Anti-malarial effect of a combination of risedronate and azithromycin against Plasmodium yoelii nigeriensis infection in Swiss mice.

Combination therapy is used to retard the selection of malaria parasite strains resistant to individual components of a combination of drugs. This approach has proved to be a success in the combination of sulphadoxine and pyrimethamine, which targets two different steps in the folate pathway of malaria parasites. However, after the success of this therapeutic combination, the efficacy of other combinations of drugs that target different enzymes in a particular metabolic pathway has, apparently, not been reported. In the current study, the antimalarial effect of a combination of risedronate (RIS), which is known for its anti-osteoporosis activity, and azithromycin (AZT) was investigated. Peter's suppression test was carried out on mice infected with 1 × 107P. yoelii infected erythrocytes. Drug efficacy was analyzed by comparing the percent reduction in parasitaemia on day 4 post-infection. RIS was observed to be a blood schizonticidal agent against P. yoelii infection which showed ED50 7.0 (4.04-12.13) mg/kg/day x 4. Normalized isobologram showed additive action between RIS 1 mg/kg/day x 4 and AZT 10 mg/kg/day x 4, and antagonistic action for the rest of the combinations (RIS 1 + AZT 20, RIS 1 + AZT 40, RIS 5 + AZT 10, RIS 5 + AZT 20, RIS 5 + AZT 40, RIS 10 + AZT 10, RIS 10 + AZT 20 and RIS 10 + AZT 40 mg/kg/day x 4). Furthermore, a combination of RIS with AZT showed inferior efficacy as compared to AZT treatment alone. This antagonistic interaction may be due to the high accumulation of AZT in WBCs, which will reduce its serum bio-availability, whereas RIS has anti-parasitic activity by increasing WBCs.

Synthesis, antiprotozoal, antimicrobial, ß-hematin inhibition, cytotoxicity and methemoglobin (MetHb) formation activities of bis(8-aminoquinolines).

In continuing our search of potent antimalarials based on 8-aminoquinoline structural framework, three series of novel bis(8-aminoquinolines) using convenient one to four steps synthetic procedures were synthesized. The bisquinolines were evaluated for in vitro antimalarial (Plasmodiumfalciparum), antileishmanial (Leishmaniadonovani), antimicrobial (a panel of pathogenic bacteria and fungi), cytotoxicity, ß-hematin inhibitory and methemoglobin (MetHb) formation activities. Several compounds exhibited superior antimalarial activities compared to parent drug primaquine. Selected compounds (44, 61 and 79) when tested for in vivo blood-schizontocidal antimalarial activity (Plasmodiumberghei) displayed potent blood-schizontocial activities. The bisquinolines showed negligible MetHb formation (0.2-1.2%) underlining their potential in the treatment of glucose-6-phosphate dehydrogenase deficient patients. The bisquinoline analogues (36, 73 and 79) also exhibited promising in vitro antileishmanial activity, and antimicrobial activities (43, 44 and 76) against a panel of pathogenic bacteria and fungi. The results of this study provide evidence that bis(8-aminoquinolines), like their bis(4-aminoquinolines) and artemisinin dimers counterparts, are a promising class of antimalarial agents.

A short-term model for preliminary screening of potential anti-tubercular compounds.

Screening of new agents for anti-tubercular activity is a challenging task due to the virulent and slow growing nature of mycobacteria. In this study, we explored the use of Mycobacterium smegmatis as an alternate model to virulent strains. We observed that the effect of standard anti-tubercular drugs was similar in mice infected with M. smegmatis and Mycobacterium tuberculosis. The total duration of the experiment, including incubation time, was 10 days in the M. smegmatis-infected mice model compared with 2 months in M. tuberculosis-infected mice. This model of anti-tubercular screening is a simple, easy to carry out, less time-consuming, safer and economical alternative for preliminary in vivo screening of potential anti-tubercular agents.

Macromolecular prodrugs. XII. Primaquine conjugates: synthesis and preliminary antimalarial evaluation.

