ABSTRACT
The function of CD5 protein in T cells is well documented, but regulation of its surface-level expression has yet to be fully understood. However, variation in its surface expression is associated with various immunopathological conditions and haematological malignancies. Briefly, expression of an alternate exon E1B of a human endogenous retroviruses (HERV) origin directly downregulates the conventional transcript variant (E1A), as its expression leads to the retention of the resultant protein at the intracellular level (cCD5). A separate promoter governs the expression of E1B and may be influenced by different transcription factors. Hence, we performed in silico transcription factor binding site (TFBS) analysis of the 3 kb upstream region from TSS of exon E1B and found five putative DREs (Dioxin Response elements) with good similarity scores. Further, we observed the upregulation in E1B expression after the exposure of BaP (a dioxin) and the reduction of E1A expression and their respective protein, i.e. sCD5 and cCD5. The binding of AHR at the predicted DRE sites was confirmed by ChIP qPCR and AHR specific inhibitor and gene silencing studies suggested the involvement of AHR in exonal switch. This study indicates that the polycyclic aromatic hydrocarbon decreases the sCD5 expression by upregulating alternative exon expression, which may adversely affect the overall T cell functions.
Subject(s)
Benzo(a)pyrene , CD5 Antigens , Exons , Gene Expression Regulation , Receptors, Aryl Hydrocarbon , Humans , CD5 Antigens/metabolism , CD5 Antigens/genetics , Receptors, Aryl Hydrocarbon/metabolism , Receptors, Aryl Hydrocarbon/genetics , Exons/genetics , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Promoter Regions, Genetic/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Protein Binding , Endogenous Retroviruses/genetics , Endogenous Retroviruses/metabolism , Binding Sites , Jurkat CellsABSTRACT
CD5, a T-cell receptor (TCR) negative regulator, is reduced on the surface of CD8+ lymphocytes in the tumor microenvironment (TME). Reduced surface CD5 expression (sCD5) occurs due to the preferential transcription of HERV-E derived exon E1B, i.e., anon-conventional formofthe cd5gene instead of its conventional exon E1A. A tumor employs several mechanisms to evade anti-tumor response, and hypoxia is one such mechanism that prevails in the TME and modulates the infiltrated T lymphocytes. We identified hypoxia response elements (HREs) upstream of E1B. We showed binding of HIF-1α onto these HREs and increased E1B mRNA expression in hypoxic T cells. This results in decreased sCD5 expression and increased cytoplasmic accumulation in T cells. We also validated our study in a solid tumor, i.e., colorectal cancer (CRC) patient samples. This hypoxia-driven mechanism reduces the surface CD5 expression on infiltrated T-cells in solid tumors.
Subject(s)
Neoplasms , Humans , Neoplasms/genetics , Hypoxia/genetics , Protein Isoforms/genetics , Exons , Phenotype , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Cell Hypoxia/genetics , Cell Line, Tumor , Tumor MicroenvironmentABSTRACT
Visceral leishmaniasis (VL) is an infectious disease caused by Leishmania donovani parasite in Indian subcontinent and is life-threatening. It primarily inflicts the malnourished population. There is little therapeutic advancement in the last one decade or more, as the available drugs show adverse effects, complex long treatment, high cost and drug resistance. Here, in a concerted approach, we intended to address the malnutrition as well as the parasite load with a single modality. Our earlier findings show the protective effects of retinoic acid (RA) in controlling the parasite load in infected macrophages (mφ) and restores their M1 phenotype. RA also restores the levels of cellular cholesterol in infected mφ. In this process, we observed loss of ergosterol in the parasite upon treatment with RA. Hence, we hypothesized that RA, besides boosting the parasiticidal mechanism in mφ, may also target the sterol pathway in the parasite by targeting sterol 24-C methyltransferase (SMT). SMT plays an essential role in the formation of ergosterol, required for growth and viability in Leishmania species. Therefore, we predicted as well as validated the 3D structure of SMT protein and performed the quality check. RA showed -9.9 free binding energy towards SMT which is higher than any of its derivatives. The molecular dynamics showed stable conjugate and the in vitro testing showed a reduction by â¼ twofold in the parasite number upon RA treatment. Importantly, it showed a loss of ergosterol possibly due to the inhibition of SMT protein. Our finding showed direct parasiticidal function of RA which is of significance in terms of therapeutic advancement.Communicated by Ramaswamy H. Sarma.
