Blocking Microglial Proliferation by CSF-1R Inhibitor Does Not Alter the Neuroprotective Effects of Adoptive Regulatory T Cells in 3xTg Alzheimer's Disease Mice.

Alzheimer's disease (AD) is a chronic neurodegenerative disease that causes cognitive impairment. Neuroinflammation induced by activated microglia exacerbates AD. Regulatory T cells (Tregs) play roles in limiting neuroinflammation by converting microglial polarization. Therefore, adoptive Treg therapy is considered an attractive option for neurodegenerative disorders. However, the mechanism underlying Treg therapy via microglial modulation is not fully understood. In this study, we sought to determine whether adoptively transferred Tregs were effective when microglia proliferation was inhibited by using GW2580, which is an inhibitor of CSF1R. We found that inhibition of microglial proliferation during Treg transfer did not alter the therapeutic effects of Tregs on cognitive deficits and the accumulation of Aß and pTAU in 3xTg-AD mice. The expression of pro- and anti-inflammatory markers in the hippocampus of 3xTg mice showed that GW2580 did not affect the inhibition of neuroinflammation by Treg transfer. Additionally, adoptively transferred Tregs were commonly detected in the brain on day 7 after transfer and their levels decreased slowly over 100 days. Our findings suggest that adoptively transferred Tregs can survive longer than 100 days in the brain, suppressing microglial activation and thus alleviating AD pathology. The present study provides valuable evidence to support the prolonged efficacy of adoptive Treg therapy in AD.

Therapeutic Effects of Aß-Specific Regulatory T Cells in Alzheimer's Disease: A Study in 5xFAD Mice.

The aging global population is placing an increasing burden on healthcare systems, and the social impact of Alzheimer's disease (AD) is on the rise. However, the availability of safe and effective treatments for AD remains limited. Adoptive Treg therapy has been explored for treating neurodegenerative diseases, including AD. To facilitate the clinical application of Treg therapy, we developed a Treg preparation protocol and highlighted the therapeutic effects of Tregs in 5xFAD mice. CD4+CD25+ Tregs, isolated after Aß stimulation and expanded using a G-rex plate with a gas-permeable membrane, were adoptively transferred into 5xFAD mice. Behavioral analysis was conducted using Y-maze and passive avoidance tests. Additionally, we measured levels of Aß, phosphorylated tau (pTAU), and nitric oxide synthase 2 (NOS2) in the hippocampus. Real-time RT-PCR was employed to assess the mRNA levels of pro- and anti-inflammatory markers. Our findings indicate that Aß-specific Tregs not only improved cognitive function but also reduced Aß and pTAU accumulation in the hippocampus of 5xFAD mice. They also inhibited microglial neuroinflammation. These effects were observed at doses as low as 1.5 × 103 cells/head. Collectively, our results demonstrate that Aß-specific Tregs can mitigate AD pathology in 5xFAD mice.

Alpha-Synuclein-Specific Regulatory T Cells Ameliorate Parkinson's Disease Progression in Mice.

Parkinson's disease (PD) is a long-term neurodegenerative disease characterized by dopaminergic neuronal loss and the aggregation of alpha-synuclein (α-syn) in the brain. Cell therapy using regulatory T cells (Tregs) has therapeutic potential on PD progression in a mouse model; however, several challenges were associated with its applications. Here, we propose a strategy for α-syn specific Treg expansion (α-syn Treg). We presented α-syn to T cells via dendritic cells. This method increased the mobility of Tregs towards the site of abundant α-syn in vitro (p < 0.01; α-syn Tregs versus polyclonal Tregs (poly Tregs)) and in vivo. Consequently, α-syn Tregs showed noteworthy neuroprotective effects against motor function deficits (p < 0.05, p < 0.01; α-syn Tregs versus poly Tregs), dopaminergic neuronal loss (p < 0.001; α-syn Tregs versus poly Tregs), and α-syn accumulation (p < 0.05; α-syn Tregs versus poly Tregs) in MPTP-induced PD mice. Furthermore, the adoptive transfer of α-syn Tregs exerted immunosuppressive effects on activated microglia, especially pro-inflammatory microglia, in PD mice. Our findings suggest that α-syn presentation may provide a significant improvement in neuroprotective activities of Tregs and suggest the effective clinical application of Treg therapy in PD.

Drug conjugates for targeting regulatory T cells in the tumor microenvironment: guided missiles for cancer treatment.

