Th1-Biased Immunomodulation and Therapeutic Potential of Artemisia annua in Murine Visceral Leishmaniasis

Background

In the absence of vaccines and limitations of currently available chemotherapy, development of safe and efficacious drugs is urgently needed for visceral leishmaniasis (VL) that is fatal, if left untreated. Earlier we reported in vitro apoptotic antileishmanial activity of n-hexane fractions of Artemisia annua leaves (AAL) and seeds (AAS) against Leishmania donovani. In the present study, we investigated the immunostimulatory and therapeutic efficacy of AAL and AAS.

Methodology/Principal Findings

Ten-weeks post infection, BALB/c mice were orally administered AAL and AAS for ten consecutive days. Significant reduction in hepatic (86.67% and 89.12%) and splenic (95.45% and 95.84%) parasite burden with decrease in spleen weight was observed. AAL and AAS treated mice induced the strongest DTH response, as well as three-fold decrease in IgG1 and two-fold increase in IgG2a levels, as compared to infected controls. Cytometric bead array further affirmed the elicitation of Th1 immune response as indicated by increased levels of IFN-γ, and low levels of Th2 cytokines (IL-4 and IL-10) in serum as well as in culture supernatant of lymphocytes from treated mice. Lymphoproliferative response, IFN-γ producing CD4⁺ and CD8⁺ T lymphocytes and nitrite levels were significantly enhanced upon antigen recall in vitro. The co-expression of CD80 and CD86 on macrophages was significantly augmented. CD8⁺ T cells exhibited CD62L^low and CD44^hi phenotype, signifying induction of immunological memory in AAL and AAS treated groups. Serum enzyme markers were in the normal range indicating inertness against nephro- and hepato-toxicity.

Conclusions/Significance

Our results establish the two-prong antileishmanial efficacy of AAL and AAS for cure against L. donovani that is dependent on both the direct leishmanicidal action as well as switching-on of Th1-biased protective cell-mediated immunity with generation of memory. AAL and AAS could represent adjunct therapies for the treatment of leishmaniasis, either alone or in combination with other antileishmanial agents.     

Conformational Switching in PolyGln Amyloid Fibrils Resulting from a Single Amino Acid Insertion

The established correlation between neurodegenerative disorders and intracerebral deposition of polyglutamine aggregates motivates attempts to better understand their fibrillar structure. We designed polyglutamines with a few lysines inserted to overcome the hindrance of extreme insolubility and two D-lysines to limit the lengths of β-strands. One is 33 amino acids long (PolyQKd-33) and the other has one fewer glutamine (PolyQKd-32). Both form well-dispersed fibrils suitable for analysis by electron microscopy. Electron diffraction confirmed cross-β structures in both fibrils. Remarkably, the deletion of just one glutamine residue from the middle of the peptide leads to substantially different amyloid structures. PolyQKd-32 fibrils are consistently 10–20% wider than PolyQKd-33, as measured by negative staining, cryo-electron microscopy, and scanning transmission electron microscopy. Scanning transmission electron microscopy analysis revealed that the PolyQKd-32 fibrils have 50% higher mass-per-length than PolyQKd-33. This distinction can be explained by a superpleated β-structure model for PolyQKd-33 and a model with two β-solenoid protofibrils for PolyQKd-32. These data provide evidence for β-arch-containing structures in polyglutamine fibrils and open future possibilities for structure-based drug design.     

Structure–activity relationship of dihydroxy-4-methylcoumarins as powerful antioxidants: Correlation between experimental & theoretical data and synergistic effect

The chain-breaking antioxidant activities of eight coumarins [7-hydroxy-4-methylcoumarin (1), 5,7-dihydroxy-4-methylcoumarin (2), 6,7-dihydroxy-4-methylcoumarin (3), 6,7-dihydroxycoumarin (4), 7,8-dihydroxy-4-methylcoumarin (5), ethyl 2-(7,8-dihydroxy-4-methylcoumar-3-yl)-acetate (6), 7,8-diacetoxy-4-methylcoumarin (7) and ethyl 2-(7,8-diacetoxy-4-methylcoumar-3-yl)-acetate (8)] during bulk lipid autoxidation at 37 °C and 80 °C in concentrations of 0.01–1.0 mM and their radical scavenging activities at 25 °C using TLC–DPPH test have been studied and compared. It has been found that the o-dihydroxycoumarins 3–6 demonstrated excellent activity as antioxidants and radical scavengers, much better than the m-dihydroxy analogue 2 and the monohydroxycoumarin 1. The substitution at the C-3 position did not have any effect either on the chain-breaking antioxidant activity or on the radical scavenging activity of the 7,8-dihydroxy- and 7,8-diacetoxy-4-methylcoumarins 6 and 8. The comparison with DL-α-tocopherol (TOH), caffeic acid (CA) and p-coumaric acid (p-CumA) showed that antioxidant efficiency decreases in the following sequence:     

