Role of aquaglyceroporin (AQP1) gene and drug uptake in antimony-resistant clinical isolates of Leishmania donovani

Antimonial-containing drugs are the first line of treatment against Leishmaniasis. Resistance to antimonials in Leishmania is proposed to be due to reduced uptake of trivalent antimony (SbIII) through the aquaglyceroporin (AQP1). We investigated the uptake of SbIII and involvement of aquaglyceroporin in developing antimony resistance phenotype in Leishmania donovani clinical isolates. SbIII accumulation, copy number of AQP1 gene, and transcript levels were compared in antimony-sensitive versus -resistant isolates. Antimony-resistant field isolates showed reduced uptake of SbIII. The copy number of AQP1 gene showed higher copy number in the antimony-resistant isolates when compared with the sensitive isolates and did not correlate to the reduced uptake of SbIII. Downregulation of AQP1 RNA levels was not consistently found in the antimony-resistant isolates. Our studies indicate that while downregulation of AQP1 may be one of the mechanisms of antimony resistance, it is however not an invariable feature

Evaluation of analgesic activity of methanolic extract of bougainvillea spectabilis leaves in experimental animal models

Background: Anti-inflammatory activity of leaves of Bougainvillea spectabilis (family Nyctaginaceae) has already been demonstrated in experimental animals. As pain is one of the important components of inflammation, we had set forward a study this find out possible analgesic activity of the same in animal models Objective: Evaluation of analgesic effects of, Bougainvillea spectabilis in mice models. Methods: 215 gm of fresh dried leaves of Bougainvillea spectabilis (BS) were collected from the local area during the flowering season and air dried. Following Methanol extraction, under reduced pressure solvent was removed on a rotary evaporator. The lyophilized extract was collected and the yield was 8 gm. That was used as an emulsion prepared in propylene glycol and orally administered (20 and 50 mg/kg). Central and peripheral analgesic activities of Bougainvillea spectabilis (BS) were evaluated by tail flick, tail immersion test and writhing test (acetic acid induced) respectively. Study Design: This is an experimental study designed on animal models. Results: Bougainvillea spectabilis (BS) had shown no analgesic action in central anal gesic model at different hours as the reaction time was less than 10 seconds at all time interval. With regard to peripheral analgesic activity, maximal activity was observed at 50 mg/kg b.w. The mean writhes ± standard deviation were 42.7±0.9 and 40±0.5 respectively in BS (20 mg/kg) and BS (50 mg/kg) in comparison to standard drug aspirin (33.3±0.4), control mice being 55.3±0.4. Conclusion: Our data indicates that Bougainvillea spectabilis (50 mg/kg) has a significant peripheral analgesic activity. Without isolating the active principles it's extremely difficult to pinpoint the mechanisms contributing to the observed analgesic activities of Bougainvillea spectabilis and extrapolate that in clinical practice

Evaluation of anti-leishmanial activity of artemisinin combined with amphotericin B or miltefosine in Leishmania donovani promastigotes

Background: The increasing incidence of drug resistance in Leishmaniasis necessitates evaluation of combination chemotherapy. Miltefosine and amphotericin B are established anti-leishmanial drugs, while artemisinin has shown significant leishmanicidal activity in experimental models. In this study, we have evaluated the additive/synergistic effect of artemisinin with amphotericin B or miltefosine.Methods: Leishmania parasites were isolated from the bone marrow aspirate of a patient with visceral leishmaniasis. Parasites were typed as Leishmania donovani by restriction fragment length polymorphism of internal transcribed spacer 1 region of Leishmania genome. Promastigotes were incubated in a fixed ratio combination of artemisinin (0-500 µM) and amphotericin B (0-100 nM) or miltefosine (0-100 µM) and cell viability was assessed. An isobologram was constructed to evaluate the additive/synergistic effect, wherein it was considered additive if the mean sum fractional inhibitory concentration (mean ΣFIC) at the IC50 level was <2, but ≥1 and synergism, if the mean ΣFIC was <1.Results: The isobologram showed an additive effect for three combinations of artemisinin-amphotericin B and artemisinin-miltefosine, the mean ΣFICs ranging from 1.02 to 1.44 and 1.08 to 1.33 along with a synergistic effect with one combination, the mean ΣFICs being 0.58 and 0.81 respectively.Conclusions: This study supports the combination use of artemisinin-amphotericin B and artemisinin-miltefosine, worthy of future pharmacological consideration