Subject(s)
Leishmania donovani , Leishmaniasis, Visceral , Humans , Tretinoin/pharmacology , Ergosterol/pharmacology , Ergosterol/metabolism , Ergosterol/therapeutic use , Leishmaniasis, Visceral/drug therapy , Leishmaniasis, Visceral/parasitology , CholesterolABSTRACT
Visceral leishmaniasis (VL) is a neglected disease with a broad spectrum of clinical manifestations and involvement of visceral organs. Organ-specific immune response against the Leishmania donovani (Ld) complex is not yet understood due to the unavailability of an appropriate experimental model. In reference to our recent work on comparing the hamster model with VL patients, it is now possible to understand immune profiling in different visceral organs. This may offer an answer to varying parasite loads in different visceral organs in the same host. Herein, we analysed a panel of immune markers (Th-2/Th-1) in visceral organs of Ld-infected hamsters and quantified parasitic load in the same tissues using qPCR assay. In spleen, liver, bone marrow and lymph node (mesenteric) from Ld-infected hamsters, the parasite burden was quantified along with mRNA expression of a panel of Th-2 and Th-1 type immune markers, namely IL-10, IL-4, Arginase-I, GATA-3, SOCS-3, IL-12, IFN-γ, iNOS, T-bet and SOCS-5. A clear dichotomy was absent between Th-2 and Th-1 type immune markers and the major players of this immune response were IFN-γ, IL-10, T-bet, GATA-3, SOCS-5 and SOCS-3.
Subject(s)
Leishmania donovani , Leishmaniasis, Visceral , Cricetinae , Animals , Humans , Interleukin-10 , Cytokines , MesocricetusABSTRACT
Infection with Leishmania donovani reduces cellular cholesterol and thus deprives the host cells by inhibiting its synthesis and uptake. Changes in cholesterol levels increase the chance of attachment and internalization of L. donovani in macrophages (MÏ). Retinoic acid (RA), an important micronutrient, restores the lysosomal uptake of cholesterol in L. donovani-infected MÏ. Importantly, mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) increases the cellular cholesterol level by increasing expression of sterol regulatory element-binding protein 2 (SREBP2). Whether the efficacy of RA in L. donovani-infected MÏ is mediated by mTOR is not yet established. Moreover, there are contradicting reports suggesting potential activation and inhibition of mTOR in L. donovani-infected MÏ. Intrigued by this, we attempted to understand the RA-mediated restoration of cholesterol as well as the possible roles of mTORC1, if any. Our findings suggest that L. donovani infection impairs the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), uptake of low-density lipoprotein receptor (LDLR), and secretion of ATP-binding cassette transporter (ABCA1) in MÏ. L. donovani infection possibly impairs mTORC1 formation, as it inhibits the expression of regulatory-associated protein of mammalian target of rapamycin (RAPTOR). Importantly, all these are restored upon RA supplementation. RA also restores the levels of SREBP2 in L. donovani-infected MÏ, resulting in increased cellular cholesterol and thus reducing the parasite burden. When mTORC1 was inhibited, RA exerted a similar response in L. donovani-infected MÏ; i.e., it restored cholesterol levels and reduced the parasite burden. In summary, RA restores cholesterol levels in L. donovani-infected MÏ and reduces the parasite burden in an mTOR-independent manner. IMPORTANCE People who reside in regions where leishmaniasis is endemic and who lack proteins, iron, zinc, and vitamin A in their diet are more prone to develop visceral leishmaniasis (VL) as a full-blown disease. Vitamin A deficiency favors the development of a parasitic infection in the human host, and the WHO recommends administering 200,000-IU doses to VL patients on admission. Additionally, Leishmania entry and its survival inside the host are achieved by utilizing host cholesterol, as all trypanosomatids lack de novo synthesis of sterol. We have already shown that RA regulates cellular cholesterol levels associated with an efficient immune response. A deficiency of retinoic acid (RA) favors the parasite in Leishmania donovani-infected macrophages by downregulating the immune response. In the present work, we observed that RA restores cellular cholesterol levels in Leishmania donovani-infected macrophages. This study proposes using RA as an immune potentiator along with standard therapy.
