An indirect enzyme-linked immunosorbent assay for rapid and quantitative assessment of Type III virulence phenotypes of Pseudomonas aeruginosa isolates

BACKGROUND: The presence of a Type III secretion system in clinical isolates of Pseudomonas aeruginosa is associated with severe disease and poor outcomes in infections caused by this pathogen. We describe an indirect enzyme-linked immunosorbent assay that rapidly and quantitatively detects two exotoxins, ExoU and ExoT, and two structural components, PopD and PcrV, of the P. aeruginosa Type III secretion system after in-vitro growth in a calcium-free minimal medium. METHODS: We used this assay to characterize the Type III secretion phenotype of 74 clinical isolates of P. aeruginosa. Findings were compared with results of standard immunoblotting and correlated with Type III secretion-dependent virulence of isolates toward cultured epithelial cells. RESULTS: Results of the ELISA assay were concordant with immunoblot detection of the secreted antigens for 73 of 74 isolates. The Type III secretion phenotype assessed by this immunoassay predicted bacterial virulence toward epithelial cells in vitro for all but five of the clinical isolates. CONCLUSION: The availability of an ELISA assay for rapid detection of Type III secreted virulence factors will facilitate large clinical studies to examine whether the Type III secretion phenotype of a P. aeruginosa isolate predicts the course of clinical disease in a patient and should be taken into account in determining optimal treatment strategies for infected patients

Experimental Visceral Leishmaniasis: Role of trans-Aconitic Acid in Combined Chemotherapy

We previously reported the effectiveness of trans-aconitic acid (TAA) as an antileishmanial compound. Inhibitory effects of TAA along with other antileishmanial compounds on transformation and in vitro multiplication in macrophage cultures of Leishmania donovani have been assessed. The efficacy of TAA in combined chemotherapy of experimental visceral leishmaniasis has also been evaluated along with those of commonly used antileishmanial compounds such as sodium stibogluconate, pentamidine, and allopurinol. TAA (2 mM) inhibited transformation of L. donovani amastigotes to promastigotes by 95.2%, whereas in combination with pentamidine (5 ,ug/ml), allopurinol (10 ,ug/ml), and sodium stibogluconate (50 ,ug of Sb per ml), it inhibited transformation by about 100, 99, and 98.5%, respectively. Sodium stibogluconate (20 ,ug of Sb per ml), pentamidine (2 ,ug/ml), and allopurinol (5 ,ug/ml) suppressed the amastigote burden in peritoneal macrophage cultures from BALB/c mice by 32.6, 56.1, and 46.3%, respectively. When these three drugs were used along with TAA (5 mM), the parasite loads were reduced by 100, 100, and 88.1%, respectively. TAA (5 mM) alone suppressed the amastigote burden by 59.5%. In experimental visceral leishmaniasis in hamsters (1-month model), TAA at a dose of 200 mg/kg of body weight per day suppressed the spleen parasite load by 73.5%, and TAA in combination with sodium stibogluconate (50 mg of Sb per kg per day), pentamidine (8 mg/kg/day), and allopurinol (15 mg/kg/day) inhibited the spleen parasite load by 98, 98.9, and 97%, respectively. Individually, these three drugs inhibited the parasite load by 35, 20, and 22%, respectively. TAA (400 mg/kg/day) inhibited the spleen parasite load by 99.8%, but an inhibitory effect of -100% was noted when TAA was supplemented with an antileishmanial drug. TAA was administered in experimental animals through oral, intraperitoneal, and intramuscular routes; the intramuscular route was most effective. A parasitic protozoan, Leishmania donovani, is the causative agent of visceral leishmaniasis or kala-azar. Kala-azar patients are generally treated with sodium stibogluconate, pentamidine, allopurinol, etc

Leishmania donovani promastigote: Effect of Adrenergic Agonists and Antagonist on Growth and Lipophosphoglycan Synthesis