Post kala-azar dermal leishmaniasis: an unresolved mystery

Post kala-azar dermal leishmaniasis (PKDL), a cutaneous sequela of visceral leishmaniasis (VL), develops in some patients alongside but more commonly after apparent cure from VL. In view of the pivotal role of PKDL patients in the transmission of VL, here we review clinical, epidemiological, parasitological, and immunological perspectives of this disease, focusing on five hypotheses to explain the development of PKDL: (i) the role of antimonial drugs; (ii) UV-induced skin damage; (iii) reinfection; (iv) organ specific failure of memory T cell responses; and (v) genetic susceptibility of the host. This review will enable researchers and clinicians to explore the unresolved mystery of PKDL and provide a framework for future application of ‘omic’ approaches for the control and eventual elimination of VL

Assessing aquaglyceroporin gene status and expression profile in antimony-susceptible and -resistant clinical isolates of Leishmania donovani from India

Objectives: Clinical resistance to pentavalent antimonials results from an interplay between uptake, efflux and sequestration in Leishmania. Aquaglyceroporins (AQPs) have been shown to facilitate uptake of trivalent metalloids. Down-regulation of AQP1 in Leishmania results in resistance to trivalent antimony, whereas overexpression of AQP1 in drug-resistant parasites can reverse the resistance. The present work investigates the role of AQP1 in monitoring antimonial resistance in Indian leishmaniasis. Methods and results: Susceptibility to trivalent antimony as determined in vitro with intracellular amastigotes from both visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients correlated well with the clinical response. Higher accumulation of trivalent antimony (SbIII) was observed in all susceptible isolates compared with resistant isolates. Reduced accumulation of SbIII correlated, with a few exceptions, with down-regulation of AQP1 RNA as determined by real-time PCR. Cloning and sequencing of the AQP1 gene from both VL and PKDL isolates showed sequence variation in four of the clinical isolates. None of the isolates had an alteration of Glu152 and Arg230, which have been previously shown to affect metalloid transport. Transfection of the AQP1 gene in a sodium antimony gluconate-resistant field isolate conferred susceptibility to the resistant isolate. Conclusions: Our studies indicate genetic variation in VL and PKDL isolates. Down-regulation of AQP1 correlates well with clinical drug resistance in a majority of Indian VL and PKDL isolates. AQP1 gene expression at both the genetic and transcriptional level showed positive correlation with SbIII accumulation, with some exceptions

Proton-Induced X-ray Emission (PIXE) Analysis and DNA-chain Break study in rat hepatocarcinogenesis: A possible chemopreventive role by combined supplementation of vanadium and beta-carotene

Combined effect of vanadium and beta-carotene on rat liver DNA-chain break and Proton induced X-ray emission (PIXE) analysis was studied during a necrogenic dose (200 mg/kg of body weight) of Diethyl Nitrosamine (DENA) induced rat liver carcinogenesis. Morphological and histopathological changes were observed as an end point biomarker. Supplementation of vanadium (0.5 ppm ad libitum) in drinking water and beta-carotene in the basal diet (120 mg/Kg of body weight) were performed four weeks before DENA treatment and continued till the end of the experiment (16 weeks). PIXE analysis revealed the restoration of near normal value of zinc, copper, and iron, which were substantially altered when compared to carcinogen treated groups. Supplementation of both vanadium and beta-carotene four weeks before DENA injection was found to offer significant (64.73%, P < 0.001) protection against generation of single-strand breaks when compared with the carcinogen control counter parts. A significant stabilization of hepatic architecture of the cells was observed as compared to carcinogen control in vanadium plus beta-carotene treated group. This study thus suggests that vanadium, a prooxidant but potential therapeutic agent yield safe and effective pharmacological formulation with beta-carotene, an antioxidant, in the inhibition of experimental rat hepatocarcinogenesis

Anti-tumor effect of fruit rind of Myristica malabarica in an Ehrlich ascites carcinoma model

Background: Among the various modalities of anti-cancer treatment, cancer chemotherapy plays a very vital role. The alarming side effects being its main drawback leads to relentless research for newer agents. A new natural agent with promising anti-cancer properties from in-vitro studies leads to this study. Here we have evaluated the anti-tumor activity of a crude extract of fruit rind of Myristica malabarica in an Ehrlich ascites carcinoma model in mice.Methods: A murine model of cancer was established with i.p. inoculation of Ehrlich Ascites carcinoma (EAC) cells; animals were divided into five groups (including normal control) to observe the inhibitory effect of a crude extract of the fruit rind of Myristica malabarica/rampatri (0-100mg/kg b.w. i.p.) as compared with methotrexate (0.4mg/kg bw., i.p.). Blood and ascitic fluid were collected on the 10th day for analysis.Results: In the EAC model, there was an increase in tumor volume, tumor weight, and tumor packed cell volume, which was decreased by rampatri (50 and 100mg/kg bw) along with an increase in the mean survival time (MST). Rampatri caused minimal alterations in hematological parameters, renal functions remained unchanged but an increase in hepatic SGOT was demonstrated.Conclusions: The crude extract of rampatri (containing Malabaricones) exhibited significant anti-tumor activity with minimal effect on hematological and renal functions

