Formulation and Characterization of Hesperidin-Loaded Transethosomal Gel for Dermal Delivery to Enhance Antibacterial Activity: Comprehension of In Vitro, Ex Vivo, and Dermatokinetic Analysis.

In this study, hesperidin was loaded into a transethosome and was developed employing the rotary evaporator method. The formulation was optimized using the Box-Behnken design (BBD). The optimized HSD-TE formulation has a spherical shape, vesicle size, polydispersity index, entrapment efficiency, and zeta potential within the range of 178.98 nm; the PDI was 0.259 with a zeta potential of -31.14 mV and % EE of 89.51%, respectively. The in vitro drug release shows that HSD-TE exhibited the release of 81.124 ± 3.45% in comparison to HSD suspension. The ex vivo skin permeation showed a 2-fold increase in HSD-TE gel permeation. The antioxidant activity of HSD-TE was found to be 79.20 ± 1.77% higher than that of the HSD solution. The formulation showed 2-fold deeper HSD-TE penetration across excised rat skin membranes in confocal laser microscopy scanning, indicating promising in vivo prospects. In a dermatokinetic study, HSD-TE gel was compared to HSD conventional gel where TE significantly boosted HSD transport in the epidermis and dermal layers. The formulation showed greater efficacy than free HSD in the inhibition of microbial growth, as evidenced by antibacterial activity on the Gram-negative and positive bacteria. These investigations found that the HSD-TE formulation could enhance the topical application in the management of cutaneous bacterial infections.

Zinc Oxide Nanoparticles Blunt Potassium-Bromate-Induced Renal Toxicity by Reinforcing the Redox System.

Potassium bromate (PB) is a general food additive, a significant by-product during water disinfection, and a carcinogen (Class II B). The compound emits toxicity depending on the extent of its exposure and dose through consumable items. The current study targeted disclosing the ameliorative efficacy of zinc oxide nanoparticles (ZnO NPs) prepared by green technology in PB-exposed Swiss albino rats. The rats were separated into six treatment groups: control without any treatment (Group I), PB alone (Group II), ZnO alone (Group III), ZnO NP alone (Group IV), PB + ZnO (Group V), and PB + ZnO NPs (Group VI). The blood and kidney samples were retrieved from the animals after following the treatment plan and kept at -20 °C until further analysis. Contrary to the control (Group I), PB-treated rats (Group II) exhibited a prominent trend in alteration in the established kidney function markers and disturbed redox status. Further, the analysis of the tissue and nuclear DNA also reinforced the biochemical results of the same treatment group. Hitherto, Groups III and IV also showed moderate toxic insults. However, Group VI showed a significant improvement from the PB-induced toxic insults compared to Group II. Hence, the present study revealed the significant therapeutic potential of the NPs against PB-induced nephrotoxicity in vivo, pleading for their usage in medicines having nephrotoxicity as a side effect or in enhancing the safety of the industrial use of PB.

Postnatal deletion of Spns2 prevents neuroinflammation without compromising blood vascular functions.

Protein Spinster homolog 2 (Spns2) is a sphingosine-1-phosphate (S1P) transporter that releases S1P to regulate lymphocyte egress and trafficking. Global deletion of Spns2 (Spns2-/-) has been shown to reduce disease severity in several autoimmune disease models. To examine whether Spns2 could be exploited as a drug target, we generated and characterized the mice with postnatal knockout of Spns2 (Spns2-Mx1Cre). Our results showed that Spns2-Mx1Cre mice had significantly low number of lymphocytes in blood and lymphoid organs similar to Spns2-/- mice. Lymph but not plasma S1P levels were significantly reduced in both groups of knockout mice. Our lipidomic results also showed that Spns2 releases different S1P species into lymph. Interestingly, lymphatic vessels in the lymph nodes (LNs) of Spns2-/- and Spns2-Mx1Cre mice exhibited morphological defects. The structures of high endothelial venules (HEV) in the LNs of Spns2-Mx1Cre mice were disorganized. These results indicate that lack of Spns2 affects both S1P secretion and LN vasculatures. Nevertheless, blood vasculature of these Spns2 deficient mice was not different to controls under homeostasis and vascular insults. Importantly, Spns2-Mx1Cre mice were resistant to multiple sclerosis in experimental autoimmune encephalomyelitis (EAE) models with significant reduction of pathogenic Th17 cells in the central nervous system (CNS). This study suggests that pharmacological inhibition of Spns2 may be exploited for therapeutic applications in treatment of neuroinflammation.