New primaquine conjugates 5-7 with glucosamine and two polymers of polyaspartamide type, poly[alpha,beta-(N-2-hydroxyethyl-DL-aspartamide)] (PHEA) and poly[alpha,beta-(N-3-hydroxypropyl-DL-aspartamide)] (PHPA), were synthesized, characterized and screened for their antimalarial activity. The conjugates differed in the type of covalent bonding, length of the spacer between the polymeric carrier and drug, molecular mass and drug-loading. Blood-schizontocidal activity of the prepared conjugates was tested against Plasmodium berghei infection in Swiss mice. Polymeric conjugates showed better antimalarial activity than the glucosamine conjugate.

Effect of morphine on Mycobacterium smegmatis infection in mice and macrophages.

The immunomodulatory effects of opioids are known in various infections. However, little is known about the effects of opioids in tuberculosis (TB). In the present study, we report the effects of morphine in Mycobacterium smegmatis infection in mice and macrophages. Morphine exerted a dose-dependent suppression of infection in vivo: 50 and 100 mg/kg morphine exerted significant (P<0.05) suppression whereas 5 mg/kg morphine showed no effect. Analogous to the in vivo effects, incubation of M. smegmatis-infected mouse peritoneal macrophages with morphine (100 µM) showed significant reduction in intramacrophage CFU counts. However, morphine did not show any direct antimycobacterial activity in broth dilution assay upto 100 µM concentration. Further, morphine-induced intramacrophage killing of M. smegmatis was abrogated by naloxone and aminoguanidine indicating the involvement of opioid-receptor activation and nitric oxide production in protective effects of morphine. In conclusion, morphine suppressed the progression of experimental TB in both mice and macrophage models.

Effects of morphine during Mycobacterium tuberculosis H37Rv infection in mice.

The effects of opiates in various infections are well known; however, very little is known about tuberculosis infection. Therefore, in the present study, we report for the first time, the effects of morphine during murine tuberculosis. Mice were infected intravenously with Mycobacterium tuberculosis H37Rv, administered morphine (0.1-100 mg/kg subcutaneously on day 0 and day +15) and sacrificed on day +30 for CFU enumeration in lungs and spleen. Morphine exerted maximum suppression of infection at 5 mg/kg, and sometimes completes elimination of infection; naloxone, silica and aminoguanidine blocked the protective effect of morphine. In vitro, morphine lacked direct antimycobacterial activity up to 1x10(-4) M concentration, as assessed by radiometric BACTEC method. In macrophage model of infection, morphine showed maximal killing at 1x10(-7) M concentration, the activity was blocked by naloxone and aminoguanidine. These observations suggest that morphine exerts a dose-dependent effect in murine tuberculosis, the protective effect being naloxone-reversible and may involve macrophage-mediated protective mechanisms. These results may be helpful in developing new opioid-like chemical entities against tuberculosis infection.

Synthesis and Biological Evaluation of 8-Quinolinamines and Their Amino Acid Conjugates as Broad-Spectrum Anti-infectives.

In the search of therapeutic agents for emerging drug-resistant parasites, the synthesis of newer classes of 8-quinolinamines has emerged as a successful chemotherapeutic approach. We report synthesis of 8-quinolinamines bearing 5-alkoxy, 4-methyl, and 2-tert-butyl groups in the quinoline framework and their amino acid conjugates as broad-spectrum anti-infectives. 8-Quinolinamines exhibited potent in vitro antimalarial activity [IC50 = 20-4760 ng/mL (drug-sensitive Plasmodium falciparum D6 strain) and IC50 = 22-4760 ng/mL (drug-resistant P. falciparum W2 strain)]. The most promising analogues have cured all animals at 25 mg/kg/day against drug-sensitive Plasmodium berghei and at 50 mg/kg/day against multidrug-resistant Plasmodium yoelii nigeriensis infections in Swiss mice. The in vitro antileishmanial activities (IC50 = 0.84-5.0 µg/mL and IC90 = 1.95-7.0 µg/mL) comparable to standard drug pentamidine were exhibited by several of the synthesized 8-quinolinamines. At the same time, very promising antifungal activities (Candida albicans-IC50 = 4.93-19.38 µg/mL; Candida glabrata-IC50 = 3.96-19.22 µg/mL; Candida krusei-IC50 = 2.89-18.95 µg/mL; Cryptococcus neoformans-IC50 = 0.67-18.64 µg/mL; and Aspergillus fumigatus-IC50 = 6.0-19.32 µg/mL) and antibacterial activities (Staphylococcus aureus-IC50 = 1.33-18.9 µg/mL; methicillin-resistant S. aureus-IC50 = 1.38-15.34 µg/mL; and Mycobacterium intracellulare-IC50 = 3.12-20 µg/mL) were also observed. None of the 8-quinolinamines exhibited cytotoxicity and therefore are a promising structural class of compounds as antiparasitic and antimicrobials.