Within the tumor microenvironment (TME), regulatory T cells (Tregs) play a key role in suppressing anticancer immune responses; therefore, various strategies targeting Tregs are becoming important for tumor therapy. To prevent the side effects of nonspecific Treg depletion, such as immunotherapy-related adverse events (irAEs), therapeutic strategies that specifically target Tregs in the TME are being investigated. Tumor-targeting drug conjugates are efficient drugs in which a cytotoxic payload is assembled into a carrier that binds Tregs via a linker. By allowing the drug to act selectively on target cells, this approach has the advantage of increasing the therapeutic effect and minimizing the side effects of immunotherapy. Antibody-drug conjugates, immunotoxins, peptide-drug conjugates, and small interfering RNA conjugates are being developed as Treg-targeting drug conjugates. In this review, we discuss key themes and recent advances in drug conjugates targeting Tregs in the TME, as well as future design strategies for successful use of drug conjugates for Treg targeting in immunotherapy.

An Automated Cell Detection Method for TH-positive Dopaminergic Neurons in a Mouse Model of Parkinson's Disease Using Convolutional Neural Networks.

Quantification of tyrosine hydroxylase (TH)-positive neurons is essential for the preclinical study of Parkinson's disease (PD). However, manual analysis of immunohistochemical (IHC) images is labor-intensive and has less reproducibility due to the lack of objectivity. Therefore, several automated methods of IHC image analysis have been proposed, although they have limitations of low accuracy and difficulties in practical use. Here, we developed a convolutional neural network-based machine learning algorithm for TH+ cell counting. The developed analytical tool showed higher accuracy than the conventional methods and could be used under diverse experimental conditions of image staining intensity, brightness, and contrast. Our automated cell detection algorithm is available for free and has an intelligible graphical user interface for cell counting to assist practical applications. Overall, we expect that the proposed TH+ cell counting tool will promote preclinical PD research by saving time and enabling objective analysis of IHC images.

Pharmacokinetics and Tissue Distribution of Bee Venom-Derived Phospholipase A2 Using a Sandwich ELISA after Subcutaneous Injection of New Composition Bee Venom in Rats.

Bee venom is a traditional drug used to treat the nervous system, musculoskeletal system, and autoimmune diseases. A previous study found that bee venom and one of its components, phospholipase A2, can protect the brain by suppressing neuroinflammation and can also be used to treat Alzheimer's disease. Thus, new composition bee venom (NCBV), which has an increased phospholipase A2 content of up to 76.2%, was developed as a treatment agent for Alzheimer's disease by INISTst (Republic of Korea). The aim of this study was to characterize the pharmacokinetic profiles of phospholipase A2 contained in NCBV in rats. Single subcutaneous administration of NCBV at doses ranging from 0.2 mg/kg to 5 mg/kg was conducted, and pharmacokinetic parameters of bee venom-derived phospholipase A2 (bvPLA2) increased in a dose-dependent manner. Additionally, no accumulation was observed following multiple dosings (0.5 mg/kg/week), and other constituents of NCBV did not affect the pharmacokinetic profile of bvPLA2. After subcutaneous injection of NCBV, the tissue-to-plasma ratios of bvPLA2 for the tested nine tissues were all <1.0, indicating a limited distribution of the bvPLA2 within the tissues. The findings of this study may help understand the pharmacokinetic characteristics of bvPLA2 and provide useful information for the clinical application of NCBV.

Melittin derived peptide-drug conjugate, M-DM1, inhibits tumor progression and induces effector cell infiltration in melanoma by targeting M2 tumor-associated macrophages.

Background: Melanoma has the highest mortality rate among all the types of skin cancer. In melanoma, M2-like tumor-associated macrophages (TAMs) are associated with the invasiveness of tumor cells and a poor prognosis. Hence, the depletion or reduction of M2-TAMs is a therapeutic strategy for the inhibition of tumor progression. The aim of this study was to evaluate the therapeutic effects of M-DM1, which is a conjugation of melittin (M), as a carrier for M2-like TAMs, and mertansine (DM1), as a payload to induce apoptosis of TAMs, in a mouse model of melanoma. Methods: Melittin and DM1 were conjugated and examined for the characterization of M-DM1 by high-performance liquid chromatography and electrospray ionization mass spectrometry. Synthesized M-DM1 were examined for in vitro cytotoxic effects. For the in vivo study, we engrafted murine B16-F10 into right flank of C57BL/6 female mice and administered an array of treatments (PBS, M, DM1, or M-DM1 (20 nmol/kg)). Subsequently, the tumor growth and survival rates were analyzed, as well as examining the phenotypes of tumor-infiltrating leukocytes and expression profiles. Results: M-DM1 was found to specifically reduce M2-like TAMs in melanoma, which potentially leads to the suppression of tumor growth, migration, and invasion. In addition, we also found that M-DM1 improved the survival rates in a mouse model of melanoma compared to M or DM1 treatment alone. Flow cytometric analysis revealed that M-DM1 enhanced the infiltration of CD8+ cytotoxic T cells and natural killer cells (NK cells) in the tumor microenvironment. Conclusion: Taken together, our findings highlight that M-DM1 is a prospective agent with enhanced anti-tumor effects.