Mfsd2b and Spns2 are essential for maintenance of blood vessels during development and in anaphylactic shock.

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that is secreted by several cell types. We recently showed that Mfsd2b is an S1P transporter from hematopoietic cells that contributes approximately 50% plasma S1P. Here we report the characterization of compound deletion of Mfsd2b and Spns2, another S1P transporter active primarily in endothelial cells. Global deletion of Mfsd2b and Spns2 (global double knockout [gDKO]) results in embryonic lethality beyond embryonic day 14.5 (E14.5), with severe hemorrhage accompanied by defects of tight junction proteins, indicating that Mfsd2b and Spns2 provide S1P for signaling, which is essential for blood vessel integrity. Compound postnatal deletion of Mfsd2b and Spns2 using Mx1Cre (ctDKO-Mx1Cre) results in maximal 80% reduction of plasma S1P. ctDKO-Mx1Cre mice exhibit severe susceptibility to anaphylaxis, indicating that S1P from Mfsd2b and Spns2 is indispensable for vascular homeostasis. Our results show that S1P export from Mfsd2b and Spns2 is essential for developing and mature vasculature.

Deletion of Mfsd2b impairs thrombotic functions of platelets.

We recently discovered that Mfsd2b, which is the S1P exporter found in blood cells. Here, we report that Mfsd2b is critical for the release of all S1P species in both resting and activated platelets. We show that resting platelets store S1P in the cytoplasm. After activation, this S1P pool is delivered to the plasma membrane, where Mfsd2b is predominantly localized for export. Employing knockout mice of Mfsd2b, we reveal that platelets contribute a minor amount of plasma S1P. Nevertheless, Mfsd2b deletion in whole body or platelets impairs platelet morphology and functions. In particular, Mfsd2b knockout mice show significantly reduced thrombus formation. We show that loss of Mfsd2b affects intrinsic platelet functions as part of remarkable sphingolipid accumulation. These findings indicate that accumulation of sphingolipids including S1P by deletion of Mfsd2b strongly impairs platelet functions, which suggests that the transporter may be a target for the prevention of thrombotic disorders.

Impact of Human Leukocyte Antigen-Associated Polymorphisms on Variability of HIV-1 Accessory and Regulatory Proteins.

HIV-1 escapes by acquiring mutations that differentially influence the course of infection. Unlike HIV-1 structural and enzymatic proteins, it remains elusive what extent the host immune-mediated selection pressure influences the variability of the accessory (Vif, Vpu, Vpr, and Nef) and regulatory (Tat and Rev) proteins. To address this, we analyzed the viral sequences encoding accessory and regulatory proteins from 446 human leukocyte antigen (HLA)-typed, chronically HIV-1 subtype B-infected, and treatment-naive individuals in Japan. We observed that Vpu and Vpr were the most and least polymorphic proteins with the average Shannon entropy scores of 0.63 and 0.38, respectively. Phylogenetically corrected methods identified a total of 161 HLA-associated polymorphisms; whereby Nef and Vpu had the highest (26.6%) and lowest (1.2%) proportion of amino acid sites associated with HLA-class I alleles, respectively. These results add further insight on the role of HLA-mediated selection pressure on HIV-1 sequence polymorphisms of HIV-1 accessory and regulatory proteins.

Erythrocytes efficiently utilize exogenous sphingosines for S1P synthesis and export via Mfsd2b.