Guanylthiourea derivatives as potential antimalarial agents: Synthesis, in vivo and molecular modelling studies.

Guanylthiourea (GTU) derivatives were identified as possible anti-malarial agents, recently, using in vitro studies on Plasmodium falciparum. This article gives an account of the in vivo anti-malarial activity of GTU derivatives against experimental rodent malaria. A total of 20 synthesized GTU derivatives were evaluated for in vivo antimalarial activity, out of which six showed encouraging results; one compound appeared to have curative potential. Molecular docking and molecular dynamics analysis were carried out to understand the molecular level interactions.

Serum amyloid P-component in murine tuberculosis: induction kinetics and intramacrophage Mycobacterium tuberculosis growth inhibition in vitro.

Serum amyloid P-component (SAP), a pentraxin, is known to play an important role in innate immunity to microbial infections; however, nothing is known about it during tuberculosis (TB). Mice intratracheally infected with Mycobacterium tuberculosis Erdman, showed peak SAP levels (442+/-58.2 microg/ml) on day 21, which declined to background levels by day 60. Their serum interleukin-6 levels paralleled SAP levels, whereas, their serum transforming growth factor-beta levels were paradoxical. During the acute phase of infection, the SAP levels positively correlated with the lung mycobacterial load. Purified mouse SAP (1-50 microg/ml) treatment of M. tuberculosis-infected alveolar macrophages (AMs), in vitro, inhibited their intracellular mycobacterial growth; maximum inhibition (1.1 log10 CFU reduction) occurred at 10 microg/ml, and a 4-day treatment appeared optimal. Treatment of AMs with both rabbit anti-mouse SAP polyclonal antibody and mannose-derived simple sugars, separately, blocked the SAP-induced inhibition of mycobacterial growth. The mycobacterial growth inhibition appeared to be nitric oxide (NO)-dependent as NO synthase inhibitors, both aminoguanidine and N(G)-monomethyl-L-arginine, annulled it. Further, SAP treatment of infected AMs induced significant (P<0.05) elaboration of nitrite (72.1+/-8.3 nM/ml), compared to the controls, and these AMs showed augmented expression of inducible NO synthase. This first study demonstrates that during murine TB the SAP levels were increased, and purified mouse SAP inhibited the intra-AM M. tuberculosis growth, in vitro, apparently via NO-dependent mechanism(s). SAP may thus contribute both to the pathogenesis and pulmonary innate immunity in TB.

Leishmania donovani amastigote component-induced colony-stimulating factor production by macrophages: modulation by morphine.

The neuroimmunomodulatory effects of opiates during microbial infections are now well known; however, not much is known during leishmaniasis. Here, we report the effects of morphine on purified approximately 12-kDa component of Leishmania donovani amastigote antigen (LDAA-12)-induced colony-stimulating factor (CSF) production by mouse peritoneal macrophages (PMs) in vitro. Low concentrations (1 x 10(-9) and 1 x 10(-11) M) of morphine significantly (P < 0.05) augmented the production of CSFs, whereas high concentrations (1 x 10(-3) and 1 x 10(-5) M) inhibited CSF production. Morphine exerted a similar concentration-dependent biphasic effect on the LDAA-12-induced elaboration of granulocyte (G)-macrophage (M)-CSF (GM-CSF) and M-CSF by PMs in their conditioned medium, as quantified by using enzyme-linked immunosorbent assay. Furthermore, selective agonists of mu-(DAGO) and delta-(DPDPE) opioid receptors also, respectively, augmented and inhibited the production of CSFs. Pretreatment of PMs with naloxone (1 x 10(-5) M) significantly (P < 0.05) blocked the augmenting effect of morphine. In contrast, at 1 x 10(-5) M, naloxone lacked any effect on the inhibitory effect of morphine; however, its 100-fold higher concentration partially blocked it. This study, apparently for the first time, demonstrates that morphine, via surface opioid receptors, biphasically modulates the LDAA-12-induced CSF production by PMs, in vitro. These results thus show the implications of opiate abuse on the outcome of therapeutic interventions in areas where both visceral leishmaniasis and drug abuse are rampant.