Subject(s)
Leishmania donovani , Leishmaniasis, Visceral , Humans , Leishmania donovani/metabolism , Tretinoin/pharmacology , Tretinoin/metabolism , Leishmaniasis, Visceral/metabolism , Leishmaniasis, Visceral/parasitology , Macrophages , TOR Serine-Threonine Kinases/metabolism , Cholesterol/metabolism , Sterols/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolismABSTRACT
Human leukemic T cells show decreased surface CD5 (sCD5) and increased cytoplasmic CD5 (cCD5). When we examined their expressions in the Jurkat T cells, it showed increased sCD5 and decreased cCD5, which is in sharp contrast with the pattern of CD5 expression observed for human leukemic T cells. Furthermore, this opposite pattern was due to the absence of an exonal switch between E1A and E1B. This study suggests that Jurkat cell does not retain all characteristics of T-ALL cells; thus, we should carefully interpret the data obtained using Jurkat T cell as a model cell line of T-ALL.
Subject(s)
Leukemia-Lymphoma, Adult T-Cell , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Cell Line , Humans , Jurkat CellsABSTRACT
Visceral leishmaniasis (VL) is a fatal form among all forms of leishmaniasis and is caused by visceralization of the Leishmania donovani (Ld) parasite to the critical organs. Mild to severe malnutrition is common in VL patients and the deficiency of retinoic acid (RA), an important micronutrient, results in a compromised state of immune response in macrophages (mφ) leading to the increased parasite load. In the continuation of our earlier work, we observed loss of cellular cholesterol in infected mφ in the absence of RA i.e., upon inhibition of RALDH pathway. Moreover, the Leishmania utilizes host cholesterol for the establishment of infection and causes a decrease in the expressions of Niemann-Pick C2 (npc2) and Niemann-Pick C1 (npc1) genes involved in the uptake of extracellular cholesterol. This results in reduced levels of cellular cholesterol in infected mφ. Intrigued by this, as the first sign of our hypothesis, we investigated the presence of RA Response Element (RARE) sequences in the upstream of npc1 and npc2 genes. To functionally confirm this, we measured their expressions and the levels of cellular cholesterol in Ld infected mφ in the absence (i.e., using an inhibitor of RALDH pathway) and presence of RA. We found restoration of the levels of cellular cholesterol in infected mφ under the supplementation of RA resulting in the decreased parasite load. Hence, the supplementation of RA with the standard therapy and/or preventive use of RA could be potentially an advancement in the treatment and cure of VL patients.
Subject(s)
Leishmania donovani , Leishmaniasis, Visceral , Cholesterol/metabolism , Humans , Macrophages/metabolism , Niemann-Pick C1 Protein , Tretinoin/metabolism , Tretinoin/pharmacology , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolismABSTRACT
The CD6 molecule, a cell surface marker, is involved in immunological synapse formation between T cell and antigen-presenting cell and T lymphocyte activation for adequate immune response. Geriatric individuals fail to mount a satisfactory immunological response against pathogens thus, insights into the functionality of CD6 may provide information for competence building in elderly immune cells. However, limited information is available regarding the status of CD6 in geriatric individuals. In this study, various isoforms of CD6 were analysed in aged mononuclear cells (MNCs) and compared with young individuals. In geriatric individuals, protein and mRNA expressions of CD6 molecule/isoforms were found to be decreased compared to their young counterparts. Furthermore, geriatric MNCs failed to show any change in CD6 levels and its isoforms upon polyclonal activation compared to young MNCs, marked by reduced Ca++ release and IL-2 expression. We suggest an overall decrease in CD6 levels in geriatric MNCs and T cells with suboptimal T cell activation in aged individuals.
