IFN-λ Regulates Neutrophil Biology to Suppress Inflammation in Herpes Simplex Virus-1-Induced Corneal Immunopathology.

HSV-1 infection of the cornea causes a severe immunoinflammatory and vision-impairing condition called herpetic stromal keratitis (SK). The virus replication in corneal epithelium followed by neutrophil- and CD4+ T cell-mediated inflammation plays a dominant role in SK. Although previous studies demonstrate critical functions of type I IFNs (IFN-α/ß) in HSV-1 infection, the role of recently discovered IFN-λ (type III IFN), specifically at the corneal mucosa, is poorly defined. Our study using a mouse model of SK pathogenesis shows that HSV-1 infection induces a robust IFN-λ response compared with type I IFN production at the corneal mucosal surface. However, the normal progression of SK indicates that the endogenous IFN responses are insufficient to suppress HSV-1-induced corneal pathology. Therefore, we examined the therapeutic efficacy of exogenous rIFN-λ during SK progression. Our results show that rIFN-λ therapy suppressed inflammatory cell infiltration in the cornea and significantly reduced the SK pathologic condition. Early rIFN-λ treatment significantly reduced neutrophil and macrophage infiltration, and IL-6, IL-1ß, and CXCL-1 production in the cornea. Notably, the virucidal capacity of neutrophils and macrophages measured by reactive oxygen species generation was not affected. Similarly, ex vivo rIFN-λ treatment of HSV-1-stimulated bone marrow-derived neutrophils significantly promoted IFN-stimulated genes without affecting reactive oxygen species production. Collectively, our data demonstrate that exogenous topical rIFN-λ treatment during the development and progression of SK could represent a novel therapeutic approach to control HSV-1-induced inflammation and associated vision impairment.

Retraction notice to "Overlapping signal sequences control nuclear localization and endoplasmic reticulum retention of GRP58" [Biochemical and Biophysical Research Communications 377 (2) (2008) 407-412].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). The University of Maryland, Baltimore conducted an internal investigation which found that the article was compromised and a preponderance of evidence supports retraction of the publication in order to correct the scientific record and ensure its integrity. The Editor-in-Chief has decided to retract this article. This article has been found to contain manipulated and enhanced figures, namely figures 1D and 1E, 4A and 4B.

Fabrication of 3-O-sn-Phosphatidyl-L-serine Anchored PLGA Nanoparticle Bearing Amphotericin B for Macrophage Targeting.

PURPOSE: To fabricate, characterize and evaluate 3-O-sn-Phosphatidyl-L-serine (PhoS) anchored PLGA nanoparticles for macrophage targeted therapeutic intervention of VL. MATERIALS AND METHODS: PLGA-AmpB NPs were prepared by well-established nanoprecipitation method and decorated with Phos by thin film hydration method. Physico-chemical characterization of the formulation was done by Zetasizer nano ZS and atomic force microscopy. RESULTS: The optimized formulation (particle size, 157.3 ± 4.64 nm; zeta potential, - 42.51 ± 2.11 mV; encapsulation efficiency, ∼98%) showed initial rapid release up to 8 h followed by sustained release until 72 h. PhoS generated 'eat-me' signal driven augmented macrophage uptake, significant increase in in-vitro (with ∼82% parasite inhibition) and in-vivo antileishmanial activity with preferential accumulation in macrophage rich organs liver and spleen were found. Excellent hemo-compatibility justified safety profile of developed formulation in comparison to commercial formulations. CONCLUSION: The developed PhoS-PLGA-AmpB NPs have improved efficacy, and necessary stability which promisingly put itself as a better alternative to available commercial formulations for optimized treatment of VL.

Syndecan-1 identifies and controls the frequency of IL-17-producing naïve natural killer T (NKT17) cells in mice.