Enhanced Antitumor Immunity Contributes to the Radio-Sensitization of Ehrlich Ascites Tumor by the Glycolytic Inhibitor 2-Deoxy-D-Glucose in Mice

Two-deoxy-D-glucose (2-DG), an inhibitor of glycolysis differentially enhances the radiation and chemotherapeutic drug induced cell death in cancer cells in vitro, while the local tumor control (tumor regression) following systemic administration of 2-DG and focal irradiation of the tumor results in both complete (cure) and partial response in a fraction of the tumor bearing mice. In the present studies, we investigated the effects of systemically administered 2-DG and focal irradiation of the tumor on the immune system in Ehrlich ascites tumor (EAT) bearing Strain “A” mice. Markers of different immune cells were analyzed by immune-flow cytometry and secretary cytokines by ELISA, besides monitoring tumor growth. Increase in the expression of innate (NK and monocytes) and adaptive CD4⁺cells, and a decrease in B cells (CD19) have been observed after the combined treatment, suggestive of activation of anti-tumor immune response. Interestingly, immature dendritic cells were found to be down regulated, while their functional markers CD86 and MHC II were up regulated in the remaining dendritic cells following the combination treatment. Similarly, decrease in the CD4⁺ naïve cells with concomitant increase in activated CD4⁺ cells corroborated the immune activation. Further, a shift from Th2 and Th17 to Th1 besides a decrease in inflammatory cytokines was also observed in the animals showing complete response (cure; tumor free survival). This shift was also complimented by respective antibody class switching followed by the combined treatment. The immune activation or alteration in the homeostasis favoring antitumor immune response may be due to depletion in T regulatory cells (CD4⁺CD25⁺FoxP3⁺). Altogether, these results suggest that early differential immune activation is responsible for the heterogenous response to the combined treatment. Taken together, these studies for the first time provided insight into the additional mechanisms underlying radio-sensitization by 2-DG in vivo by unraveling its potential as an immune-modulator besides direct effects on the tumor.     

Phosphate starvation response controls genes required to synthesize the phosphate analog arsenate

Environmental arsenic poisoning affects roughly 200 million people worldwide. The toxicity and mobility of arsenic in the environment is significantly influenced by microbial redox reactions, with arsenite (As^III) being more toxic than arsenate (As^V). Microbial oxidation of As^III to As^V is known to be regulated by the AioXSR signal transduction system and viewed to function for detoxification or energy generation. Here, we show that As^III oxidation is ultimately regulated by the phosphate starvation response (PSR), requiring the sensor kinase PhoR for expression of the As^III oxidase structural genes aioBA. The PhoRB and AioSR signal transduction systems are capable of transphosphorylation cross‐talk, closely integrating As^III oxidation with the PSR. Further, under PSR conditions, As^V significantly extends bacterial growth and accumulates in the lipid fraction to the apparent exclusion of phosphorus. This could spare phosphorus for nucleic acid synthesis or triphosphate metabolism wherein unstable arsenic esters are not tolerated, thereby enhancing cell survival potential. We conclude that As^III oxidation is logically part of the bacterial PSR, enabling the synthesis of the phosphate analog As^V to replace phosphorus in specific biomolecules or to synthesize other molecules capable of a similar function, although not for total replacement of cellular phosphate.     

Astaxanthin attenuates hepatic steatosis in high-fat diet-fed rats by suppressing microRNA-21 via transactivation of nuclear factor erythroid 2-related factor 2

Journal of Physiology and Biochemistry - This study examined whether astaxanthin (ASX) could alleviate hepatic steatosis in rats fed a high-fat diet (HFD) by modulating the nuclear factor erythroid...     

Impact on degradation of antibiotics from poultry litter using Autothermal Thermophilic Aerobic Digestion (ATAD)

Tetracycline (TC) is one of the common antibiotics which is widely used in livestock growth promotion. The prevalent application TC may pave way to progression of antibiotic resistant bacteria. The main objective of this study is to determine the effect of Autothermal Thermophilic Aerobic Digestion (ATAD) on the fate of TC residues found in digested poultry litter. For the determination of TC in poultry litter, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) were done. TLC result revealed that the R_f value of standard TC on TLC plate was 0.97 which correlates with the R_f value of TC at 0, 12, 24 and 36 h of digested poultry litter sample and not at 48, 60 and 72 h. HPLC chromatogram revealed that the limits of detection and the recovery were 5 µg/kg and 96% for standard TC. Linear correlation curves were obtained over the series of 100–500 µg/mL with correlation coefficient of 0.996 and the calibration curve was Y = 0.001X + 0.066. These results confirmed the degradation of TC in ATAD digestion of poultry litter by abiotic processes.     

Molecular and allergenic characterization of recombinant tropomyosin from mud crab Scylla olivacea

Molecular Biology Reports - Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this...