Enhanced Therapeutic Effect of Optimized Melittin-dKLA, a Peptide Agent Targeting M2-like Tumor-Associated Macrophages in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by a high possibility of metastasis. M2-like tumor-associated macrophages (TAMs) are the main components of the tumor microenvironment (TME) and play a key role in TNBC metastasis. Therefore, TAMs may be a potential target for reducing TNBC metastasis. Melittin-dKLA, a peptide composed of fused melittin and pro-apoptotic peptide d(KLAKLAK)2 (dKLA), showed a potent therapeutic effect against cancers by depleting TAMs. However, melittin has a strong adverse hemolytic effect. Hence, we attempted to improve the therapeutic potential of melittin-dKLA by reducing toxicity and increasing stability. Nine truncated melittin fragments were synthesized and examined. Of the nine peptides, the melittin-dKLA8-26 showed the best binding properties to M2 macrophages and discriminated M0/M1/M2. All fragments, except melittin, lost their hemolytic effects. To increase the stability of the peptide, melittin-dKLA8-26 fragment was conjugated with PEGylation at the amino terminus and was named PEG-melittin-dKLA8-26. This final drug candidate was assessed in vivo in a murine TNBC model and showed superior effects on tumor growth, survival rates, and lung metastasis compared with the previously used melittin-dKLA. Taken together, our study showed that the novel PEG-melittin-dKLA8-26 possesses potential as a new drug for treating TNBC and TNBC-mediated metastasis by targeting TAMs.

Adoptive therapy with amyloid-ß specific regulatory T cells alleviates Alzheimer's disease.

Rationale: Neuroinflammation is a primary feature of Alzheimer's disease (AD), for which an increasing number of drugs have been specifically developed. The present study aimed to define the therapeutic impact of a specific subpopulation of T cells that can suppress excessive inflammation in various immune and inflammatory disorders, namely, CD4+CD25+Foxp3+ regulatory T cells (Tregs). Methods: To generate Aß antigen-specific Tregs (Aß+ Tregs), Aß 1-42 peptide was applied in vivo and subsequent in vitro splenocyte culture. After isolating Tregs by magnetic bead based purification method, Aß+ Tregs were adoptively transferred into 3xTg-AD mice via tail vein injection. Therapeutic efficacy was confirmed with behavior test, Western blot, quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry staining (IHC). In vitro suppression assay was performed to evaluate the suppressive activity of Aß+ Tregs using flow cytometry. Thy1.1+ Treg trafficking and distribution was analyzed to explore the infused Tregs migration into specific organs in an antigen-driven manner in AD mice. We further assessed cerebral glucose metabolism using 18F-FDG-PET, an imaging approach for AD biological definition. Subsequently, we evaluated the migration of Aß+ Tregs toward Aß activated microglia using live cell imaging, chemotaxis, antibody blocking and migration assay. Results: We showed that Aß-stimulated Tregs inhibited microglial proinflammatory activity and modulated the microglial phenotype via bystander suppression. Single adoptive transfer of Aß+ Tregs was enough to induce amelioration of cognitive impairments, Aß accumulation, hyper-phosphorylation of tau, and neuroinflammation during AD pathology. Moreover, Aß-specific Tregs effectively inhibited inflammation in primary microglia induced by Aß exposure. It may indicate bystander suppression in which Aß-specific Tregs promote immune tolerance by secreting cytokines to modulate immune responses during neurodegeneration. Conclusions: The administration of Aß antigen-specific regulatory T cells may represent a new cellular therapeutic strategy for AD that acts by modulating the inflammatory status in AD.

A Randomized Double-Blind, Placebo-Controlled Study on Anti-Stress Effects of Nelumbinis Semen.