Subject(s)
Aging/metabolism , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Immunosenescence , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation , T-Lymphocytes/metabolism , Age Factors , Aged , Aged, 80 and over , Aging/genetics , Aging/immunology , Antigens, CD/genetics , Antigens, Differentiation, T-Lymphocyte/genetics , Cells, Cultured , Female , Humans , Leukocytes, Mononuclear/immunology , Male , Middle Aged , T-Lymphocytes/immunologyABSTRACT
Visceral leishmaniasis is a neglected tropical disease caused by Leishmania (L.) donovani parasite in the Indian subcontinent. Macrophages (mÏ) are the harboring cells for parasite and their interactions dictate the pathogenesis of this disease. Polyunsaturated fatty acids are an integral part of the mÏ cell membrane and are derived from linoleic acid (LA), which is a principal essential fatty acid. Here, we have investigated the effect of the simultaneous presence of LA during L. donovani infection in mÏ. Treatment with LA suppresses the parasitic load in mÏ (kDNA expression) and promotes the Th-1 type immune response (IL-12, iNOS). However, no significant change in kDNA expressions was observed when L. donovani promastigotes were treated with LA. Intrigued by this observation, we explored mechanism(s) by which LA promoted the protective type immune response in infected mÏ. Interestingly, LA decreased the release of L. donovani derived extracellular vesicle later characterized as microvesicles. Moreover, these microvesicles were suppressive concerning their bias toward the Th-2 type of immune responses (IL-10, Arginase) in mÏ. We suggest that LA plays a protective role in the immune response against L. donovani infection by inhibiting the release to Leishmania derived microvesicles and thus promoting Th-1 type immune response in mÏ.
Subject(s)
Leishmania donovani , Leishmaniasis, Visceral , Animals , Linoleic Acid , Macrophages , Mice , Mice, Inbred BALB C , Parasite LoadABSTRACT
People suffering from malnutrition show compromised levels of ω-6 fatty acid and malnutrition is frequently observed among visceral leishmaniasis (VL) patients as disease inflicts primarily the socioeconomic destitute communities. Dietary linoleic acid (LA, 18:2; ω-6 fatty acid) is the principal source of essential fatty acid and its derivatives i.e. eicosanoids possess immune-modulatory activities. However, its role in VL is not yet established. LA was measured in VL human subjects (serum) as well as in Leishmania(L.)donovani infected hamsters (serum and visceral organs). Organ-specific mRNA expressions of various enzymes of the LA metabolic pathway were measured in visceral organs of infected hamsters. Our findings showed a decrease in the concentrations of LA in the serum samples of VL patients, suggesting malnutrition among these patients. However, in L. donovani infected hamsters, its level was not altered in the early infection (15 days) and then increased at late infection (60 days). Importantly, the supplementation of LA restored the Th-1 type of immune response and significantly reduced the parasite load within infected macrophages in vitro. This protective response of LA was mediated through 5-lipoxygenase pathway not via the cyclooxygenase pathway. Preventive usage of LA to mÏ followed by L. donovani infection also showed the strengthening of Th-1 immune response and significantly fewer parasite loads. Our findings demonstrate the protective role of LA in the containment of the parasite load. Incorporating LA rich oils in daily food habits across highly inflicted regions may be a significant advancement towards the eradication of the disease.
Subject(s)
Immunity, Cellular/drug effects , Leishmania donovani/immunology , Leishmaniasis, Visceral/drug therapy , Linoleic Acid/pharmacology , Th1 Cells/immunology , Adolescent , Adult , Animals , Female , Humans , Leishmaniasis, Visceral/immunology , Leishmaniasis, Visceral/pathology , Male , MesocricetusABSTRACT
BACKGROUND: Current understanding of visceral leishmaniasis (VL) depends upon the experimental model. Different species of mouse and hamster have been used as model for VL. It is already evident that the mouse model of VL is not a true reflection of the pathology of human visceral leishmaniasis (HuVL). On the other hand, hamster is reported to be a better model of VL to study the progressive as well as chronic pathology of the disease. OBJECTIVE: To compare immuno-clinicopathological features of experimental VL (ExVL) and HuVL by Leishmania donovani. METHODS: Hamsters were infected (15 and 60 days) and their immunological, clinical and biochemical parameters were compared with the cases of HuVL. RESULTS: Splenomegaly and hepatomegaly were observed in infected hamster post-infection, which are hallmarks of symptomatic HuVL cases. Clinical, biochemical and pathological manifestations of infected hamsters were consistent with that of HuVL cases, except parameters such as body weight, uric acid, alkaline phosphatase and random glucose. The absence of clear dichotomy between pro- and anti-inflammatory cytokines was also observed after infection at different sites of infection. CONCLUSION: Our results suggest that the golden hamster (Mesocricetus auratus), infected via the intracardiac route, constitutes a very good model for the study of experimental Leishmania donovani infections. However, certain differences in clinical presentations of infected hamsters (via intracardiac route) with HuVL suggest further optimization of this animal model like route of infection such as intradermal, which is more close to natural infection.
