Stepwise representation of various procedures involved in the neutralisation and short term splenic cell culture assay on VL subjects.

<p>Stepwise representation of various procedures involved in the neutralisation and short term splenic cell culture assay on VL subjects.</p

Effect of anti-IFN-γ + anti-TNF-α antibody on cytokine production in splenic aspirate cultures of human VL.

<p>Splenic supernatants from anti-IFN-γ + anti-TNF-α antibody treated cultures were used for estimation of cytokines employing ELISA. Value shown <b>(A)</b> IL-4 (pg/ml) and <b>(B)</b> IL-10 (pg/ml) are cytokine concentrations. Statistical analyses were executed using Wilcoxon matched-pair test. Solid black dots represent splenic aspirate cells devoid of CD4⁺ or CD14⁺ subset whereas open circles show splenic biopsy treated with control magnetic beads.</p

Dual-Sensitive Fluorescent Nanoprobes for Simultaneously Monitoring <i>In Situ</i> Changes in pH and Matrix Metalloproteinase Expression in Stiffness-Tunable Three-Dimensional <i>In Vitro</i> Scaffolds

A tumor microenvironment often presents altered physicochemical characteristics of the extracellular matrix (ECM) including changes in matrix composition, stiffness, protein expression, pH, temperature, or the presence of certain stromal and immune cells. Of these, overexpression of matrix metalloproteinases (MMPs) and extracellular acidosis are the two major hallmarks of cancer that can be exploited for tumor detection. The change in matrix stiffness and the release of certain cytokines (TNF-α) in the tumor microenvironment play major roles in inducing MMP-9 expression in cancerous cells. This study highlights the role of mechanical cues in upregulating MMP-9 expression in cancerous cells using stiffness-tunable matrix compositions and dual-sensitive fluorescent nanoprobes. Ionically cross-linked 3D alginate/gelatin (AG) scaffolds with three stiffnesses were chosen to reflect the ECM stiffnesses corresponding to healthy and pathological tissues. Moreover, a dual-sensitive nanoprobe, an MMP-sensitive peptide conjugated to carbon nanoparticles with intrinsic pH fluorescence properties, was utilized for in situ monitoring of the two cancer hallmarks in the 3D scaffolds. This platform was further utilized for designing a 3D core–shell platform for spatially mapping tumor margins and for visualizing TNF-α-induced MMP-9 expression in cancerous cells

Effect of anti-IFN-γ + anti-TNF-α antibody on number of viable amastigotes.

<p>Splenic biopsies were treated with monoclonal neutralising antibodies in a 72hr culture. Following three days of incubation number of viable parasite was determined using titration culture. Results obtained were presented on vertical scatter dot plot <b>(A)</b> Splenic cultures (n = 27) were plated after treatment with neutralising antibodies against human IFN-γ and TNF-α (solid grey circles) or with their isotype controls (open circles). <b>(B)</b> Apart of splenic biopsy (n = 18) was also treated with anti-IL-10 monoclonal antibody (solid grey circles) and its isotype control IgG_2B (open circles), as a benchmark for this neutralising assays in splenic cells. Number of viable amastigotes in cultures is presented on log scale and in scientific format. Antibody treated samples were compared with their corresponding isotype control treated cultures using Wilcoxon matched pair test. Statistical significances are shown with a P value **P<0.01.</p

Aggregate clinical features of all the VL patients enrolled in the study.

<p>Aggregate clinical features of all the VL patients enrolled in the study.</p

Effect of E-64 on different oxidative stress markers in <i>S. cervi</i>.

<p>Adult female worms (n = 10) were incubated with different concentration of E-64 separately in 20 ml maintenance medium at 37°C. Worms incubated in only maintenance medium served as Control. After 8 h of incubation different enzyme activity were checked according to the method given in materials and methods section. The level of GSH is expressed as μM/mg protein. Protein carbonyl content is expressed in terms of μmol/mg protein. The level of lipid peroxidation is expressed in terms of μmol MDA/mg protein. NADPH oxidase activity is expressed as U/ml. Values in parentheses indicate percentage inhibition/activation in comparison to control. Data expressed is Mean ± SEM of at least three values (n = 3). ^***P<0.0001, ^**P<0.001, ^*P<0.05. Values with P<0.05 were considered significant.</p

The expression of <i>ced-3</i> and <i>ced-9</i> in the control and parasites exposed to E-64.

<p>GAPDH was used as a loading control in the experiment.</p

Effect of E-64 on the activity of different enzymes in <i>S. cervi.</i>

<p>Adult female worms (n = 10) were incubated with different concentration of E-64 separately in 20 ml maintenance medium at 37°C. Worms incubated in only maintenance medium served as Control. After 8 h of incubation different enzyme activity were checked according to the method given in materials and methods section. Activities are indicated as U/ml. Values in parentheses indicate percentage inhibition/activation in comparison to control. Data expressed is Mean ± SEM of at least three values (n = 3). ^***P<0.0001, ^**P<0.001, ^*P<0.05. Values with P<0.05 were considered significant.</p

Interaction of E-64 with GSH was drawn using ChemDraw Ultra 7.0.

<p>(A) <i>In vitro</i> effect of E-64 on GSH using DTNB as substrate (B) Possible interaction of E-64 with GSH molecule.</p

The effect of E-64 on viability of adult parasites and microfilariae.

<p>(A) Adult female worms (n = 10) of equal size were incubated in 20 ml maintenance at specified experimental conditions and viability was assessed by MTT assay after 8 h of exposure. Worms incubated in maintenance medium only served as control. (B) Microfilariae (mf) recovered from the uterus of gravid female of control and treated set. Adult female parasites were dissected longitudinally and the released mf was visualized under microscope at 40× (Motic B1 series). Data expressed is Mean ± SEM of at least three values (n = 3). ^***P<0.0001, ^**P<0.001, ^*P<0.05. Values with P<0.05 were considered significant.</p