Randomized trials of potential antibiotic treatment in severe melioidosis.

<p>Recommendations for diagnosis, treatment and continuing care of melioidosis based on recent experience and best practice. Antibioti<i>c</i> treatment for severe infection is either intravenous Ceftazidime or Meropenem for several weeks, followed by up to 20 weeks oral treatment with a combination of trimethoprim-sulphamethoxazole and doxycycline.</p

Schematic representation showing the virulence and pathogenesis of melioidosis infection.

<p><i>B</i>. <i>pseudomallei</i> is transmitted from its environmental reservoir to lung epithelial cells, where it initially attaches, possibly through bacterial components such as the capsule and type IV pili. Following invasion of epithelial cells, the T3SS-3 effectors assist in vacuolar escape and intracellular motility due to BimA-mediated actin polymerization. The activation of TLR-2, TLR-4, and TLR-5 by bacterial LPS and flagella results in recruitment of innate immune cells, such as neutrophils, macrophages, and natural killer cells. IRAK-M, interleukin-1-associated kinase 3; TLR, toll-like receptor; LPS, lipopolysaccharide; CD14, cluster of differentiation; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; IL, interleukin; TNF-α, tumor necrosis factor alpha; NLRC-4, NRL family CARD domain-containing protein 4; NLRP3, NACHT, LRR, and PYD domain-containing protein 3; ACS, acetyl-CoA synthetases; PCR, polymerase chain reaction.</p

Ultrastructural examination of <i>B</i>. <i>pseudomallei</i> used for infection.

<p>(A) Scanning electron microscopy (SEM) image showing Bp before the infection with U-937 cells. (B) Bp-infected cells were examined by a transmission electron microscopy (TEM) with large number of bacilli (Bp) presented in the cytoplasm. Light micrograph showing <i>B</i>. <i>pseudomallei</i> infection of mouse spleen; section stained by Haematoxylin and Eosin (H&E) imaged with different magnification. (C–D) Large abscesses (Abs) with focal areas of necrosis, surrounded by a rim of meshed fibrous tissue (f) are evident after 2 weeks IP challenge with 1.7x105 CFU/ml. The bacterial invasion is more in spleen and liver than kidney. Bp, <i>B</i>. <i>pseudomallei</i>; abs, abscess; f, fibrous tissue; rs, rough surface; s, septa; sb, single bacilli; n, nucleus; ph, phagocytosis.</p

Global distribution of melioidosis outbreak, incidence, and their reported cases (an overview).

<p>Global distribution of melioidosis outbreak, incidence, and their reported cases (an overview).</p

Global geographical distribution of melioidosis.

<p>The areas of high endemicity in Southeast Asia (Thailand, Vietnam, Cambodia, Malaysia, and Singapore)/Northern Australia. Possible endemic, sporadic areas, environmental isolates only, and unconfirmed travel history/only serology evidence are indicated. However, sporadic cases have been reported throughout the world in Pakistan, India, Bangladesh, Indonesia, Philippines, Sri Lanka, Papua New Guinea, Madagascar, France, Mexico, Brazil, Colombia, Venezuela, Ecuador, the Middle East (Iran), and the Caribbean.</p

Purification and characterization of anticancer protein CP-P.

<p>(A) The clear supernatant of <i>Calotropis procera</i> root-bark was separated by gel-filtration (Superdex G-75) chromatography into five major peaks CP-1–CP-5, (B) the most active peak (CP-3) was further separated by reverse-phase high performance liquid chromatography (RP-HPLC) by using a C18 column which produced two peaks namely CP-F1 and CP-F2, (C) the active fraction (CP-F1) was resolved by a C8 column and gave a single peak named <i>Calotropis procera</i> protein “CP-P”, (D) CP-P mass was analyzed by a perspective biosystem matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/MS).</p

The activities of antioxidant enzymes levels in breast tissue of control and experimental animals.

<p>Values are mean ± SD (n = 3 replicates)</p>*<p>P<0.001,</p><p>≠ P<0.01,</p>§<p>P<0.05,</p><p>NS - Not Significant (with respect to control), Abbreviations: NC - normal control, BCC - breast cancer control, BC - breast cancer, CYC - cyclophosphamide, CP-P - <i>Calotropis procera</i> protein, (a) - Group II and IV compared between Group I, (b) - Group III, IV and V compared between Group II.</p>1<p>units/mg protein required to inhibit 50% of adrenaline autooxidation,</p>2<p>µ moles of H₂O₂ consumed/min/mg protein,</p>3<p>µg of thio ester formed/min/mg protein,</p>4<p>µg of GSH/mg protein,</p>5 & 6<p>mg/g wet tissue.</p

The antiproliferative effect of CP-P evaluated against MCF-7 and MDA-MB-231 human breast cancer cells.