Subject(s)
Cytokines/immunology , Disease Models, Animal , Leishmaniasis, Visceral/immunology , Adolescent , Adult , Animals , Cricetinae , DNA Primers/genetics , DNA, Protozoan/genetics , Female , Hepatomegaly/immunology , Hepatomegaly/parasitology , Humans , Leishmania donovani , Male , Mesocricetus , Splenomegaly/immunology , Splenomegaly/parasitology , Young AdultABSTRACT
Infection with L. donovani affects mainly visceral organs. Importantly, the parasitic load differs in different visceral organs; therefore there is a need to understand the organ specific immune regulation, particularly in the spleen and liver. Comparative studies between these organs in Leishmania infected hamster (Mesocricetus auratus) are lacking. Our study highlights the importance of eicosanoids in the organ specific pathology of visceral leishmaniasis. Among other immune cells, macrophages (mφ) which harbor Leishmania parasite are major producers of eicosanoids. In this study, we intend to explore linkage between organ specific immune response and eicosanoids. We suggest that eicosanoids (early immune modulators) and their organ specific expressions, possibly tune the outcome of mφ differently at different sites. We have observed that liver showed better containment of parasitic load than spleen, where we have found higher expression of 5-lipoxygenase (5-LO) enzyme along with IL-12 and iNOS. However, in spleen, enzymes of the PGE2 pathway i.e. PGE2 synthases (cytosolic and microsomal) along with IL-10 were predominantly higher. To further corroborate our findings, in vitro assays were carried out using purified eicosanoids (LTB4 and PGE2) and the inhibitors of these pathways. Findings establish that the 5-lipoxygenase pathway (i.e. LTB4) is anti-parasitic and its inhibition increases the parasitic load (qPCR based kDNA detection). On the contrary, PGES pathway (i.e. PGE2) supports establishment of infection in mφ. Taken together, 5-LO pathway plays a protective role in liver during L. donovani infection. However, the PGES pathway favors the parasite growth, particularly in the spleen at a later stage.
Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , Leishmania donovani/physiology , Leishmaniasis, Visceral/parasitology , Liver/parasitology , Parasite Load/methods , Prostaglandin-E Synthases/metabolism , Spleen/parasitology , Animals , Cricetinae , Dinoprostone/metabolism , Eicosanoids/metabolism , Female , Humans , Immunologic Factors , Interleukin-10/metabolism , Leishmaniasis, Visceral/immunology , Leishmaniasis, Visceral/metabolism , Liver/metabolism , Macrophages/metabolism , Macrophages/parasitology , Spleen/metabolismABSTRACT
CD6 is a transmembrane glycoprotein, mainly expressed by all T cells and a subset of B cells. It has been shown that the human CD6 gene has six reported transcript isoforms, including full length and Δ3 isoforms. These are CD6a, CD6b, CD6c, CD6d, CD6e and CD6Δ3. In every isoform, particular exon(s) are known to be alternatively spliced out. In our findings, we observed a novel transcript isoform of CD6, where exon 9 is spliced out. Semi-qPCR and nucleotide sequencing confirmed the presence of a novel isoform of CD6 i.e. CD6f in human mononuclear cells. Quantitative expression showed significant high expression of CD6f over the full length transcript variant i.e. CD6a. Interestingly, their expressions get further increased upon polyclonal activation.