Introduction: Depression is a serious and common mental disease that causes low mood and loss of interest in activities. Nelumbinis semen (NS) has been widely used as a treatment for depression for hundreds of years in many Asian countries. Water extract of nelumbinis semen (WNS) is a standardized herbal medicine made from NS. Methods: The objective of the present research was to perform a randomized, double-blind, placebo-controlled trial to estimate the efficacy of WNS for improving depressive and stress symptoms using Beck depression inventory (BDI) and the stress response inventory (SRI) in 45 adults diagnosed with major depression or other forms of depressive disorders. They were randomized to either a placebo-treated group, a 2.4 g per day WNS-treated group, or a 4.8 g per day WNS-treated group. BDI and SRI were determined in order to evaluate changes in depression before and after two weeks of WNS treatment. Results: The average BDI and SRI of the 2.4 g WNS-treated group were significantly (p < 0.05) improved compared to those of the placebo-treated group. Their BDI subscale A (negative attitudes towards self) and subscale C (somatic disturbances), SRI E, and depression subscale of SRI were substantially shorter (p < 0.05). In addition, an analysis of collected EEG data of participants showed a significant increase in alpha/beta activity in the 4.8 g WNS-treated group, which might be explained as an advancement of their depression symptoms (p < 0.05). Conclusions: These results suggest that WNS treatment can decrease depression. Our study provides preliminary evidence for the safety of WNS and its potential to decrease depression.

[6]-Shogaol Attenuates Oxaliplatin-Induced Allodynia through Serotonergic Receptors and GABA in the Spinal Cord in Mice.

Although oxaliplatin is a well-known anti-cancer agent used for the treatment of colorectal cancer, treated patients often experience acute cold and mechanical allodynia as side effects. Unfortunately, no optimal treatment has been developed yet. In this study, [6]-shogaol (10 mg/kg, i.p.), which is one of the major bioactive components of Zingiber officinale roscoe (Z. officinale), significantly alleviated allodynia induced by oxaliplatin (6 mg/kg, i.p.) injection. Cold and mechanical allodynia were assessed by acetone drop and von Frey filament tests, respectively. The analgesic effect of [6]-shogaol was blocked by the intrathecal injection of 5-HT1A, 5-HT3, and GABAB receptor antagonists, NAN-190 (1 µg), MDL-72222 (15 µg), and CGP 55845 (10 µg), respectively. Furthermore, oxaliplatin injection lowered the GABA concentration in the superficial laminae of the spinal dorsal horn, whereas [6]-shogaol injection significantly elevated it. The GAD (glutamic acid decarboxylase) 65 concentration also increased after [6]-shogaol administration. However, pre-treatment of NAN-190 completely inhibited the increased GABA induced by [6]-shogaol in the spinal dorsal horn, whereas MDL-72222 partially blocked the effect. Altogether, these results suggest that [6]-shogaol could attenuate oxaliplatin-induced cold and mechanical allodynia through 5-HT1A and 5-HT3 receptor antagonists located in the GABAergic neurons in the spinal dorsal horn in mice.

Neuroprotective effects of ex vivo-expanded regulatory T cells on trimethyltin-induced neurodegeneration in mice.

BACKGROUND: Trimethyltin (TMT) is a potent neurotoxicant that leads to hippocampal neurodegeneration. Regulatory T cells (Tregs) play an important role in maintaining the immune balance in the central nervous system (CNS), but their activities are impaired in neurodegenerative diseases. In this study, we aimed to determine whether adoptive transfer of Tregs, as a living drug, ameliorates hippocampal neurodegeneration in TMT-intoxicated mice. METHODS: CD4+CD25+ Tregs were expanded in vitro and adoptively transferred to TMT-treated mice. First, we explored the effects of Tregs on behavioral deficits using the Morris water maze and elevated plus maze tests. Biomarkers related to memory formation, such as cAMP response element-binding protein (CREB), protein kinase C (PKC), neuronal nuclear protein (NeuN), nerve growth factor (NGF), and ionized calcium binding adaptor molecule 1 (Iba1) in the hippocampus were examined by immunohistochemistry after killing the mouse. To investigate the neuroinflammatory responses, the polarization status of microglia was examined in vivo and in vitro using real-time reverse transcription polymerase chain reaction (rtPCR) and Enzyme-linked immunosorbent assay (ELISA). Additionally, the inhibitory effects of Tregs on TMT-induced microglial activation were examined using time-lapse live imaging in vitro with an activation-specific fluorescence probe, CDr20. RESULTS: Adoptive transfer of Tregs improved spatial learning and memory functions and reduced anxiety in TMT-intoxicated mice. Additionally, adoptive transfer of Tregs reduced neuronal loss and recovered the expression of neurogenesis enhancing molecules in the hippocampi of TMT-intoxicated mice. In particular, Tregs inhibited microglial activation and pro-inflammatory cytokine release in the hippocampi of TMT-intoxicated mice. The inhibitory effects of TMT were also confirmed via in vitro live time-lapse imaging in a Treg/microglia co-culture system. CONCLUSIONS: These data suggest that adoptive transfer of Tregs ameliorates disease progression in TMT-induced neurodegeneration by promoting neurogenesis and modulating microglial activation and polarization.