Invariant natural killer T (iNKT) cells recognize glycolipids as antigens and diversify into NKT1 (IFN-γ), NKT2 (IL-4), and NKT17 (IL-17) functional subsets while developing in the thymus. Mechanisms that govern the balance between these functional subsets are poorly understood due, partly, to the lack of distinguishing surface markers. Here we identify the heparan sulfate proteoglycan syndecan-1 (sdc1) as a specific marker of naïve thymic NKT17 cells in mice and show that sdc1 deficiency significantly increases thymic NKT17 cells at the expense of NKT1 cells, leading to impaired iNKT cell-derived IFN-γ, both in vitro and in vivo. Using surface expression of sdc1 to identify NKT17 cells, we confirm differential tissue localization and interstrain variability of NKT17 cells, and reveal that NKT17 cells express high levels of TCR-ß, preferentially use Vß8, and are more highly sensitive to ɑ-GalCer than to CD3/CD28 stimulation. These findings provide a novel, noninvasive, simple method for identification, and viable sorting of naïve NKT17 cells from unmanipulated mice, and suggest that sdc1 expression negatively regulates homeostasis in iNKT cells. In addition, these findings lay the groundwork for investigating the mechanisms by which sdc1 regulates NKT17 cells.

Bioinspired Calcium Phosphate Nanoparticles Featuring as Efficient Carrier and Prompter for Macrophage Intervention in Experimental Leishmaniasis.

PURPOSE: To develop a biocompatible and bioresorbable calcium phosphate (CaP) nanoparticles (NPs) bearing Amphotericin B (AmB) with an aim to provide macrophage specific targeting in visceral leishmaniasis (VL). MATERIALS & METHODS: CaP-AmB-NPs were architectured through emulsion precipitation method. The developed formulation was extensively characterized for various parameters including in-vitro and in-vivo antileishmanial activity. Moreover, plasma pharmacokinetics, tissue biodistribution and toxicity profile were also assessed. RESULTS: Optimized CaP-AmB-NPs exhibited higher entrapment (71.1 ± 6.68%) of AmB. No trend related to higher hemolysis was apparent in the developed formulation as evidenced in commercially available colloidal and liposomal formulations. Cellular uptake of the developed CaP-AmB-NPs was quantified through flow cytometry in J774A.1 cell line, and a 23.90 fold rise in uptake was observed. Fluorescent microscopy also confirmed the time dependent rise in uptake. In-vivo multiple dose toxicity study demonstrated no toxicity upto 5 mg/kg dose of AmB. Plasma kinetics and tissue distribution studies established significantly higher concentration of AmB in group treated with CaP-AmB-NPs in liver and spleen as compared to CAmB, LAmB and AmB suspension group. In-vivo animal experimental results revealed that the CaP-AmB-NPs showed higher splenic parasite inhibition compared to CAmB and LAmB in leishmania parasite infected hamsters. CONCLUSIONS: The investigated CaP-AmB-NPs are effective in provoking macrophage mediated uptake and collectively features lower toxicity and offers a suitable replacement for available AmB-formulations for the obliteration of intra-macrophage VL parasite.

Oncogene PKCε controls INrf2-Nrf2 interaction in normal and cancer cells through phosphorylation of INrf2.

The INrf2 (Keap1)-Nrf2 cell sensor complex has a crucial role in protection against chemical- and radiation-induced oxidative stress and cellular transformation. INrf2, in association with Cul3-Rbx1, ubiquitylates and degrades Nrf2. Exposure to stressors leads to stabilization of Nrf2 and the coordinated activation of cytoprotective proteins and cellular protection. However, the molecular signal(s) that regulate control of Nrf2 by INrf2 remain elusive. In this report, we demonstrate that phosphorylation of INrf2 at Ser599 and Ser602 by the oncoprotein PKCε is essential for INrf2-Nrf2 interaction, and the subsequent ubiquitylation and degradation of Nrf2. Inhibition of PKCε, knockdown of PKCε and the INrf2S602A mutant all failed to phosphorylate INrf2, leading to loss of the INrf2-Nrf2 interaction, Nrf2 degradation and enhanced cytoprotection and drug resistance. Molecular modeling analyses revealed that phosphorylation of S599 exposes the deeply buried S602 for phosphorylation and enhanced INrf2-Nrf2 interaction. Analysis of human lung and liver tumor protein arrays showed lower PKCε and higher Nrf2 levels, which presumably promoted cancer cell survival and drug resistance. In conclusion, phosphorylation of INrf2 by PKCε leads to regulation of Nrf2, with significant implications for the survival of cancer cells, which often express lower levels of PKCε.