Subject(s)
Alternative Splicing , Antigens, CD/genetics , Antigens, Differentiation, T-Lymphocyte/genetics , Exons , Gene Expression Regulation , Adult , Base Sequence , Female , Humans , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Male , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
People suffering from malnutrition become susceptible to the infection like Leishmania sp., as it results in a compromised immune response. Retinoic acid (RA), an important constituent of nutrition, shows an immune-modulatory activity. However, its role in the containment of infection is not yet ascertained, particularly in case of visceral leishmaniasis (VL). VL patients (n = 10) and healthy endemic controls (n = 9) were recruited to measure the serum levels of RA. An in vitro model of Leishmania infection using the murine mφ cell line J774.1 was used to investigate the RA-synthesizing enzymes (RALDH-1 and RALDH-2). Parasite loads among infected mφ were measured by quantitative expression of kDNA in the presence of an inhibitor of the RALDH-2 enzyme. We found a significant decrease in the serum levels of RA in VL cases. Importantly, we observed decreased levels of RALDH-1 and RALDH-2 among L. donovani-infected mφ along with simultaneous decrease as well as increase in the Th-1 and Th-2-associated factors, respectively. Furthermore, the pretreatment of mφ with an RALDH-2 inhibitor improved parasite in vitro infection. Our findings show impaired RA pathway among infected mφ and indicate that an intact RA pathway is critical for anti-Leishmania immune response. Graphical abstract á .
Subject(s)
Immunologic Factors/blood , Leishmania donovani/physiology , Leishmaniasis, Visceral/parasitology , Macrophages/parasitology , Retinal Dehydrogenase/metabolism , Tretinoin/blood , Animals , Cell Line , Down-Regulation , Gene Expression Regulation , Humans , Leishmaniasis, Visceral/blood , Leishmaniasis, Visceral/immunology , Macrophages/enzymology , Male , Mice , Tretinoin/immunology , Tretinoin/metabolismABSTRACT
In the present study, we have synthesized chitin-glucan-aldehyde-quercetin (chi-glu-ald-que) conjugate via condensation reaction. Synthesis of chitin-glucan-aldehyde (chi-glu-ald) complex was facilitated by the oxidation of chitin-glucan (chi-glu) complex. Formation of conjugate was confirmed by Proton nuclear magnetic resonance spectroscopy (1H NMR) and Fourier-transform infrared spectroscopy (FT-IR). Morphological studies showed that after grafting of quercetin, several changes on surface were depicted and a more crystalline nature was observed. The chi-glu-ald-que conjugate displayed strong antioxidant activity. It showed 69% of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical, DPPH* scavenging activity at 1â¯mg/mL and 72% of 2, 2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical cation, ABTS*+ scavenging activity at 1â¯mg/mL concentration, which are much higher than that of chi-glu complex. The anticancer activity of chi-glu-ald-que conjugate was performed in Macrophage cancer cell lines (J774) and biocompatibility was performed in Peripheral blood mononuclear cells (PBMCs). The chi-glu-ald-que conjugate showed excellent cytotoxicity against J774 cell lines but no cytotoxicity towards PBMCs.
Subject(s)
Aldehydes/pharmacology , Antineoplastic Agents/pharmacology , Antioxidants/pharmacology , Chitin/pharmacology , Glucans/pharmacology , Quercetin/pharmacology , Aldehydes/chemistry , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antioxidants/chemical synthesis , Antioxidants/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Chitin/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Glucans/chemistry , Mice , Molecular Structure , Particle Size , Quercetin/chemistry , Structure-Activity Relationship , Surface Properties , Tumor Cells, CulturedABSTRACT
Inorganic phosphate (Pi) is shown to be involved in excretion of methylglyoxal (MG) in the promastigote form of Leishmania donovani parasite. Absence of Pi leads to its accumulation inside the parasite. Accumulation of MG is toxic to the parasite and utilizes glyoxylase as well as excretory pathways for its detoxification. In addition, Pi is also reported to regulate activities of ectoenzymes and energy metabolism (glucose to pyruvate) etc. Thus, it is known to cumulatively affect the growth of Leishmania parasite. Hence the transporters, which allow the movement of Pi across the membrane, can prove to be a crucial drug target. Therefore, we characterized two phosphate transporters in Leishmania (i) H+ dependent myo-inositol transporter (LdPHO84), and (ii) Na+ dependent transporter (LdPHO89), based on similar studies done previously on other lower organisms and trypanosomatids. We tried to understand the secondary structure of these two proteins and confirm modulation in their expression with the change in Pi concentration outside. Moreover, their modes of action were also measured in the presence of specific inhibitors (LiF, CCCP). Further analysis on the physiological role of these transporters in various stages of the parasite life cycle needs to be entrenched.