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that exerts its activity via activation of five different G protein-coupled receptors, designated as S1P1-5. This potent lipid mediator is synthesized from the sphingosine precursor by two sphingosine kinases (SphK1 and 2) and must be exported to exert extracellular signaling functions. We recently identified Mfsd2b as the S1P transporter in the hematopoietic system. However, the sources of sphingosine for S1P synthesis and the transport mechanism of Mfsd2b in erythrocytes remain to be determined. Here, we show that erythrocytes efficiently take up exogenous sphingosine and that a de novo synthesis pathway in part provides sphingosines to erythrocytes. The uptake of sphingosine in erythrocytes is facilitated by the activity of SphK1. By converting sphingosine into S1P, SphK1 indirectly increases the influx of sphingosine, a process that is irreversible in erythrocytes. Our results explain for the abnormally high amount of sphingosine accumulation in Mfsd2b knockout erythrocytes. Furthermore, we show that Mfsd2b utilizes a proton gradient to facilitate the release of S1P. The negatively charged residues D95 and T157 are essential for Mfsd2b transport activity. Of interest, we also discovered an S1P analog that inhibits S1P export from erythrocytes, providing evidence that sphingosine analogs can be used to inhibit S1P export by Mfsd2b. Collectively, our results highlight that erythrocytes are efficient in sphingosine uptake for S1P production and the release of S1P is dependent on Mfsd2b functions.

Deficiency of MFSD7c results in microcephaly-associated vasculopathy in Fowler syndrome.

Several missense mutations in the orphan transporter FLVCR2 have been reported in Fowler syndrome. Affected subjects exhibit signs of severe neurological defects. We identified the mouse ortholog Mfsd7c as a gene expressed in the blood-brain barrier. Here, we report the characterizations of Mfsd7c-KO mice and compare these characterizations to phenotypic findings in humans with biallelic FLVCR2 mutations. Global KO of Mfsd7c in mice resulted in late-gestation lethality, likely due to CNS phenotypes. We found that the angiogenic growth of CNS blood vessels in the brain of Mfsd7c-KO embryos was inhibited in cortical ventricular zones and ganglionic eminences. Vascular tips were dilated and fused, resulting in glomeruloid vessels. Nonetheless, CNS blood vessels were intact, without hemorrhage. Both embryos and humans with biallelic FLVCR2 mutations exhibited reduced cerebral cortical layers, enlargement of the cerebral ventricles, and microcephaly. Transcriptomic analysis of Mfsd7cK-KO embryonic brains revealed upregulation of genes involved in glycolysis and angiogenesis. The Mfsd7c-KO brain exhibited hypoxia and neuronal cell death. Our results indicate that MFSD7c is required for the normal growth of CNS blood vessels and that ablation of this gene results in microcephaly-associated vasculopathy in mice and humans.

JunB is essential for IL-23-dependent pathogenicity of Th17 cells.

CD4+ T-helper cells producing interleukin-17 (IL-17), known as T-helper 17 (TH17) cells, comprise heterogeneous subsets that exhibit distinct pathogenicity. Although pathogenic and non-pathogenic TH17 subsets share a common RORγt-dependent TH17 transcriptional programme, transcriptional regulatory mechanisms specific to each of these subsets are mostly unknown. Here we show that the AP-1 transcription factor JunB is critical for TH17 pathogenicity. JunB, which is induced by IL-6, is essential for expression of RORγt and IL-23 receptor by facilitating DNA binding of BATF at the Rorc locus in IL-23-dependent pathogenic TH17 cells, but not in TGF-ß1-dependent non-pathogenic TH17 cells. Junb-deficient T cells fail to induce TH17-mediated autoimmune encephalomyelitis and colitis. However, JunB deficiency does not affect the abundance of gut-resident non-pathogenic TH17 cells. The selective requirement of JunB for IL-23-dependent TH17 pathogenicity suggests that the JunB-dependent pathway may be a therapeutic target for autoimmune diseases.

Identification of two unique naturally occurring Vpr sequence polymorphisms associated with clinical parameters in HIV-1 chronic infection.