Particulate Matter Exacerbates the Death of Dopaminergic Neurons in Parkinson's Disease through an Inflammatory Response.

Particulate matter (PM), a component of air pollution, has been epidemiologically associated with a variety of diseases. Recent reports reveal that PM has detrimental effects on the brain. In this study, we aimed to investigate the biological effects of ambient particles on the neurodegenerative disease Parkinson's disease (PD). We exposed mice to coarse particles (PM10: 2.5-10 µm) for short (5 days) and long (8 weeks) durations via intratracheal instillation. Long-term PM10 exposure exacerbated motor impairment and dopaminergic neuron death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse models. Short-term PM10 exposure resulted in both pulmonary and systemic inflammatory responses in mice. We further investigated the mechanism underlying PM10-induced neurotoxicity in cocultures of lung LA-4 epithelial cells and RAW264.7 macrophages. PM10 treatment elicited a dramatic increase in proinflammatory mediators in LA-4/RAW264.7 coculture. Treating BV2 microglial cells with PM10-treated conditioned medium induced microglial activation. Furthermore, 1-methyl-4-phenylpyridinium (MPP+) treatment caused notable cell death in N2A neurons cocultured with activated BV2 cells in PM10-conditioned medium. Altogether, our results demonstrated that PM10 plays a role in the neurodegeneration associated with PD. Thus, the impact of PM10 on neurodegeneration could be related to detrimental air pollution-induced systemic effects on the brain.

Loss of SP-A in the Lung Exacerbates Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating and common chronic lung disease that is pathologically characterized by the destruction of lung architecture and the accumulation of extracellular matrix in the lung. Previous studies have shown an association between lung surfactant protein (SP) and the pathogenesis of IPF, as demonstrated by mutations and the altered expression of SP in patients with IPF. However, the role of SP in the development of lung fibrosis is poorly understood. In this study, the role of surfactant protein A (SP-A) was explored in experimental lung fibrosis induced with a low or high dose of bleomycin (BLM) and CRISPR/Cas9-mediated genetic deletion of SP-A. Our results showed that lung SP-A deficiency in mice promoted the development of fibrotic damage and exacerbated inflammatory responses to the BLM challenge. In vitro experiments with murine lung epithelial LA-4 cells demonstrated that in response to transforming growth factor-ß1 (TGF-ß1), LA-4 cells had a decreased protein expression of SP-A. Furthermore, exogenous SP administration to LA-4 cells inhibited the TGF-ß1-induced upregulation of fibrotic markers. Overall, these findings suggest a novel antifibrotic mechanism of SP-A in the development of lung fibrosis, which indicates the therapeutic potential of the lung SP-A in preventing the development of IPF.

Therapeutic Effect of Melittin-dKLA Targeting Tumor-Associated Macrophages in Melanoma.

Melanoma is an immunogenic tumor and a serious type of skin cancer. Tumor-associated macrophages (TAMs) express an M2-like phenotype and are involved in all stages of melanomagenesis; it is hence a promising target for cancer immunotherapy. We herein investigated whether melittin-dKLA inhibits the growth of melanoma by inducing apoptosis of M2-like macrophages. For the in vitro study, a conditioned medium of macrophages was prepared from M0, M1, or M2-differentiated THP-1 cells with and without melittin-dKLA. The affinity of melittin for M2 macrophages was studied with FITC (fluorescein isothiocyanate)-conjugated melittin. For the in vivo study, murine melanoma cells were inoculated subcutaneously in the right flank of mice, melittin-dKLA was intraperitoneally injected at 200 nmol/kg every three days, and flow cytometry analysis of TAMs was performed. Since melittin binds preferentially to M2-like macrophages, melittin-dKLA induced more caspase 3 expression and cell death in M2 macrophages compared with M0 and M1 macrophages and melanoma cells. Melittin-dKLA significantly inhibited the proliferation and migration of M2 macrophages, resulting in a decrease in melanoma tumor growth in vivo. The CD206+ M2-like TAMs were reduced, while the CD86+ M1-like TAMs were not affected. Melittin-dKLA is therapeutically effective against melanoma by inducing the apoptosis of M2-like TAMs.