ABSTRACTBackground: Leishmania is the causative agent of a spectrum of diseases in human ranging in severity from self-healing cutaneous form to disfiguring mucocutaneous lesion to deadly visceral form of leishmaniasis. The treatment is still suffering from toxicity of the present drug regimen and the emergence of drug resistant leishmaniasis. Successful immunotherapy is yet to develop. It is beneficial to venture the parasite second messenger systems for the development of successful antileishmanial agents. The role of adrenergic receptor agonists and antagonist in the growth of L. donovani promastigotes and the synthesis of phosphorylated macromolecules were addressed in this report. Materials and Methods: The present work is entirely experimental. The effect of dibutyryl cAMP (db-cAMP), dibutyryl cGMP (db-cGMP), epinephrine, isoproterenol and propranolol on the growth of L. donovani promastigotes using defined medium were studied. Biosynthetically pulse labeling of promastigotes with [32P]-orthophosphate with or without experimental agents following SDS-PAGE and autoradiography was analyzed. Results: The growth of L. donovani promastigotes at 96 h culture was inhibited by 69%, 83, 52% and 95% with db-cAMP, epinephrine, isoproterenol and propranolol at 100 μM each, respectively in compared to control. Multiplication of parasite was stimulated by db-cGMP. The expression of lipophosphoglycan of the parasite was drastically affected in the following order with propranolol&gt;epinephrine&gt;cAMP&gt;isoproterenol at 100 μM of each agent for 6 h exposure to the promastigotes. Conclusion: The growth of the parasite and the synthesis of lipophosphoglycan were significantly more inhibited by epinephrine or propranolol in a dose dependent manner than cAMP or isoproterenol.Keywords: cAMP; cGMP; epinephrine; Leishmania donovani; LPG; propronolol. </p

Increase in MMP-9 Expressing CD11b+ cells in a CD44 Dependent Way Reduces Severity of Experimental Autoimmune Encephalomyelitis

Background: Multiple sclerosis (MS) is a dangerous neurodegenerative disorder. Various aspects of  the    disease have been studied in experimental animal model. Migration of immune cells to the central nervous system (CNS) is a predominant feature of MS. CD44 molecule has been reported to be involved in many  important biological processes including contribution in severing inflammation in experimental autoimmune encephalomyelitis (EAE). Matrix metalloprotease-9 (MMP-9) interaction with CD44 has been well known to be involved in cellular adhesion, transmigration and inflammation. In this study, we were interested to examine the role of phagocytic cells expressing MMP-9 in resolving EAE. Materials and Methods: C57BL/6 WT and CD44 KO mice were used as EAE animal model. The level of phagocytic cells expressing MMP-9 in the  secondary lymphoid organs were assessed in EAE induced WT as well as CD44 KO animals. Results: EAE severity was found in CD44 KO group compared to WT. Level of CD11b cells (marker of phagocytic cell) in the peritoneal cells expressing MMP-9 was higher in WT compared to CD44 KO. CD11b stained area found to be greater in WT lymph node compared to CD44 KO. Conclusions: This observation suggests the role of CD44 molecule in modulating the immune scenario which is related to disease severity. This study also opens avenues for the specific inflammatory roles of different immune cells in MS.Keywords: multiple sclerosis; EAE; CD44; MMP-9; CD11b; CNS. </p

Experimental Visceral Leishmaniasis: Role of trans-Aconitic Acid in Combined Chemotherapy

We previously reported the effectiveness of trans-aconitic acid (TAA) as an antileishmanial compound. Inhibitory effects of TAA along with other antileishmanial compounds on transformation and in vitro multiplication in macrophage cultures of Leishmania donovani have been assessed. The efficacy of TAA in combined chemotherapy of experimental visceral leishmaniasis has also been evaluated along with those of commonly used antileishmanial compounds such as sodium stibogluconate, pentamidine, and allopurinol. TAA (2 mM) inhibited transformation of L. donovani amastigotes to promastigotes by 95.2%, whereas in combination with pentamidine (5 ,ug/ml), allopurinol (10 ,ug/ml), and sodium stibogluconate (50 ,ug of Sb perml), it inhibited transformation by about 100, 99, and 98.5%, respectively. Sodium stibogluconate (20 ,ug of Sb per ml), pentamidine (2 ,ug/ml), and allopurinol (5 ,ug/ml) suppressed the amastigote burden in peritoneal macrophage cultures from BALB/c mice by 32.6, 56.1, and 46.3%, respectively. When these three drugs were used along with TAA (5 mM), the parasite loads were reduced by 100, 100, and 88.1%, respectively. TAA (5mM) alone suppressed the amastigote burden by 59.5%. In experimental visceral leishmaniasis in hamsters (1-month model), TAA at a dose of 200 mg/kg of body weight per day suppressed the spleen parasite load by 73.5%, and TAA in combination with sodium stibogluconate (50 mg of Sb per kg per day), pentamidine (8mg/kg/day), and allopurinol (15 mg/kg/day) inhibited the spleen parasite load by 98, 98.9, and 97%, respectively. Individually, these three drugs inhibited the parasite load by 35, 20, and 22%, respectively. TAA(400 mg/kg/day) inhibited the spleen parasite load by 99.8%, but an inhibitory effect of -100% was noted when TAA was supplemented with an antileishmanial drug. TAA was administered in experimental animals through oral, intraperitoneal, and intramuscular routes; the intramuscular route was most effective

