IFN-γ treatment of rodents infected with erythrocytic stages of Plasmodium chabaudi: Differential effects according to the immunological status

Recombinant rat interferon-γ (rrIFN-γ) was tested for its antimalarial activity in three different models of Plasmodium chabaudi-blood stage malaria. Doses ranging from 1 × 10⁴ to 1 × 10⁵ U of rrIFN-γ were used in each model. In BALB/c mice (lethal infection), prophylactic treatment with daily intraperitoneal (i.p.) injections reduced parasitemia and delayed mortality. In contrast, subcutaneous administration of rrIFN-γ was inefficient, as was currative schedule of i.p. administration. Euthymic Fischer rats, which develop an acute and resolutive infection, were partly protected by i.p. prophyactic administration of rrIFN-γ. Parasitemia was reduced without being lengthened, resulting in a marked decrease in parasite burden. Subcutaneous administration was less efficient whereas curative schedule was not. Athymic (nude) Fischer rats which present a longlasting and stable infection were treated with propylactic and curative schedules of i.p. administration of rrINF-γ. In each case, rrIFN-γ-treated nude rats, as control nude rats, were unable to resolve their chronic infection. The conditions required to obtain a beneficial effect are thus restrictive for a therapetic use in humans. Moreover, these results show that, despite the fact that IFN-γ is considered as a major component of the immune response, this cytokine alone is not sufficient to induce the totality of the effector mechanisms necessary to cure malarial infections.     

Intracellularly monitoring/imaging the release of doxorubicin from pH-responsive nanoparticles using Förster resonance energy transfer

Stimuli-responsive nanoparticles (NPs) have been receiving much attention as a drug-delivery vehicle for therapeutic applications; once internalized into cells, the intracellular fate of NPs and their drug release behavior in response to local stimuli must be understood for efficient delivery of therapeutics. In this study, we prepared pH-responsive doxorubicin (DOX)-loaded NPs, made of N-palmitoyl chitosan bearing a Cy5 moiety (Cy5-NPCS), as an anticancer delivery device. The results of our molecular dynamic simulations showed that the ability of Cy5-NPCS to self-associate offered the close proximity between the donor (DOX) and the acceptor (Cy5) required for F?rster resonance energy transfer (FRET), while the pH-driven structure transition prescribed the on-to-off switch of the energy transfer. The caveolae-mediated pathway played a major role in the internalization of NPCS NPs. Using the concept of FRET, we found that the DOX fluorescence in the cytosol was first seen when NPCS NPs were present in the slightly acidic early endosomes. Following NPCS NPs trafficking into a more acidic organelle (late endosomes/lysosomes), a more evident release of DOX into the cytosol was observed; the released DOX was then gradually accumulated in the cell nuclei, leading to a significant cytotoxicity. Understanding the fate of NPs with respect to their intracellular localization and drug release behavior is crucial for the rational design of drug carriers.     

Nanostructural control of the release of macromolecules from silica sol–gels

The therapeutic use of biological molecules such as growth factors and monoclonal antibodies is challenging in view of their limited half-life in vivo. This has elicited the interest in delivery materials that can protect these molecules until released over extended periods of time. Although previous studies have shown controlled release of biologically functional BMP-2 and TGF-β from silica sol–gels, more versatile release conditions are desirable. This study focuses on the relationship between room temperature processed silica sol–gel synthesis conditions and the nanopore size and size distribution of the sol–gels. Furthermore, the effect on release of large molecules with a size up to 70 kDa is determined. Dextran, a hydrophilic polysaccharide, was selected as a large model molecule at molecular sizes of 10, 40 and 70 kDa, as it enabled us to determine a size effect uniquely without possible confounding chemical effects arising from the various molecules used. Previously, acid catalysis was performed at a pH value of 1.8 below the isoelectric point of silica. Herein the silica synthesis was pursued using acid catalysis at either pH 1.8 or 3.05 first, followed by catalysis at higher values by adding base. This results in a mesoporous structure with an abundance of pores around 3.5 nm. The data show that all molecular sizes can be released in a controlled manner. The data also reveal a unique in vivo approach to enable release of large biological molecules: the use more labile sol–gel structures by acid catalyzing above the pH value of the isoelectric point of silica; upon immersion in a physiological fluid the pores expand to reach an average size of 3.5 nm, thereby facilitating molecular out-diffusion.     