HIV-1 viral protein R (Vpr) plays important roles in HIV-1 replication. Despite the identification of a number of HLA class I-associated immune escape mutations; it is yet known whether immune-driven Vpr polymorphisms are associated with disease outcome. Hereby, we comprehensively analyzed Vpr sequence polymorphisms and their association with disease outcome and host HLA genotypes, by using plasma viral RNA isolated from 444 HLA-typed, treatment-naïve, chronically HIV-1 infected individuals. Vpr amino acid residues at positions 13, 37, 45, 55, 63, 77, 84, 85, 86, and 93 were significantly associated with patients' plasma viral load and/or CD4 count. Further analysis revealed Ala at position 55 was significantly associated with lower plasma viral load; and Thr at position 63 was significantly associated with lower plasma viral load and higher CD4 count. Also, the number of amino acid residues at the two positions, located in a functionally important α-helical domain, correlated inversely with plasma viral load and positively with CD4 count. Moreover, a phylogenetically corrected method revealed residues at positions 55 and 63 are associated with patients' HLA genotypes. Taken together, our results suggest that Vpr polymorphisms at functionally important and immune-reactive sites may contribute, at least in part, to viral replication and disease outcome in vivo. J. Med. Virol. 89:123-129, 2017. © 2016 Wiley Periodicals, Inc.

Region-Specific Vulnerability to Oxidative Stress, Neuroinflammation, and Tau Hyperphosphorylation in Experimental Diabetes Mellitus Mice.

Recent epidemiological evidence suggests that diabetes mellitus (DM) is a risk factor for Alzheimer's disease (AD). One of the pathological hallmarks of AD is hyperphosphorylated tau protein, which forms neurofibrillary tangles. Oxidative stress and the activation of inflammatory pathways are features that are associated with both DM and AD. However, the brain region specificity of AD-related neurodegeneration, which mainly occurs in the hippocampus while the cerebellum is relatively unaffected, has not yet been clarified. Therefore, we used experimental DM mice (caused by an intraperitoneal injection of streptozotocin [STZ]) to determine whether these neurodegeneration-associated mechanisms were associated with region-specific selective vulnerability or tau phosphorylation. The hippocampus, midbrain, and cerebellum of aged (14 to 18 months old) non-transgenic (NTg) and transgenic mice overexpressing wild-type human tau (Tg601 mice) were evaluated after a treatment with STZ. The STZ injection increased reactive oxygen species, lipid peroxidation markers such as 4-hydroxynonenal and malondialdehyde in the hippocampus, but not in the midbrain or cerebellum. The STZ treatment also increased the number of Iba-1-positive and CD68-positive microglial cells, astrocytes, and IL-1ß, IL-6, IL-10, and IL-18 levels in the hippocampus, but not in the midbrain or cerebellum. Tau hyperphosphorylation was also enhanced in the hippocampus, but not in the midbrain or cerebellum. When the effects of STZ were compared between Tg601 and NTg mice, microglial proliferation and elevations in IL-6 and phosphorylated tau were higher in Tg601 mice. These results suggest that neuroinflammation and oxidative stress in STZ-treated mice are associated with tau hyperphosphorylation, which may contribute to selective neurodegeneration in human AD.

Short-term treadmill exercise increased tau insolubility and neuroinflammation in tauopathy model mice.

Physical exercise has been identified as a preventive measure for Alzheimer's disease (AD), one of the neuropathological hallmarks of which, neurofibrillary tangles, consist of hyperphosphorylated insoluble tau. Previous studies demonstrated that long-term treadmill exercise reduced tau hyperphosphorylation and insolubility; however, whether short-term treadmill exercise (STE) alters tau modifications currently remains unknown. In the present study, we attempted to characterize the effects of STE on tau solubility and determine its relationship with neuroinflammation using tauopathy model mice (Tg601), which express wild-type human tau. The results obtained showed that 3 weeks of non-shock treadmill exercise in Tg601 and non-transgenic female mice markedly increased insoluble tau. An analysis of phosphorylation patterns indicated that changes in tau solubility were related to an increase in phosphorylation at the tau C-terminal end. The results of immunohistochemical analyses revealed that STE increased the number of Iba-1-positive microglial cells in the hippocampus. Elevations in the levels of the lipid peroxidation markers, 4-hydroxy-trans-2-noneal and malondialdehyde, indicated the presence of oxidative stress. Moreover, higher levels of cytokines, IL-1ß and IL-18, and chemokines, CXCL-1 and CXCL-12, supported neuroinflammation.

