Characterization of spinal cord tissue-derived extracellular vesicles in neuroinflammation.

Extracellular vesicles (EVs) are released by all cells, can cross the blood-brain barrier, and have been shown to play an important role in cellular communication, substance shuttling, and immune modulation. In recent years EVs have shifted into focus in multiple sclerosis (MS) research as potential plasma biomarkers and therapeutic vehicles. Yet little is known about the disease-associated changes in EVs in the central nervous system (CNS). To address this gap, we characterized the physical and proteomic changes of mouse spinal cord-derived EVs before and at 16 and 25 days after the induction of experimental autoimmune encephalomyelitis (EAE), a neuroinflammatory model of MS. Using various bioinformatic tools, we found changes in inflammatory, glial, and synaptic proteins and pathways, as well as a shift in the predicted contribution of immune and glial cell types over time. These results show that EVs provide snapshots of crucial disease processes such as CNS-compartmentalized inflammation, re/de-myelination, and synaptic pathology, and might also mediate these processes. Additionally, inflammatory plasma EV biomarkers previously identified in people with MS were also altered in EAE spinal cord EVs, suggesting commonalities of EV-related pathological processes during EAE and MS and overlap of EV proteomic changes between CNS and circulating EVs.

BAFF blockade in experimental autoimmune encephalomyelitis reduces inflammation in the meninges and synaptic and neuronal loss in adjacent brain regions.

Multiple sclerosis (MS) has traditionally been viewed as a chronic inflammatory disease affecting the white matter of the central nervous system. However, over the past two decades, increasing evidence has highlighted the role of gray matter pathology in MS-related disability. Numerous studies have linked the presence of leptomeningeal inflammation to a more severe disease course, underscoring its potential importance as a driver of gray matter pathology in MS. The major components of leptomeningeal inflammation include T cells, B cells, macrophages, follicular dendritic cells, and plasma cells. Since BAFF [B cell-activating factor of the tumor necrosis factor (TNF) family] promotes B cell survival and maturation and is a co-stimulator of T cells, we used anti-BAFF antibody 10F4 as a BAFF antagonist to study its effect on meningeal inflammation and adjacent brain regions in a relapsing-remitting PLP-EAE (rr-EAE) model of multiple sclerosis in SJL/J mice. rr-EAE mice were treated either with anti-BAFF antibody 10F4 or with IgG control antibody. We performed ultra-high field (11.7 T) MRI to identify areas of meningeal inflammation and track them over time in both treatment groups. We also performed histopathological analysis in brain sections of these mice to study the effects of the BAFF antagonist on leptomeningeal inflammation, and hippocampal and cortical neurons and synapses. We observed that BAFF antagonist treatment reduced B cells, T cells, and myeloid cells in regions of meningeal inflammation. Additionally, we noted that BAFF treatment protected against EAE-induced synaptic and neuronal loss in the adjacent cortex and in the CA1, CA3, and dentate gyrus regions of the hippocampus likely due to its effects on meningeal inflammation.

Early Changes in Exo- and Endocytosis in the EAE Mouse Model of Multiple Sclerosis Correlate with Decreased Synaptic Ribbon Size and Reduced Ribbon-Associated Vesicle Pools in Rod Photoreceptor Synapses.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system that finally leads to demyelination. Demyelinating optic neuritis is a frequent symptom in MS. Recent studies also revealed synapse dysfunctions in MS patients and MS mouse models. We previously reported alterations of photoreceptor ribbon synapses in the experimental auto-immune encephalomyelitis (EAE) mouse model of MS. In the present study, we found that the previously observed decreased imunosignals of photoreceptor ribbons in early EAE resulted from a decrease in synaptic ribbon size, whereas the number/density of ribbons in photoreceptor synapses remained unchanged. Smaller photoreceptor ribbons are associated with fewer docked and ribbon-associated vesicles. At a functional level, depolarization-evoked exocytosis as monitored by optical recording was diminished even as early as on day 7 after EAE induction. Moreover compensatory, post-depolarization endocytosis was decreased. Decreased post-depolarization endocytosis in early EAE correlated with diminished synaptic enrichment of dynamin3. In contrast, basal endocytosis in photoreceptor synapses of resting non-depolarized retinal slices was increased in early EAE. Increased basal endocytosis correlated with increased de-phosphorylation of dynamin1. Thus, multiple endocytic pathways in photoreceptor synapse are differentially affected in early EAE and likely contribute to the observed synapse pathology in early EAE.

Anti-HSV-2 activity of Terminalia chebula Retz extract and its constituents, chebulagic and chebulinic acids.