Antigen presenting cells targeting and stimulation potential of lipoteichoic acid functionalized lipo-polymerosome: a chemo-immunotherapeutic approach against intracellular infectious disease.

Antigen presenting cells (APC) are well-recognized therapeutic targets for intracellular infectious diseases, including visceral leishmaniasis. These targets have raised concerns regarding their potential for drug delivery due to overexpression of a variety of receptors for pathogen associated molecular pathways after infection. Since, lipoteichoic acid (LTA), a surface glycolipid of Gram-positive bacteria responsible for recognition of bacteria by APC receptors that also regulate their activation for pro-inflammatory cytokine secretion, provides additive and significant protection against parasite. Here, we report the nanoarchitechture of APC focused LTA functionalized amphotericin B encapsulated lipo-polymerosome (LTA-AmB-L-Psome) delivery system mediated by self-assembly of synthesized glycol chitosan-stearic acid copolymer (GC-SA) and cholesterol lipid, which can activate and target the chemotherapeutic agents to Leishmania parasite resident APC. Greater J774A and RAW264.7 macrophage internalization of FITC tagged LTA-AmB-L-Psome compared to core AmB-L-Psome was observed by FACSCalibur cytometer assessment. This was further confirmed by higher accumulation in macrophage rich liver, lung and spleen during biodistribution study. The LTA-AmB-L-Psome overcame encapsulated drug toxicity and significantly increased parasite growth inhibition beyond commercial AmB treatment in both in vitro (macrophage-amastigote system; IC50, 0.082 ± 0.009 µg/mL) and in vivo (Leishmania donovani infected hamsters; 89.25 ± 6.44% parasite inhibition) models. Moreover, LTA-AmB-L-Psome stimulated the production of protective cytokines like interferon-γ (IFN-γ), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase and nitric oxide with down-regulation of disease susceptible cytokines, like transforming growth factor-ß (TGF-ß), IL-10, and IL-4. These data demonstrate the potential use of LTA-functionalized lipo-polymerosome as a biocompatible lucrative nanotherapeutic platform for overcoming toxicity and improving drug efficacy along with induction of robust APC immune responses for effective therapeutics of intracellular diseases.

Overexpressed Macrophage Mannose Receptor Targeted Nanocapsules- Mediated Cargo Delivery Approach for Eradication of Resident Parasite: In Vitro and In Vivo Studies.

PURPOSE: Since, Leishmania protozoans are obligate intracellular parasites of macrophages, an immunopotentiating macrophage-specific Amphotericin B (AB) delivery system would be ideally appropriate to increase its superiority for leishmaniasis treatment and to eliminate undesirable toxicity. Herein, we report AB entrapped mannose grafted chitosan nanocapsules (MnosCNc-AB) that results in effective treatment of visceral leishmaniasis, while also enhancing L. donovani specific T-cell immune responses in infected host. METHODS: MnosCNc-AB were prepared via synthesized mannosylated chitosan deposition on interface of oil/water nanoemulsion intermediate and were characterized. J774A.1 macrophage uptake potential, antileishmanial activity and immunomodulatory profile were evaluated in hamster. Tissue localization, biodistribution and toxicity profile were also investigated. RESULTS: MnosCNc-AB had nanometric size (197.8 ± 8.84 nm), unimodal distribution (0.115 ± 0.04), positive zeta potential (+31.7 ± 1.03 mV) and 97.5 ± 1.13% cargo encapsulation efficiency. Superior macrophage internalization of mannosylated chitosan nanocapsules compared to unmodified chitosan nanocapsules was observed by fluorescence-based assessment, further confirmed by rapid blood clearance and, greater localization and higher accumulation in macrophage rich liver and spleen. While, MnosCNc-AB mediated cargo distribution to kidney decreased. Augmented in vitro antileishmanial activity and in vivo pro-inflammatory mediator's expression were observed with MnosCNc-AB, led to significant reduction (∼90%) in splenic parasite burden. CONCLUSIONS: Results demonstrated that mannose ligand grafted chitosan nanocapsules could improve selective delivery of AB into macrophages via interactions with overexpressed mannose receptors thus reduce undesirable toxicity. Study provides evidence for MnosCNc-AB potential to leishmaniasis therapeutics and presents valuable therapeutic strategies for combating chronic macrophage-resident microbial infections.