Antibodies against 9-O-acetylated sialoglycans: a potent marker to monitor clinical status in childhood acute lymphoblastic leukemia

Background: Although childhood acute lymphoblastic leukemia (ALL) is highly responsive to chemotherapy, reliable techniques are needed to determine treatment outcome and predict impending relapse. In ALL, the cell surface over expression of 9-O-acetylated sialoglycans (9-OAcSGs) on lymphoblasts and concomitant high antibody titers in patients' sera was reported. Objectives: The present study was aimed to evaluate whether anti-9-OAcSG titers can be harnessed to monitor the clinical outcome of ALL. Design and methods: Anti-9-OAcSGs were analyzed by ELISA in children receiving either UK ALL X (n = 69, Group I) in India or UK ALL 97 (n = 47, Group II) in UK along with age-matched normal healthy controls at different time points over a period of &gt; 2 years. An attempt was also made to investigate subclass distribution of disease-specific IgG. Moreover, 17 patients having a higher sample size were longitudinally monitored. Results: Antibody levels were raised at disease presentation, decreased with remission induction, and importantly, reappeared with clinical relapse. Sera from patients with other hematological disorders and normal controls showed negligible levels of circulating anti-9-OAcSGs. In patients of both Groups I and II, the assay showed high sensitivity (98.92% and 96.77%) and specificity (92.1% and 95.91%), respectively. IgG subclass analyses during different phases of treatment revealed that 9-OAcSG-specific IgG1 could serve as a better prognostic marker in ALL. Conclusions: This study demonstrated the potential of this disease-specific antibody as an alternate marker in diagnosis and long-term assessment of ALL patients, suggesting its application in detection and prediction of impending relapse. Therefore, the expression of anti-9-OAcSGs, irrespective of their treatment protocol, may serve as an economical yet effective index for monitoring of childhood ALL

Effectiveness of malabaricone-A in P-glycoprotein over-expressing cancer cell lines

Background: A major impediment in treatment for cancers is resistance to chemotherapy and is primarily attributed to over-expression of efflux pumps. This study aimed to establish the cytotoxicity of malabaricone-A (MAL-A) in P-glycoprotein/multidrug resistance (P-gp/MDR) over-expressing hematopoietic cancer cell lines.Methods: Leukemia and multiple myeloma cell lines were indirectly evaluated for their P-gp/MDR status by examining Calcein-AM fluorescence and cell viability was assessed by the MTS-PMS assay.Results: The fluorescence of calcein was significantly decreased in three cell lines LP-1, RPMI-8226 and CEM-ADR 5000 and reversal with verapamil endorsed their P-gp/MDR activity. The mean IC50 of MAL-A in these MDR+ cell lines (5.40±1.41 to 12.33±0.78 µg/ml) was comparable with the MDR- leukemic (9.72±1.08 to 19.26±0.75 µg/ml) and multiple myeloma cell lines (9.65±0.39 to 18.05±0.17 μg/ml).Conclusions: Irrespective of their P-gp activity, the cytotoxicity of MAL-A was comparable, making it worthy of future pharmacological consideration in multidrug resistance

Role of ABC transporter MRPA, γ-glutamylcysteine synthetase and ornithine decarboxylase in natural antimony-resistant isolates of Leishmania donovani

Objectives: The resistance of clinical isolates of Leishmania donovani to sodium antimony gluconate (SAG), the mainstay of treatment in Indian visceral leishmaniasis, has become a critical issue in India. The present work investigates the mechanism of resistance to SAG in parasites isolated from patients who are unresponsive to SAG. Methods and results: Susceptibility to SAG as determined in vitro with intracellular amastigotes correlated well with the clinical response. The ABC transporter gene MRPA was amplified in resistant field isolates as part of an extrachromosomal circle. Co-amplification of the pterin reductase gene (PTR1) and MRPA suggests amplification of the H locus in SAG-resistant isolates. Amplification of MRPA was correlated to increased RNA as determined by real-time PCR. MRPA is an ABC-thiol transporter, and cysteine and glutathione were increased in the resistant isolates. Ornithine decarboxylase (a rate limiting enzyme in polyamine biosynthesis), and γ -glutamylcysteine synthetase (a rate limiting enzyme in glutathione biosynthesis), the two building blocks of the main cellular thiol trypanothione, were overexpressed in some of the resistant isolates. Conclusions: A variety of resistance mechanisms to SAG, most of them consistent with a model based on the study of resistance in vitro, were present in clinical isolates from the same geographical region