<p>(A) <i>In vitro</i> cytotoxicity of CP-P, with or without CYC, upon MCF-7 and MDA-MB-231 cells at different doses (800–12.5 µg/ml). The CP-P alone, or when combined with CYC, leads to significant (*P<0.01) reduction in cell proliferation even at 12.5 and 50 µg/ml doses versus untreated control. The MCF-7 cells were more sensitive against the combination treatment. Values are mean ± SD of three replicates. Untreated MCF-7 cells served as a control, CP-P treatment evidenced cell death (CD) at 100 µg/ml concentrations on MDA-MB-231 cells. The CP-P alone, or in combination with CYC, caused the most significant (*P<0.01) leakage of LDH. The combination treatment induced more cell-death and lytic effects in both cell lines than CP-P or CYC alone. (B) Light micrographs showing the MCF-7 cell morphology before and after treatment. (i) There was no effect on untreated MCF-7 cells as a control, (ii) protein (CP-P) or drug (CYC) alone treated cells induced severe cell membrane damage and inhibited MCF-7 cell growth, (iv) We also observed that MCF-7 cells treated with CP-P in combination with CYC showed remarkable morphological changes like cell shrinkage, formation of membrane blebs, nuclear and cytoplasmic condensation when observed under microscope (Symbol denotes: NC-Normal Control, MC-morphological changes, CD-Cell Death, MD - membrane damage). (C) Electron micrographs of MCF-7 cells exposed to CP-P alone, or with CYC, showing the cellular and morphological changes. The transmission electron microscopy (TEM) analysis was performed on MCF-7 human breast cancer cells exposed to the CP-P alone, or with CYC treatment to observe morphological changes. Electron micrographs of MCF-7 cells, (i) untreated control MCF-7 cells which showed normal morphology. (ii) <i>Calotropis procera</i> protein (CP-P) treatment exhibited cell death at 100 µg/ml concentrations, a portion of the nucleus dismantled within the cytoplasm and a large number of vacuoles were clearly visible in proteins treated cells. (iii) CYC alone treatment caused blebbing as revealed by the presence of large vacuoles on MCF-7 treated cells. (iv) These results further confirmed that the combination treatment of CP-P+CYC induce MCF-7 cells death at 12.5 µg/ml doses. MCF-7 cells treated with CP-P+CYC showed apoptotic morphology including chromatin condensation into dense granules or blocks. The cells showed loss of microvilli and blebbing of cell membrane which are characteristics of cells undergoing apoptosis.</p

The number of apoptotic cells in breast tumor sections was detected by TUNEL assay.

<p>(<b>A</b>) The apoptotic effect of CP-P alone or in a combination of CYC treatment was analyzed by flow cytometry to compare the various cell cycle phases. The CP-P alone, or in combination with CYC, induced promising apoptotic effects. As a result, fewer MCF-7 cells accumulated in the Sub-G1 phase compared to the control. (<b>B</b>) TUNEL positive cells were quantified, revealing that apoptotic cells in breast tumors increased significantly in CP-P or CYC drug alone, or in combination, groups versus untreated cells. CP-P and CYC drug-treated groups were compared to breast cancer control groups. The combination treatment induced more positive cells than others. Values are mean ± S.D. (n = 3) replicates, **P<0.01 values indicate significance to control. (<b>C</b>) CP-P inhibits TNF-α -induced nuclear degradation/phosphorylation of IκBα. MCF-7 cells were either untreated or pretreated with 50 µg/ml CP-P for 6 h at 37°C and then treated with 1 nM TNF-α for 30 min. Cytoplasmic extracts were prepared and analyzed by Western blotting using antibodies against anti- IκBα and phospho-specific IκBα. The same membrane was reblotted with GAPDH antibody to verify equal loading. The results shown are representative of two independent experiments. (<b>D</b>) CP-P inhibits TNF-α-induced nuclear phosphorylation/translocation of p65. MCF-7 cells were either untreated or pretreated with 50 µg/ml CP-P for 6 h at 37°C and then incubated with 1 nM TNF-α for 30 min. Nuclear extracts were prepared and analyzed by Western blotting using antibodies against anti-p65 and phospho-specific p65. The same membrane was reblotted with PARP antibody to verify equal loading. The results shown are representative of two independent experiments. (<b>E</b>) Effect of CP-P on TNF-induced phosphorylation of IKK-α and IKK-β. MCF-7 cells were incubated with 50 µg/ml CP-P for 6 h at 37°C and then treated with 1 nM TNF-α for 15 min. Whole cell extracts were prepared and analyzed by Western blot analysis using anti-phospho-specific IKK-α/β antibody. The same membrane was reblotted with anti-IKK-α antibody to verify equal loading. (<b>F</b>) CP-P inhibits TNF-α -induced expression of NF-κB-dependent proliferative and antiapoptotic proteins. MCF-7 cells were incubated with 50 µg/ml CP-P for 6 h at 37°C and then treated with 1 nM TNF-α for 24 h. Whole-cell extracts were prepared and analyzed by Western blot using the indicated antibodies (Cyclin D1 and Bcl-2). The same membrane was reblotted with β-actin antibody to verify equal loading. Results are representative of two independent experiments.</p

Antitumor effect of CP-P (0.1 mg/kg, oral administration)+CYC (0.1 mg/kg, intra peritoneal injection) treated on DMBA-induced mammary tumors in rats after 110 days.

<p>(A) Microscopic images showed that the DMBA-induced breast tumor development after 90 days on either side of the mammary gland are marked by a circle and an arrow pointed out. (B) Breast tumor volume was measured and calculated as 30 millimeter in diameter in breast cancer control panel and the tumor incidence was compared within the groups. (C) Percentage reduction of tumor volume was measured after 20 days treatment of CP-P (0.1 mg/kg, oral administration)+CYC (0.1 mg/kg, intra peritoneal injection). (D) Body weight loss before and after the treatment. Body weight changes of different treatments compared within the five different groups (Symbol denotes: NC-Normal Control, BBC - Breast Cancer Control).</p