Self assembled ionically sodium alginate cross-linked amphotericin B encapsulated glycol chitosan stearate nanoparticles: applicability in better chemotherapy and non-toxic delivery in visceral leishmaniasis.

OBJECTIVES: To investigate the applicability, localization, biodistribution and toxicity of self assembled ionically sodium alginate cross-linked AmB loaded glycol chitosan stearate nanoparticles for effective management of visceral leishmaniasis. METHODS: Here, we fabricated Amphotericin B (AmB) encapsulated sodium alginate-glycol chitosan stearate nanoparticles (AmB-SA-GCS-NP) using strong electrostatic interaction between oppositely charged polymer and copolymer by ionotropic complexation method. The tagged FAmB-SA-GCS-NP was compared with tagged FAmB for in vitro macrophagic uptake in J774A macrophages and in vivo localization in liver, spleen, lung and kidney tissues. The AmB-SA-GCS-NP and plain AmB were compared for in vitro and in vivo antileishmanial activity, pharmacokinetics, organ distribution and toxicity profiling. RESULTS: The morphology of SA-GCS-NP revealed as nanocrystal (size, 196.3 ± 17.2 nm; PDI, 0.216 ± 0.078; zeta potential, (-) 32.4 ± 5.1 mV) by field emission scanning electron microscopy and high resolution transmission electron microscopy. The macrophage uptake and in vivo tissue localization studies shows tagged FAmB-SA-GCS-NP has significantly higher (~1.7) uptake compared to tagged FAmB. The biodistribution study of AmB-SA-GCS-NP showed more localized distribution towards Leishmania infected organs i.e. spleen and liver while lesser towards kidney. The in vitro (IC50, 0.128 ± 0.024 µg AmB/ml) and in vivo (parasite inhibition, 70.21 ± 3.46%) results of AmB-SA-GCS-NP illustrated significantly higher (P < 0.05) efficacy over plain AmB. The monomeric form of AmB within SA-GCS-NP, observed by UV-visible spectroscopy, favored very less in vitro and in vivo toxicities compared to plain AmB. CONCLUSION: The molecular organization, toxicity studies, desired localization and biodistribution of cost effective AmB-SA-GCS-NP was found to be highly effective and can be proved as practical delivery platform for better management of leishmaniasis.

NRH:quinone oxidoreductase 2 (NQO2) protein competes with the 20 S proteasome to stabilize transcription factor CCAAT enhancer-binding protein α (C/EBPα), leading to protection against γ radiation-induced myeloproliferative disease.

NRH:quinone oxidoreductase 2 (NQO2) is a flavoprotein that protects cells against radiation and chemical-induced oxidative stress. Disruption of the NQO2 gene in mice leads to γ radiation-induced myeloproliferative diseases. In this report, we showed that the 20 S proteasome and NQO2 both interact with myeloid differentiation factor CCAAT-enhancer-binding protein α (C/EBPα). The interaction of the 20 S proteasome with C/EBPα led to the degradation of C/EBPα. NQO2, in the presence of its cofactor NRH, protected C/EBPα against 20 S degradation. Deletion and site-directed mutagenesis demonstrated that NQO2 and 20 S competed for the same binding region of S(268)GAGAGKAKKSV(279) in C/EBPα. Exposure of mice and HL-60 cells to γ radiation enhanced the levels of NQO2, which led to an increased NQO2 interaction with C/EBPα and decreased 20 S interaction with C/EBPα. NQO2 stabilization of C/EBPα was independent of NQO1, even though both interacted with the same C/EBPα domain. NQO2(-/-) mice, deficient in NQO2, failed to stabilize C/EBPα. This contributed to the development of γ radiation-induced myeloproliferative disease in NQO2(-/-) mice.

Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression.