Effect of Plasmodium yoelii YM Infection on Vaccination with 19 kDa Carboxylterminus of the Merozoite Surface Protein 1 （MSP1 19）

The 19 kDa carboxylterminal fragment of merozoite surfaceprotein 1 (MSP119) is a leading malaria vaccine candidate[1]. Immunization of monkeys [ 2 , 3 ] or mice [ 4 , 5 ]with recombinant MSP119 confers protection against chal-lenge infection. Studies in m…     

Synergistic effects of SDF-1α chemokine and hyaluronic acid release from degradable hydrogels on directing bone marrow derived cell homing to the myocardium

Poor cell engraftment in the myocardium is a limiting factor towards the use of bone marrow derived cells (BMCs) to treat myocardial infarction (MI). In order to enhance the engraftment of circulating BMCs in the myocardium following MI, we have developed in situ forming hyaluronic acid (HA) hydrogels with degradable crosslinks to sustain the release of recombinant stromal cell-derived factor-1 alpha (rSDF-1α) and HA to the injured myocardium. Both rSDF-1α and the crosslinkable HA macromer stimulate BMC chemotaxis up to 4-fold in?vitro through CXCR4 and CD44 receptor signaling, respectively. Moreover, the HA macromer binds rSDF-1α with a dissociation constant of 36?±?5?μM through electrostatic interaction. When formed into hydrogels via photoinitiated crosslinking, release of encapsulated rSDF-1α and crosslinked HA was sustained for over 7 days, and these molecules significantly increased BMC chemotaxis in?vitro. When applied to the heart following experimental MI in mice, the HA gel containing rSDF-1α significantly increased the number of systemically infused BMCs in the heart by ～8.5 fold after 7 days, likely through both systemic and local effects of released molecules. We conclude that sustained release of rSDF-1α and HA from our engineered HA hydrogels enhances BMC homing to the remodeling myocardium better than delivery of rSDF-1α alone.     

Gram-negative bacterial membrane vesicle release in response to the host-environment: different threats,same trick?

Bacteria are confronted with a multitude of stressors when occupying niches within the host. These stressors originate from host defense mechanisms, other bacteria during niche competition or result from physiological challenges such as nutrient limitation. To counteract these stressors, bacteria have developed a stress-induced network to mount the adaptations required for survival. These stress-induced adaptations include the release of membrane vesicles from the bacterial envelope. Membrane vesicles can provide bacteria with a plethora of immediate and ultimate benefits for coping with environmental stressors. This review addresses how membrane vesicles aid Gram-negative bacteria to cope with host-associated stress factors, focusing on vesicle biogenesis and the physiological functions. As many of the pathways, that drive vesicle biogenesis, confer we propose that shedding of membrane vesicles by Gram-negative bacteria entails an integrated part of general stress responses.     

Adenovirus-mediated gene transfer of TGF-β1 to the renal glomeruli leads to proteinuria

The mechanism of proteinuria in many common kidney diseases involves glomerular hemodynamic effects and local expression of angiogenic, fibrogenic, and vasoactive factors. Transforming growth factor (TGF)-β has been associated with many diseases involving proteinuria and renal fibrosis. TGF-β has been shown to induce podocyte dedifferentiation in vitro, but its in vivo effects on the glomerular filtration barrier are not well described. In this study, we used an adenovirus vector to transfer active TGF-β1 to the glomeruli of rat kidneys. Transient TGF-β1 overexpression induced significant proteinuria, podocyte foot process effacement, nephrin down-regulation, and nephrinuria. The expression of synaptopodin was also significantly down-regulated by TGF-β1. Increased glomerular expression of Snail, suggestive of an in vivo dedifferentiation process, was associated with a loss of podocyte epithelial markers. The expression of angiopoietin-1 and angiopoietin-2 was significantly increased in TGF-β1-transfected glomeruli, and TGF-β1 increased the expression of the angiopoietin receptor, Tie2, in podocyte cell culture. TGF-β1 down-regulated nephrin and synaptopodin expression in podocytes in cell culture; this effect was reversed by the blockade of both angiopoietin and Tie2 activities. These findings suggest that locally produced TGF-β1 can cause podocyte dedifferentiation marked by a loss of synaptopodin, nephrin, and foot process effacement, partly regulated by angiopoietins. This process represents a novel pathway that may explain proteinuria in a variety of common renal diseases.     