BACKGROUND: Development of new and effective therapeutics for sexually transmitted herpes simplex virus-2 (HSV-2) infection is important from public health perspective. With an aim to identify natural products from medicinal plants, in the present study, the potential of Terminalia chebula Retz was investigated for its activity against HSV-2. METHODS: Fruits of Terminalia chebula Retz were used to prepare 50% ethanolic extract. In addition, chebulagic acid and chebulinic acid both purified from T. chebula were also used. The extract as well as purified compounds were first used to determine their in vitro cytotoxicity on Vero cells by MTT assay. T. chebula extract, chebulagic acid, chebulinic acid along with acyclovir were subsequently assessed for direct anti-viral activity, and their ability to inhibit attachment and penetration of HSV-2 to the Vero cells. In addition, their anti-HSV-2 activity was also determined by in vitro post-infection plaque reduction assay. RESULTS: Cytotoxicity assay using Vero cells revealed CC50 = 409.71 ± 47.70 µg/ml for the extract whereas chebulagic acid and chebulinic acid showed more than 95% cell viability up to 200 µg/ml. The extract from T. chebula (IC50 = 0.01 ± 0.0002 µg/ml), chebulagic (IC50 = 1.41 ± 0.51 µg/ml) and chebulinic acids (IC50 = 0.06 ± 0.002 µg/ml) showed dose dependent potent in vitro direct anti-viral activity against HSV-2. These also effectively prevented the attachment as well as penetration of the HSV-2 to Vero cells. In comparison, acyclovir showed poor direct anti-viral activity and failed to significantly (p > 0.05) prevent the attachment as well as penetration of HSV-2 to Vero cells when tested upto 50 µg/ml. However, in post-infection plaque reduction assay, T. chebula extract, chebulagic and chebulinic acids showed IC50 values of 50.06 ± 6.12, 31.84 ± 2.64, and 8.69 ± 2.09 µg/ml, respectively, which were much lower than acyclovir (71.80 ± 19.95 ng/ml). CONCLUSIONS: The results presented herein suggest that T. chebula extract, chebulagic and chebulinic acids have higher direct antiviral activity against HSV-2 and efficacy to inhibit virus attachment and penetration to the host cells as compared to acyclovir. However, acyclovir is more potent to inhibit post-infection virus replication. Hence, T. chebula may be a useful candidate for developing alternative therapy for prevention of sexually transmitted HSV-2 infection. ᅟ.

Multimodal techniques and strategies for chemical and metabolic imaging at the single-cell level.

Single-cell chemical and metabolic imaging technologies provide unprecedented insights into individual cell dynamics, advancing our understanding of cellular processes, molecular interactions, and metabolic activities. Advances in fluorescence, Raman, optoacoustic (photoacoustic), or mass spectrometry methods have paved the way to characterize metabolites, signaling molecules, and other moieties within individual cells. These modalities can also lead to single-cell imaging capabilities by targeting endogenous cell contrast or by employing exogenous contrast generation techniques, including contrast agents that target specific cell structure or function. In this review, we present key developments, summarize recent applications in single-cell interrogation and imaging, and illustrate their advantages, limitations, and outlook.

Herbal Gel Formulation Developed for Anti-Human Immunodeficiency Virus (HIV)-1 Activity Also Inhibits In Vitro HSV-2 Infection.

Herpes simplex virus-2 (HSV-2) infection is the most common cause of genital ulcers. The impact of ulcers also demonstrates a strong link to the human immunodeficiency virus (HIV) infection. Complications, drug resistance, and side-effects of anti-viral drugs make the treatment of HSV-2 infection challenging. Herbal medicines have shown potential against HSV-2 and HIV infections. In this context, polyherbal gel formulation comprising 50% ethanolic extracts from Acacia catechu, Lagerstroemia speciosa, Terminalia chebula and Phyllanthus emblica has been developed. The gel formulation significantly exhibited virucidal activity against both HIV-1 and HSV-2 infections with IC50, 55.93 ± 5.30 µg/mL and 27.26 ± 4.87 µg/mL, respectively. It also inhibited HSV-2 attachment and penetration to the Vero cells with an IC50 = 46.55 ± 1.25 µg/mL and 54.94 ± 2.52 µg/mL respectively, which were significantly lower than acyclovir. However, acyclovir is more potent in post-infection assay with an IC50 = 0.065 ± 0.01 µg/mL whereas gel formulation showed an IC50 = 469.05 ± 16.65 µg/mL under similar conditions. Gel formulation showed no inhibitory effect on the viability of lactobacilli, human vaginal keratinocyte cells (Vk2/E6E7), and the integrity of the Caco-2 cells monolayer. Gel formulation did not lead to any significant increase in the secretion of pro-inflammatory cytokines and mutagenic index. The proposed gel formulation may be a promising candidate microbicide for the prevention of sexually transmitted HIV-1 and HSV-2.

Early auto-immune targeting of photoreceptor ribbon synapses in mouse models of multiple sclerosis.

Optic neuritis is one of the first manifestations of multiple sclerosis. Its pathogenesis is incompletely understood, but considered to be initiated by an auto-immune response directed against myelin sheaths of the optic nerve. Here, we demonstrate in two frequently used and well-validated mouse models of optic neuritis that ribbon synapses in the myelin-free retina are targeted by an auto-reactive immune system even before alterations in the optic nerve have developed. The auto-immune response is directed against two adhesion proteins (CASPR1/CNTN1) that are present both in the paranodal region of myelinated nerves as well as at retinal ribbon synapses. This occurs in parallel with altered synaptic vesicle cycling in retinal ribbon synapses and altered visual behavior before the onset of optic nerve demyelination. These findings indicate that early synaptic dysfunctions in the retina contribute to the pathology of optic neuritis in multiple sclerosis.

N-hydroxy-substituted 2-aryl acetamide analogs: A novel class of HIV-1 integrase inhibitors.

An in silico method has been used to discover N-hydroxy-substituted 2-aryl acetamide analogs as a new class of HIV-1 integrase inhibitors. Based on the molecular requirements of the binding pocket of catalytic active site, two molecules (compounds 2 and 4b) were designed as fragments. These were further synthesized and biologically evaluated. In vitro potency along with docking studies highlighted compound 4b as an active fragment which was further used to synthesize new leads as HIV-1 integrase inhibitors. Finally, six promising compounds (compounds 5b, 5c, 5e, 6-2c, 6-3b, and 6-5b) were identified by integrase inhibition assay (>50% inhibition). Based on in vitro anti-HIV-1 activity in a reporter gene-based cell assay system, compounds 5d, 6s, and 6k were found as novel HIV-1 integrase inhibitors due to its better selectivity index. Additionally, docking study revealed the importance of H-bond as well as hydrophobic interactions with Asn155, Lys156, and Lys159 which were required for their anti-HIV-1 activity.