INrf2 (Keap1) serves as a negative regulator of the cytoprotective transcription factor Nrf2. At basal levels, INrf2 functions as a substrate adaptor to sequester Nrf2 into the Cul3-Rbx1 E3 ligase complex for ubiquitylation and proteasomal degradation. In response to antioxidants, Nrf2 is released from the INrf2-Cul3-Rbx1 complex and translocates into the nucleus, where it activates ARE-mediated cytoprotective gene expression. The present studies demonstrate that INrf2, Cul3 and Rbx1 export out of the nucleus and are degraded during the early or pre-induction response to antioxidants. Mutation of Tyr85 in INrf2 stymied the nuclear export of INrf2, suggesting that tyrosine phosphorylation controls the pre-induction nuclear export and degradation in response to antioxidants. The nuclear export of Cul3-Rbx1 were also blocked when INrf2Tyr85 was mutated, suggesting that INrf2-Cul3-Rbx1 undergo nuclear export as a complex. INrf2 siRNA also inhibited the nuclear export of Cul3-Rbx1, confirming that Cul3-Rbx1 requires INrf2 for nuclear export. Newly synthesized INrf2-Cul3-Rbx1 is imported back into the nucleus during the post-induction period to ubiquitylate and degrade Nrf2. Mutation of INrf2Tyr85 had no effect on activation of Nrf2 but led to nuclear accumulation of Nrf2 during the post-induction period owing to reduced export and degradation of Nrf2. Our results also showed that nuclear export and degradation followed by the new synthesis of INrf2-Cul3-Rbx1 controls the cellular abundance of the proteins during different phases of antioxidant responses. In conclusion, the early or pre-induction nuclear export of INrf2 in response to antioxidants is controlled by tyrosine phosphorylation, whereas the nuclear export of Cul3 and Rbx1 is controlled by INrf2, allowing normal activation or repression of Nrf2.

Exploitation of lectinized lipo-polymerosome encapsulated Amphotericin B to target macrophages for effective chemotherapy of visceral leishmaniasis.

We have designed lectin functionalized Lipo-polymerosome bearing Amphotericin B (Lec-AmB-L-Psome) for specific internalization via lectin receptors overexpressed on infected macrophages of mononuclear phagocytic system (MPS) for the effective management of intramacrophage diseases such as visceral leishmaniasis. The lipo-polymerosome composed of glycol chitosan-stearic acid copolymer (GC-SA25%) and model lipid cholesterol was surface-functionalized with lectin by the EDC/NHS carbodiimide coupling method. Our designed Lec-AmB-L-Psome showed >2-fold enhanced uptake and significantly higher internalization in macrophages as compared to AmB-L-Psome. Importantly, pharmacokinetic and organ distribution studies illustrate significantly higher accumulation of Lec-AmB-L-Psome in MPS especially in liver, spleen, and lung as compared to AmB-L-Psome, Ambisome, and Fungizone. The IC50 value demonstrated that Lec-AmB-L-Psome has 1.63, 2.23, and 3.43 times higher activity than AmB-L-Psome (p < 0.01), Ambisome (p < 0.05), and Fungizone (p < 0.05), respectively. Additionally, the Lec-AmB-L-Psome showed significantly higher splenic parasite inhibition (78.66 ± 3.08%) compared to Fungizone and Ambisome that caused only 56.54 ± 3.91% (p < 0.05) and 66.46 ± 2.08% (p < 0.05) parasite inhibition, respectively, in Leishmania-infected hamsters. The toxicity profile revealed that Lec-AmB-L-Psome is a safe delivery system with diminished nephrotoxicity which is a limiting factor of Fungizone application. Taken together, these studies suggest that this surface functionalized self-assembled Lec-AmB-L-Psome can introduce a new platform to specifically target macrophages for effective management of intramacrophage diseases.

The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene?