Association between a naturally arising polymorphism within a functional region of HIV-1 Nef and disease progression in chronic HIV-1 infection.

HIV-1 Nef mediates downregulation of HLA class I (HLA-I) through a number of highly conserved sequence motifs. We investigated the in vivo implication(s) of naturally arising polymorphisms in functional motifs in HIV-1 Nef that are associated with HLA-I downregulation, including the acidic cluster, polyproline, di-arginine and Met-20 regions. Plasma samples from treatment-naive, chronically HIV-1 infected subjects were collected after obtaining informed consent, and viral RNA was extracted and amplified by nested RT-PCR. The resultant nef amplicons were sequenced directly, and subtype-B sequences with an intact open reading frame (n = 406) were included in our analyses. There was over-representation of isoleucine at position 20 (Ile-20) in our dataset when compared to sequences in the Los Alamos sequence database (17.7 vs. 6.9 %, p = 0.0309). The presence of having Ile-20 in Nef was found to be associated with higher median plasma viral load (p = 0.013), independent of associated codons or viral lineage effects, whereas no clinical association was found with polymorphisms in the other functional motifs. Moreover, introduction of a Met-20-to-Ile mutation in a laboratory strain SF2 Nef resulted in a modest, albeit not statistically significant, increase in HLA class I downregulation activity (p = 0.06). Taken together, we have identified a naturally arising polymorphism, Ile-20, within HIV-1 subtype B Nef that is associated with poorer disease outcome.

CD8(+) T cell cross-reactivity profiles and HIV-1 immune escape towards an HLA-B35-restricted immunodominant Nef epitope.

Antigen cross-reactivity is an inbuilt feature of the T cell compartment. However, little is known about the flexibility of T cell recognition in the context of genetically variable pathogens such as HIV-1. In this study, we used a combinatorial library containing 24 billion octamer peptides to characterize the cross-reactivity profiles of CD8(+) T cells specific for the immunodominant HIV-1 subtype B Nef epitope VY8 (VPLRPMTY) presented by HLA-B(*)35∶01. In conjunction, we examined naturally occurring antigenic variations within the VY8 epitope. Sequence analysis of plasma viral RNA isolated from 336 HIV-1-infected individuals revealed variability at position (P) 3 and P8 of VY8; Phe at P8, but not Val at P3, was identified as an HLA-B(*)35∶01-associated polymorphism. VY8-specific T cells generated from several different HIV-1-infected patients showed unique and clonotype-dependent cross-reactivity footprints. Nonetheless, all T cells recognized both the index Leu and mutant Val at P3 equally well. In contrast, competitive titration assays revealed that the Tyr to Phe substitution at P8 reduced T cell recognition by 50-130 fold despite intact peptide binding to HLA-B(*)35∶01. These findings explain the preferential selection of Phe at the C-terminus of VY8 in HLA-B(*)35∶01(+) individuals and demonstrate that HIV-1 can exploit the limitations of T cell recognition in vivo.

Minor contribution of HLA class I-associated selective pressure to the variability of HIV-1 accessory protein Vpu.