Subject(s)
Leishmania donovani/enzymology , Leishmania donovani/growth & development , Phosphate Transport Proteins/metabolism , Phosphates/metabolism , Protozoan Proteins/metabolism , Symporters/metabolism , Animals , Gene Expression Regulation , Leishmania donovani/drug effects , Leishmania donovani/genetics , Phosphate Transport Proteins/chemistry , Phosphate Transport Proteins/genetics , Protein Conformation , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Pyruvaldehyde/metabolism , Pyruvaldehyde/toxicity , Symporters/chemistry , Symporters/geneticsABSTRACT
BACKGROUND: Natural Killer (NK) cell plays an important role in the innate immune system and is known to produce IFN-γ at an early stage of infection that is essential to eliminate intracellular infection like Leishmania spp. It is already established that Leishmania parasite inhibits the activity of NK cells, avoiding the encounter with the early innate immune response. This, in turn, favors establishment and further dissemination of the infection. METHODS: In the present study, we have tried to measure the frequency of different phenotypic subsets of NK cells among visceral leishmaniasis (VL) patients. RESULTS: We have phenotyped three distinct three distinct subsets (CD56-CD161+, CD56+CD161-, and CD56+CD161+) of NK (CD3-) cell using their specific markers CD161 and CD56. CONCLUSION: Interestingly, we observed selective loss of CD56+CD161+ subset of circulating NK (CD3-) cells. Importantly, the other subsets (i.e., CD56-CD161+ and CD56+CD161-) of circulating NK cells remain unaffected as compared with healthy subjects.
Subject(s)
Killer Cells, Natural/immunology , Leishmaniasis, Visceral/immunology , Lymphocyte Subsets/immunology , Adolescent , Adult , CD56 Antigen/metabolism , Cells, Cultured , Female , Humans , Immunity, Innate , Immunophenotyping , Interferon-gamma/metabolism , Male , NK Cell Lectin-Like Receptor Subfamily B/metabolism , Young AdultABSTRACT
In this study, we have synthesized chitin-glucan quercetin conjugate (ChGCQ) by an easy and facile free radical grafting reaction. The structure of ChGCQ was confirmed by proton nuclear magnetic resonance (1H NMR) and Fourier transforms infrared spectroscopy (FT-IR). Curcumin was loaded into ChGCQ to study its anti-cancer efficiency. The biocompatibility of ChGCQ and curcumin loaded ChGCQ (Cu-ChGCQ) were analysed by different assays in Peripheral blood mononuclear cells (PBMCs) and cytotoxicity test was performed in a macrophage cancer cell line (J774). The result shows tremendous biocompatibility of ChGCQ and Cu-ChGCQ in peripheral blood mononuclear cells and excellent cytotoxity in macrophage cancer cell line (J774). Chitin-glucan complex (ChGC), ChGCQ and Cu-ChGCQ showed 51%, 66% and 74% of DPPH radical-scavenging activity at 1mg/ml respectively, which are much higher than that of ChGC and in ABTS*+ assay 58%, 71% and 83% show radical-scavenging activity at 1mg/ml. Antioxidant assay of Cu-ChGCQ conjugate expressed much higher antioxidant activity than ChGCQ and ChGC. In vitro drug release study of Cu-ChGCQ conjuagate showed faster drug release in acidic medium in comparison to PBS of physiological pH and anticancer activity in vitro assay showed more anticancer activity of Cu-ChGCQ in comparison to ChGCQ conjugate.