Acute-phase reactants during murine tuberculosis: unknown dimensions and new frontiers.

SETTING: Serum amyloid P-component (SAP) plays important roles in host defense during various infectious diseases; however, nothing is known in tuberculosis (TB). OBJECTIVE: To study the SAP response of Mycobacterium tuberculosis H37Rv- and H37Ra-infected mice, and to determine the effect(s) of purified mouse SAP both on their intra-alveolar macrophage (AM) uptake and intra-AM growth in vitro. DESIGN: The SAP levels of mice intratracheally infected with M. tuberculosis H37Rv and H37Ra were determined by ELISA. Mycobacterial AM uptake and intra-AM growth in vitro were determined using fluorescence microscopy and plating, respectively. RESULTS: M. tuberculosis H37Rv-infected mice showed significantly (p < 0.05) increased SAP levels (352.8+/-36.1 microg/ml) with compared mice infected with M. tuberculosis H37Ra (170+/-18.5 microg/ml). During the acute phase of both these infections, enhanced SAP levels correlated with the lung mycobacterial load. In vitro, purified mouse SAP (1-80 microg/ml) inhibited the AM uptake of both the mycobacteria in a concentration-dependent manners to a similar extent; 20 microg/ml SAP appeared optimal. Mycobacterial uptake inhibition was divalent cation- and pH-dependent, and was unaffected both by heat-inactivated and deglycosylated SAP, separately. Curiously, purified mouse SAP (1-80 microg/ml), in a concentration-dependent manner, inhibited the intra-AM growth of both M. tuberculosis H37Rv and H37Ra in vitro; the effect was 0.8 log10 CFUs greater on the latter. Both the mannose-based simple sugars and rabbit anti-mouse SAP polyclonal antibody, separately, annulled the inhibition of mycobacterial growth in vitro. CONCLUSION: This initial study demonstrates that both the SAP response of M. tuberculosis-infected mice, and the SAP-induced intra-AM mycobacterial growth inhibition in vitro were apparently dependent on mycobacterial virulence.

Bioimmunotherapy of rodent malaria: co-treatment with recombinant mouse granulocyte-macrophage colony-stimulating factor and an enkephalin fragment peptide Tyr-Gly-Gly.

We have earlier shown that recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and methionine-enkephalin co-treatment can protect mice from malaria. We now report the bioimmunotherapeutic effect of rmGM-CSF and a synthetic enkephalin fragment peptide Tyr-Gly-Gly (TGG) co-treatment on blood-induced Plasmodium berghei infection in Swiss mice. Mice were completely aparasitimic following co-treatment with rmGM-CSF (10.0 microg/kg) and TGG (2.0 mg/kg x 3 per day, intraperitoneally (i.p.)) starting from day -1 to day +4; however, in monotherapy, neither of these agents showed any detectable bioimmunotherapeutic effect. Curiously, similar co-treatment with rmGM-CSF (10.0 microg/kg) and higher doses of TGG (10.0 mg/kg) did not protect the mice. The combined bioimmunotherapeutic effect of these agents was abrogated by the separate administration each of rabbit neutralizing anti-rmGM-CSF antibody, non-selective opioid receptor antagonist naltrexone (10.0 mg/kg x 6 per day, i.p.), and silica (3.0 mg per mouse, intravenously (i.v.)). The peritoneal and splenic macrophages from the protected mice showed a significant (P<0.05) increase in their pool-size and the phagocytic activity, ex vivo. Furthermore, the protected mice, as compared to the unprotected ones, showed a significant (P<0.05) maximum increase in their serum nitrate and nitrite, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) levels in their splenic homogenates, on the day before the beginning of the resolution of parasitaemia. Selective inhibitors of both inducible (aminoguanidine) and all forms (L-N(G)-monomethyl arginine) of nitric oxide (NO) synthase, significantly (P<0.05) augmented the mortality of co-treated mice, suggesting the role of NO in protection. These data show that, in P. berghei-infected mice, co-treatment with rmGM-CSF and conditional doses of TGG can impart protection, apparently through partly NO-dependent and macrophage-mediated mechanism(s).