Host HLA class I (HLA-I) allele-associated immune responses are major forces driving the evolution of HIV-1 proteins such as Gag and Nef. The viral protein U (Vpu) is an HIV-1 accessory protein responsible for CD4 degradation and enhancement of virion release by antagonizing tetherin/CD317. Although Vpu represents one of the most variable proteins in the HIV-1 proteome, it is still not clear to what extent HLA-I influence its evolution. To examine this issue, we enrolled 240 HLA-I-typed, treatment naïve, chronically HIV-infected subjects in Japan, and analyzed plasma HIV RNA nucleotide sequences of the vpu region. Using a phylogenetically-informed method incorporating corrections for HIV codon covariation and linkage disequilibrium among HLA alleles, we investigated HLA-associated amino acid mutations in the Vpu protein as well as in the translational products encoded by alternative reading frames. Despite substantial amino acid variability in Vpu, we identified only 4 HLA-associations in all possible translational products encoded in this region, suggesting that HLA-associated immune responses had minor effects on Vpu variability in this cohort. Rather, despite its size (81 amino acids), Vpu showed 103 codon-codon covariation associations, suggesting that Vpu conformation and function are preserved through many possible combinations of primary and secondary polymorphisms. Taken together, our study suggests that Vpu has been comparably less influenced by HLA-I-associated immune-driven evolution at the population level compared to other highly variable HIV-1 accessory proteins.

Effects of naturally-arising HIV Nef mutations on cytotoxic T lymphocyte recognition and Nef's functionality in primary macrophages.

BACKGROUND: Although HIV can infect several cellular subsets, such as CD4⁺ T lymphocytes and macrophages, it remains unclear whether an HIV infection in macrophages supports cytotoxic T lymphocyte (CTL) escape. Here, we tested two naturally-arising mutations located in the well-conserved polyproline region of Nef for their effects on CTL recognition, Nef's functionality, and viral replication capacity in macrophages. These mutations were selected because they are known to cause CTL escape in the context of T lymphocytes. FINDINGS: Monocyte-derived macrophages (MDMs) infected with the wild-type virus, but not with variant viruses, were efficiently killed by CTL clones targeting Nef epitopes, VY8 (VPLRPMTY) and RY11 (RPQVPLRPMTY). The CTL-escape mutation, Arg75Thr, or Arg75Thr/Tyr85Phe double mutation, reduced the HLA class I down-regulation activity and, interestingly, increased the susceptibility of virus-infected MDMs to recognition by CTLs targeting a different epitope. The same mutations reduced the CCR5, but not CD4, down-regulation activity. Moreover, the Nef variants were impaired for Hck activation and enhancement of viral replication in MDMs. CONCLUSIONS: These results suggest that HIV-infected MDMs are killed by CTLs targeting Nef epitopes, contributing to selection and adaptation of CTL-escape viral variants.

Naturally arising HIV-1 Nef variants conferring escape from cytotoxic T lymphocytes influence viral entry co-receptor expression and susceptibility to superinfection.

HIV-1 Nef is a key factor for pathogenesis and is known to down-regulate functionally important molecules, including viral entry co-receptor CCR5 and CXCR4, from the surface of HIV-infected cells. Some of these Nef activities are mediated by the well-conserved proline-rich region of Nef, and this region is highly targeted by cytotoxic T lymphocytes (CTLs). In the present study, we asked whether Nef variants selected under CTL-mediated selective pressure in vivo may constrain these important Nef activities. The analysis of autologous nef sequences isolated from a cohort of total 235 subjects in Japan revealed that the subjects showing amino acid variations, such as Arg75Thr and Tyr85Phe, located within the proline-rich region were significantly over-represented by those having HLA-B*3501. CTL assays corroborated that these mutations conferred escape from HLA-B(∗)3501-restricted CTLs. The Arg75Thr variant Nef selectively impaired CCR5, but not CXCR4, down-regulation activity from the cell surface; whereas the Tyr85Phe variant Nef affected neither CCR5 nor CXCR4 down-regulation activity. Moreover, the cells expressing the Arg75Thr variant Nef significantly impaired protection from superinfection by CCR5-tropic, but not CXCR4-tropic, viruses. These results highlighted the importance of certain Nef-specific CTLs in modulation of viral co-receptor down-regulation activity and protection from HIV-1 superinfection, providing us with additional insight into vaccine design.