Amyloid β-protein stimulates trafficking of cholesterol and caveolin-1 from the plasma membrane to the Golgi complex in mouse primary astrocytes

The Golgi complex plays a key role in cholesterol trafficking in cells. Our earlier study demonstrated amyloid β-protein (Aβ) alters cholesterol distribution and abundance in the Golgi complex of astrocytes. We now test the hypothesis that the Aβ-induced increase in Golgi complex cholesterol is due to retrograde movement of the cholesterol carrier protein caveolin-1 from the cell plasma membrane to the Golgi complex in astrocytes. Results with mouse primary astrocytes indicated that Aβ_1-42-induced increase in cholesterol and caveolin abundance in the Golgi complex was accompanied by a reduction in cholesterol and caveolin levels in the plasma membrane. Transfected rat astrocytes (DITNC1) with siRNA directed at caveolin-1 mRNA inhibited the Aβ_1-42-induced redistribution of both cholesterol and caveolin from the plasma membrane to the Golgi complex. In astrocytes not treated with Aβ_1-42, suppression of caveolin-1 expression also significantly reduced cholesterol abundance in the Golgi complex, further demonstrating the role for caveolin in retrograde transport of cholesterol from the plasma membrane to the Golgi complex. Perturbation of this process by Aβ_1-42 could have consequences on membrane structure and cellular functions requiring optimal levels of cholesterol.     

Spindle cell lesions of the breast – An approach to diagnosis

Spindle cell lesions of the breast are among the less common entities encountered in breast pathology. They encompass a whole spectrum of benign reactive lesions to high grade malignant neoplasms. An accurate diagnosis is important to ensure that the patient receives the appropriate management. While this group of conditions broadly share the same basic morphology of a lesion composed of spindle cells, there are often recognizable differences on histology, which coupled with ancillary studies and correlation with the clinical and imaging findings, can help one to arrive at a specific diagnosis. On core biopsy however, spindle cell lesions pose significant interpretive challenges and a firm diagnosis is often not possible. We share our approach to this group of conditions, with a focus on the more common entities, highlighting their key clinical, imaging and pathological features.     

WISH cell line: from the antiviral system to a novel reporter gene assay to test the potency of human IFN-α and IFN-β

Interferons (IFNs) are potent biologically active proteins that are widely used as biopharmaceuticals, so their potency must be correctly identified. Usually, the biological activity is quantified by a bioassay based on its capacity to induce an antiviral state in target cells, but this type of assays is subject to virus manipulation-related issues and they show considerable intra- and inter-assay variability. In this work, we generated a reporter gene assay (RGA) supported on the WISH-Mx/eGFP reporter cell line to determine human type I IFN activity. WISH cells were stably transfected with the enhanced green fluorescent protein (eGFP) gene under the control of type I IFN-inducible Mx2 promoter. This system implies the use of a standardized cell line for human IFN-potency analysis such as WISH cells and the simultaneous use of the sensitive reporter gene eGFP, having also several advantages when compared to antiviral activity assays and other RGAs: it can determine the potency of hIFN-α and hIFN-β using only one cell line showing the highest expression of eGFP after 28h and being only observed in cells treated with type I IFNs due to the specificity of the Mx promoter. It is a sensitive assay and it represents a safe alternative when compared with the conventional antiviral tests. The cell line showed the same sensitivity along 57 generations, allowing its use during two months of successive culture. The inter- and intra-assay coefficients of variation were lower than 20%, demonstrating its reproducibility. In addition, this reporter cell line can be used for the conventional antiviral assay, either for hIFN-α or hIFN-β. In conclusion, we have developed an alternative reporter system for the analysis of type I IFNs, in which its performance make it a suitable candidate to replace or complement conventional bioassays that are currently employed to measure IFN potency.     

Controlled release of transforming growth factor-β3 from cartilage-extra-cellular-matrix-derived scaffolds to promote chondrogenesis of human-joint-tissue-derived stem cells

The objective of this study was to develop a scaffold derived from cartilaginous extracellular matrix (ECM) that could be used as a growth factor delivery system to promote chondrogenesis of stem cells. Dehydrothermal crosslinked scaffolds were fabricated using a slurry of homogenized porcine articular cartilage, which was then seeded with human infrapatellar-fat-pad-derived stem cells (FPSCs). It was found that these ECM-derived scaffolds promoted superior chondrogenesis of FPSCs when the constructs were additionally stimulated with transforming growth factor (TGF)-β3. Cell-mediated contraction of the scaffold was observed, which could be limited by the additional use of 1-ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) crosslinking without suppressing cartilage-specific matrix accumulation within the construct. To further validate the utility of the ECM-derived scaffold, we next compared its chondro-permissive properties to a biomimetic collagen–hyaluronic acid (HA) scaffold optimized for cartilage tissue engineering (TE) applications. The cartilage-ECM-derived scaffold supported at least comparable chondrogenesis to the collagen–HA scaffold, underwent less contraction and retained a greater proportion of synthesized sulfated glycosaminoglycans. Having developed a promising scaffold for TE, with superior chondrogenesis observed in the presence of exogenously supplied TGF-β3, the final phase of the study explored whether this scaffold could be used as a TGF-β3 delivery system to promote chondrogenesis of FPSCs. It was found that the majority of TGF-β3 that was loaded onto the scaffold was released in a controlled manner over the first 10 days of culture, with comparable long-term chondrogenesis observed in these TGF-β3-loaded constructs compared to scaffolds where the TGF-β3 was continuously added to the media. The results of this study support the use of cartilage-ECM-derived scaffolds as a growth factor delivery system for use in articular cartilage regeneration.     