Induction of colony-stimulating factors by a 30-kDa secretory protein of Mycobacterium tuberculosis H37Rv.

Colony-stimulating factors (CSFs)-induced increased hematopoietic activity is known to occur in various microbial diseases; however, not much is known during tuberculosis (TB). We investigated the CSF-inducing capability of a Mycobacterium tuberculosis H37Rv component. Swiss mice intravenously injected with purified 30-kDa secretory protein of M. tuberculosis H37Rv (Mtb30; 0.1-10 mg/kg) showed enhanced levels of serum CSFs; maximum response (142 +/- 16 colonies) occurred at 1 mg/kg. In vitro, Mtb30 (1-50 mug/mL) induced mouse peritoneal macrophages (PMs) to elaborate CSFs in the conditioned medium (CM); 25 mug/mL appeared optimal (97 +/- 11 colonies). Both in vivo and in vitro, peak CSF production occurred 24 h after stimulation which levelled-off to background levels by 72 h. Rabbit anti-Mtb30 antibody significantly (p<0.05) reduced CSF production by both Mtb30-stimulated and M. tuberculosis-infected PMs, in vitro. The induced CSFs, both in the serum and CM, appeared to be functionally similar, as they supported the formation of granulocyte (G), monocyte (M) and GM colonies, in similar proportions; the GM colonies were maximum (>79 %). Neutralizing (100%) rabbit anti-mouse interleukin-1 (IL-1) polyclonal antibody did not affect the Mtb30-induced CSF production, indicating it to be IL-1-independent; whereas, CSF production was partly dependent on tumour necrosis factor-alpha (TNF-alpha), as goat anti-mouse TNF-alpha immunoglobulin G only partly inhibited it. Mtb30-induced PM production of CSFs was de novo as it was completely blocked by cycloheximide (50 mug/mL). The CSF-inducing capability of Mtb30 appeared to be proteinaceous in nature as it was heat (70 degrees C; 1 h)-labile, was destroyed by proteases (pronase E and trypsin) and was unaffected by sodium periodate treatment. Further, compared to the controls, Mtb30 induced significantly (p<0.05) high levels of immunoreactive GM-CSF (9+/-1 and 7.5+/-0.8 ng/mL) and M-CSF (4.3+/-0.5 and 3.9+/-0.4 ng/mL) in serum and CM, respectively; G-CSF levels did not increase significantly (p>0.05). Mtb30-treated mice showed a maximum of 2.23- and 2.36-fold increase, in the splenic and femur colony forming unit-GM counts, respectively, as compared to the controls. This is the first report which demonstrates Mtb30-induced production of CSFs that is up-regulated both posttranscriptionally and functionally, and thus adds to our understanding of the molecular pathogenetic mechanisms of TB.

The dichotomy (generation of MAbs with functional heterogeneity) in antimalarial immune response in vaccinated/protected mice: a new concept in our understanding of the protective immune mechanisms in malaria.

Globally, vaccines have emerged as one of the most effective, safe, and cost-effective public health interventions, and are known to save 2-3 million lives, annually. However, despite various commendable efforts, a suitable human malaria vaccine is yet to see the light of the day. The lack of our complete understanding of the molecular mechanisms of pathogenesis and immune protection in malaria appears to be responsible for this state. Earlier, our laboratory has reported that Swiss mice vaccinated with Plasmodium yoelii nigeriensis-total parasite antigens soluble in culture medium and saponin, following a 100% lethal challenge, showed 60% protection. The monoclonal antibodies (MAbs) generated from the splenocytes of these vaccinated/protected mice, following characterization by in vitro merozoite invasion inhibition assay, ex vivo macrophage phagocytosis assay, and in vivo passive transfer of protection test, belonged to 2 distinct groups-a larger group of MAbs inhibited<58% Mz invasion and transferred 30% passive protection, whereas a smaller group of MAbs inhibited 86% Mz invasion and transferred 60% passive protection. Additionally, the MAbs of the smaller group, as compared with the larger one, mediated nearly 2.4-fold enhanced macrophage phagocytosis of infected-erythrocytes, in vitro. These results thus clearly showed a dichotomy among the generated MAbs. An exploration of the phenomenon of dichotomy in protective immunity in malaria by using various hosts and malaria parasite combinations, especially at the level of antibodies, cells, and cytokines, may add new insights to our understanding of the protective immunity, and help in the identification of biomarkers/biosignatures of immune protection and development of future human malaria vaccines.

