in the parasite population can be more effectively used in diagnostics and therapeutics, hence, in the present study polymorphism in PfLDH (Plasmodiumfalciparum specific LDH) and PvLDH (Plasmodiumvivax specific LDH) genes was analyzed using PCR-single strand confirmation polymorphism (PCR-SSCP) and sequencing. Forty-six P. falciparum and thirty-five P. vivax samples were screened from different states of India. Our findings have revealed presence of a single PfLDH genotype and six PvLDH genotypes among the studied samples. Interestingly, along with synonymous substitutions, nonsynonymous substitutions were reported to be present for the first time in the PvLDH genotypes. Further, through amino acid sequence alignment and homology modeling studies we observed that the catalytic residues were conserved in all PvLDH genotypes and the nonsynonymous substitutions have not altered the enzyme structure significantly. Evolutionary genetics studies have confirmed that PfLDH and PvLDH loci are under strong purifying selection. Phylogenetic analysis of the pLDH gene sequences revealed that P. falciparum compared to P. vivax, has recent origin. The study therefore supports PfLDH and PvLDH as suitable therapeutic and diagnostic targets as well as phylogenetic markers to understand the genealogy of malaria species.
Subject(s)
L-Lactate Dehydrogenase/genetics , Malaria, Falciparum/epidemiology , Malaria, Vivax/epidemiology , Plasmodium falciparum/genetics , Plasmodium vivax/genetics , Polymorphism, Genetic , Amino Acid Sequence , DNA, Protozoan , Evolution, Molecular , Humans , India/epidemiology , L-Lactate Dehydrogenase/chemistry , Malaria, Falciparum/parasitology , Malaria, Vivax/parasitology , Models, Molecular , Molecular Sequence Data , Phylogeny , Plasmodium falciparum/classification , Plasmodium falciparum/isolation & purification , Plasmodium vivax/classification , Plasmodium vivax/isolation & purification , Protein Conformation , Sequence Alignment , Sequence Analysis, DNA , Species SpecificityABSTRACT
BACKGROUND & OBJECTIVES: The search for effective polymorphic markers in Plasmodium vivax is highly demanding to understand its transmission in a population. Due to the limited knowledge existing for P. vivax, the search for polymorphic markers for population studies is ongoing. The MSP gene family of Plasmodia has been linked with immune evasion. To study the circulating parasite population P. vivax merozoite surface protein 3ß (PvMSP3ß) polymorphic marker was used to investigate the genetic diversity of P. vivax in natural infections. METHODS: Polymorphism of PvMSP3ß gene was determined in 46 P. vivax blood samples from six different regions of India by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) with PstI. RESULTS: Two different parasite genotypes, viz. type-A and type-B were detected among 46 samples that were positive for PCR, based on the size of the amplification. RFLP analysis with PstI showed 22 allelic groups and 15.2% samples revealed mixed infections on analysis. CONCLUSION: PvMSP3ß was found to be an effective molecular marker for P. vivax as it shows high diversity in India and multiple genotypes easily distinguishable without the need for sequencing.