Improvement of the yields of recombinant actin and myosin V–HMM in the insect cell/baculovirus system by the addition of nutrients to the high-density cell culture

Baculovirus infection of Sf9 cells at high densities, such as during mid- and late exponential phase, often results in a significant reduction of protein yield per cell, compared to the early exponential phase. Nutrient depletion has been considered as a major cause for the decreased protein yield. In this study, we report that the addition of nutrients (glucose, yeastolate ultrafiltrate, and lactalbumin hydrolysate) and small fraction of fresh medium at time of infection restores the expression level of actin and myosin V?CHMM at late exponential phase (11.3?×?10⁶?cells/ml) to that at early exponential phase (1.0?×?10⁶?cells/ml). The relative yields of actin and myosin V?CHMM were approximately equal at both phases (typically 200?mg of actin and 5?mg of myosin V?CHMM per 10¹⁰?cells), i.e., the volumetric yield of proteins from the cell culture at late exponential phase was approximately tenfold higher than at early exponential phase. The functionality of the recombinant actin and myosin V?CHMM was confirmed by measuring the rate of actin polymerization, actin-activated ATPase, and the gliding velocity of actin filaments in an in vitro motility assay.     

Interaction of Plasmodium falciparum histidine-rich protein II with human lymphocytes leads to suppression of proliferation, IFN-γ release, and CD69 expression

The presence of histidine-rich protein II (HRP II) synthesized by Plasmodium falciparum in the plasma of malaria patients for longer periods even after parasite clearance raises questions about its extracellular functions. The present study was carried out to examine its influence on host immune system. Recombinant HRP-II protein was radiolabeled with ¹²⁵I to study the specific binding with T and B cells. We found that the binding of ¹²⁵I-HRP II with human T and B cells was specific, concentration dependent, saturable, and reversible. Scatchard plot analysis revealed two classes of binding sites for both T and B cells. For the T cells, the high affinity class had dissociation constant (K _d) of 5.61×10⁻¹¹ M, and the low affinity class had a K _d of 8.58×10⁻¹¹ M. For the B cells, the high and low affinity classes had a K _d of 1.32×10⁻¹¹ and 2.84×10⁻¹¹ M, respectively. Dot-blot, autoradiography, and Western blot analysis also confirmed the specific binding of HRP II with lymphocytes. HRP II significantly inhibited (∼75%) T-cell rosette formation with sheep erythrocytes. HRP II also suppressed proliferation of T and B cells triggered by CD3 and LPS, respectively. We found a reduction in IFN-γ release in T cells preincubated with HRP II. HRP II also reduced the CD69 expression on the T cells. In conclusion, HRP-II binding to human lymphocytes leads to suppression of some of their functions.P. Das and J. S. Grewal contributed equally to this work.     

Difference of Epstein–Barr virus isolates from Japanese patients and African Burkitt’s lymphoma cell lines based on the sequence of latent membrane protein 1

Sequence variations in the Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) gene have been described in many EBV-isolates. To characterize the genomic relationship between Japanese EBV and the EBV isolates of other countries, we analyzed the LMP1 nucleotide sequences in EBV positive cell lines and clinical specimens, including five African Burkitt’s lymphoma (BL) cell lines, a Japanese BL cell line, a B-lymphoblastoid cell line, a nasopharyngeal carcinoma hybrid cell line, six gastric carcinoma tissues, two peripheral blood mononuclear cells, and a B95-8 cell line, which contained the prototype EBV genome. We determined the C-terminal nucleotide sequences of LMP1 by PCR-direct sequencing analysis and characterized the sequence variation of Japanese isolates, made a phylogenetic tree from the sequence patterns of LMP1 by a neighbor-joining method. The results indicate that the Japanese EBV isolates are greatly different from the African BL isolates but are closely related to the China 1, which is a strain of Chinese EBV isolates.     

Study of the inhibition by poly morphonuclear leukocytes of TNF-α release from human mononuclear cells and its mechanism

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