Determination of the activity of standard anti-tuberculosis drugs against intramacrophage Mycobacterium tuberculosis, in vitro: MGIT 960 as a viable alternative for BACTEC 460.

BACTEC 460 has now been phased out, so the search for an alternative is imperative. We have determined the activity of standard anti-tuberculosis drugs against intramacrophage Mycobacterium tuberculosis, in vitro, by using BACTEC 460 and MGIT 960 methods. The minimum inhibitory concentrations of isoniazid, rifampicin, ethambutol and streptomycin against intracellular M. tuberculosis H37Rv were found to be 0.2, 0.8, 8.0, and 5.0 µg/mL, respectively, by both methods. These results show a significant (p<0.001) concordance between minimum inhibitory concentrations obtained by these two different methods. MGIT 960 system uses a robust florescence quenching-based oxygen sensor, requires no radioisotope, is safe, and relatively easy to operate. Apparently, this is the first report wherein MGIT 960 has been validated for anti-tubercular susceptibility testing against intracellular M. tuberculosis H37Rv. Our preliminary data thus clearly demonstrate that the MGIT 960 method can be considered as a promising alternative to BACTEC 460 method.

Interleukin-6: a potent biomarker of mycobacterial infection.

BACKGROUND: Human tuberculosis (TB), a chronic inflammatory disease is caused by Mycobacterium tuberculosis, a facultative intramacrophage pathogen. The highly complex interactions between mycobacteria and macrophages (MΦs), characterized in part by the induction and elaboration of several cytokines including IL-1, IL-6, IL-10, IL-12 p40 and IL-12 p70 are not yet fully understood. The cytokines are known to have important bearing on the pathogenesis and host defense during TB. We thus studied different patterns of cytokines elaborated by mouse peritoneal macrophages (PMs) following their interaction with live and heat-killed, virulent and avirulent, and pathogenic and non-pathogenic mycobacteria, in vitro. MATERIALS AND METHODS: Pathogenic M. tuberculosis H37Rv (virulent) and M. tuberculosis H37Ra (avirulent), and non-pathogenic M. smegmatis were grown in complete Middle Brook 7H9 broth. For some experiments, mycobacteria were heat-killed (80°C; 20 min). The supernatants of cultured PMs, having ingested mycobacteria for 6 h, 24 h, 4 days and 7 days, were harvested for the quantification of IL-1, IL-6, IL-10, IL-12 p40 and IL-12 p70 by using a multiplex suspension cytokine array system. RESULTS: The PMs infected with heat-killed mycobacteria, as compared to their respective live counterparts, invariably elaborated significantly (p < 0.001) increased (approximately 2-3-fold) amounts of IL-6, at all the time-points studied, in vitro. Further, PMs infected with M. tuberculosis H37Ra, as compared to M. tuberculosis H37Rv, elaborated 4-5-fold more (p < 0.001) IL-6. Non-pathogenic M. smegmatis, as compared to pathogenic M. tuberculosis H37Ra and M. tuberculosis H37Rv, following infection, induced the PMs to elaborate highest (p < 0.001) amounts of IL-6 at all the time-points studied. Curiously, none of these mycobacteria-infected PMs elaborated IL-1, IL-10, IL-12 p40 and IL-12 p70, significantly. CONCLUSION: IL-6 appears to be the only major cytokine elaborated by mycobacteria-infected PMs, in vitro, and thus may function as a potent biomarker of mycobacterial infection, either stand-alone or along with other cytokines.