Use of “e” and “g” operators to a fuzzy production inventory control model for substitute items

In this paper, a fuzzy optimal control model for substitute items with stock and selling price dependent demand has been developed. Here the state variables (stocks) are assumed to be fuzzy variables. So the proposed dynamic control system can be represented as a fuzzy differential system which optimize the profit of the production inventory control model through Pontryagin’s maximum principle. The proposed fuzzy control problem has been transformed into an equivalent crisp differential system using “e” and “g” operators. The deterministic system is then solved by using Newton’s forward-backward method through MATLAB. Finally some numerical results are presented both in tabular and graphical form

Multi-item Optimal control problem with fuzzy costs and constraints using Fuzzy variational principle

An imperfect multi-item production system is considered against time dependent demands for a finite time horizon. Here production is defective. Following [Khouja and Mehrez J. Oper. Res. Soc. 45 (1994) 1405–1417], unit production cost depends on production, raw-material and maintenance costs. Produced items have same fixed life-time. Warehouse capacity is limited and used as a constraint. Available space, production, stock and different costs are assumed as crisp or imprecise. With the above considerations, crisp and fuzzy constrained optimal control problems are formulated for the minimization of total cost consisting of raw-material, production and holding costs. These models are solved using conventional and fuzzy variational principles with equality constraint condition and no-stock as end conditions. For the first time, the inequality space constraint is converted into an equality constraint introducing a pseudo state variable following Bang Bang control. [Roul et al., J. Intell. Fuzzy Syst. 32 (2017) 565–577], as stock is mainly controlled by production, for the control problems production is taken as the control variable and stock as state variable. The reduced optimal control problem is solved by generalised reduced gradient method using Lingo-11.0. The models are illustrated numerically. For the fuzzy model, optimum results are obtained as fuzzy numbers expressed by their membership functions. From fuzzy results, crisp results are derived using α-cuts

Structural Elucidation of the Cell-Penetrating Penetratin Peptide in Model Membranes at the Atomic Level: Probing Hydrophobic Interactions in the Blood–Brain Barrier

Cell-penetrating peptides (CPPs) have shown promise in nonpermeable therapeutic drug delivery, because of their ability to transport a variety of cargo molecules across the cell membranes and their noncytotoxicity. Drosophila antennapedia homeodomain-derived CPP penetratin (RQI­K­I­W­F­Q­N­R­R­M­K­WKK), being rich in positively charged residues, has been increasingly used as a potential drug carrier for various purposes. Penetratin can breach the tight endothelial network known as the blood–brain barrier (BBB), permitting treatment of several neurodegenerative maladies, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. However, a detailed structural understanding of penetratin and its mechanism of action is lacking. This study defines structural features of the penetratin-derived peptide, DK17 (DRQ­I­K­I­W­F­Q­N­R­R­M­K­WKK), in several model membranes and describes a membrane-induced conformational transition of the DK17 peptide in these environments. A series of biophysical experiments, including high-resolution nuclear magnetic resonance spectroscopy, provides the three-dimensional structure of DK17 in different membranes mimicking the BBB or total brain lipid extract. Molecular dynamics simulations support the experimental results showing preferential binding of DK17 to particular lipids at atomic resolution. The peptide conserves the structure of the subdomain spanning residues Ile6–Arg11, despite considerable conformational variation in different membrane models. <i>In vivo</i> data suggest that the wild type, not a mutated sequence, enters the central nervous system. Together, these data highlight important structural and functional attributes of DK17 that could be utilized in drug delivery for neurodegenerative disorders