The transcription factor Nrf2 is responsible for regulating a battery of antioxidant and cellular protective genes, primarily in response to oxidative stress. A member of the cap 'n' collar family of transcription factors, Nrf2 activation is tightly controlled by a series of signaling events. These events can be separated into the basal state, a preinduction response, gene induction, and finally a postinduction response, culminating in the restoration of redox homeostasis. However, despite the immensely intricate level of control the cellular environment imposes on Nrf2 activity, there are many opportunities for perturbations to arise in the signaling events that favor carcinogenesis and, therefore, implicate Nrf2 as both a tumor suppressor and a protooncogene. Herein, we highlight the ways in which Nrf2 is regulated, and discuss some of the Nrf2-inducible antioxidant (NQO1, NQO2, HO-1, GCLC), antiapoptotic (Bcl-2), metabolic (G6PD, TKT, PPARγ), and drug efflux transporter (ABCG2, MRP3, MRP4) genes. In addition, we focus on how Nrf2 functions as a tumor suppressor under normal conditions and how its ability to detoxify the cellular environment makes it an attractive target for other oncogenes either via stabilization or degradation of the transcription factor. Finally, we discuss some of the ways in which Nrf2 is being considered as a therapeutic target for cancer treatment.

Covalent functionalized self-assembled lipo-polymerosome bearing amphotericin B for better management of leishmaniasis and its toxicity evaluation.

Amphotericin B remains the preferred choice for leishmanial infection, but it has limited clinical applications due to substantial dose limiting toxicities. In the present work, AmB has been formulated in lipo-polymerosome (L-Psome) by spontaneous self-assembly of synthesized glycol chitosan-stearic acid copolymer. The optimized L-Psome formulation with vesicle size of 243.5 ± 17.9 nm, PDI of 0.168 ± 0.08 and zeta potential of (+) 27.15 ± 0.46 mV with 25.59 ± 0.87% AmB loading was obtained. The field emission scanning electron microscopy (FESEM) and high resolution transmission electron microscopy (HRTEM) images suggest nearly spherical morphology of L-Psome. An in vitro study showed comparatively sustained AmB release (66.082 ± 1.73% within 24 h) and high plasma stability compared to commercial Ambisome and Fungizone, where glycol chitosan content was found to be efficient in preventing L-Psome destabilization in the presence of plasma protein. In vitro and in vivo toxicity studies revealed less toxicity of AmB-L-Psome compared to commercialized Fungizone and Ambisome favored by monomeric form of AmB within L-Psome, observed by UV-visible spectroscopy. Experimental results of in vitro (macrophage amastigote system) and in vivo (Leishmania donovani infected hamsters) illustrated the efficacy of AmB-L-Psome to augment effective antileishmanial properties supported by upregulation of Th-1 cytokines (TNF-α, IL-12 and IFN-γ) and inducible nitric oxide synthase, and downregulation of Th-2 cytokines (TGF-ß, IL-10 and IL-4), measured by quantitative mRNA analysis by real time PCR (RT-PCR). Conclusively, developed L-Psome system could be a viable alternative to the current less stable, toxic commercial formulations and developed as a highly efficacious drug delivery system.

Assessment of the contribution of NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) to the reduction of vitamin K in wild-type and NQO1-deficient mice.

NQO1 [NAD(P)H quinone oxidoreductase 1; also known as DT-diaphorase] is a cytosolic enzyme that catalyses the two-electron reduction of various quinones including vitamin K. The enzyme may play a role in vitamin K metabolism by reducing vitamin K to vitamin K hydroquinone for utilization in the post-translational γ-glutamyl carboxylation reactions required by several proteins involved in blood coagulation. The aim of the present study was to assess the contribution of NQO1 to vitamin K reduction and haemostasis in an in vivo model. We examined the contribution of NQO1 to haemostasis by examining survival rates in mice poisoned with the anticoagulant warfarin. Supraphysiological amounts of vitamin K sufficiently reversed the effects of warfarin in both wild-type and NQO1-deficient mice. Additionally, vitamin K reductase activities distinct from VKOR (vitamin K epoxide reductase) and NQO1 were measured in vitro from both wild-type and NQO1-defecient mice. The results of the present study suggest that NQO1 does not play a major role in the production of vitamin K hydroquinone and supports the existence of multiple vitamin K reduction pathways. The properties of a NAD(P)H-dependent vitamin K reductase different from NQO1 are described.