TAMpepK Suppresses Metastasis through the Elimination of M2-Like Tumor-Associated Macrophages in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) accounts for approximately 10-15% of all breast cancer cases and is characterized by high invasiveness, high metastatic potential, relapse proneness, and poor prognosis. M2-like tumor-associated macrophages (TAMs) contribute to tumorigenesis and are promising targets for inhibiting breast cancer metastasis. Therefore, we investigated whether melittin-conjugated pro-apoptotic peptide (TAMpepK) exerts therapeutic effects on breast cancer metastasis by targeting M2-like TAMs. TAMpepK is composed of M2-like TAM binding peptide (TAMpep) and pro-apoptotic peptide d(KLAKLAK)2 (dKLA). A metastatic mouse model was constructed by injecting 4T1-luc2 cells either orthotopically or via tail vein injection, and tumor burden was quantified using a bioluminescence in vivo imaging system. We found that TAMpepK suppressed lung and lymph node metastases of breast cancer by eliminating M2-like TAMs without affecting the viability of M1-like macrophages and resident macrophages in the orthotopic model. Furthermore, TAMpepK reduced pulmonary seeding and the colonization of tumor cells in the tail vein injection model. The number of CD8+ T cells in contact with TAMs was significantly decreased in tumor nodules treated with TAMpepK, resulting in the functional activation of cytotoxic CD8+ T cells. Taken together, our findings suggest that TAMpepK could be a novel therapeutic agent for the inhibition of breast cancer metastasis by targeting M2-like TAMs.

The involvement of the noradrenergic system in the antinociceptive effect of cucurbitacin D on mice with paclitaxel-induced neuropathic pain.

Paclitaxel (sold under the brand name Taxol) is a chemotherapeutic drug that is widely used to treat cancer. However, it can also induce peripheral neuropathy, which limits its use. Although several drugs are used to attenuate neuropathy, no optimal treatment is available to date. In this study, the effect of cucurbitacins B and D on paclitaxel-induced neuropathic pain was assessed. Multiple paclitaxel injections (a cumulative dose of 8 mg/kg, i. p.) induced cold and mechanical allodynia from days 10 to 21 in mice, and the i. p. administration of 0.025 mg/kg of cucurbitacins B and D attenuated both allodynia types. However, as cucurbitacin B showed a more toxic effect on non-cancerous (RAW 264.7) cells, further experiments were conducted with cucurbitacin D. The cucurbitacin D dose-dependently (0.025, 0.1, and 0.5 mg/kg) attenuated both allodynia types. In the spinal cord, paclitaxel injection increased the gene expression of noradrenergic (α 1-and α 2-adrenergic) receptors but not serotonergic (5-HT1A and 3) receptors. Cucurbitacin D treatment significantly decreased the spinal α 1- but not α 2-adrenergic receptors, and the amount of spinal noradrenaline was also downregulated. However, the tyrosine hydroxylase expression measured via liquid chromatography in the locus coeruleus did not decrease significantly. Finally, cucurbitacin D treatment did not lower the anticancer effect of chemotherapeutic drugs when co-administered with paclitaxel in CT-26 cell-implanted mice. Altogether, these results suggest that cucurbitacin D could be considered a treatment option against paclitaxel-induced neuropathic pain.

The Responsiveness of Bee Venom Phospholipase A2 on Regulatory T Cells Correlates with the CD11c+CD206+Population in Human Peripheral Blood Mononuclear Cells.

Bee venom phospholipase A2 (bvPLA2) has been reported to have therapeutic effects such as neuroprotection, anti-inflammation, anti-nociception, anti-cancer properties, caused by increasing regulatory T cells (Tregs). The mechanism of Tregs modulation by bvPLA2 has been demonstrated by binding with the mannose receptor, CD206 in experimental models of several diseases. However, it remains unknown whether this mechanism can also be applied in human blood. In this study, we collected peripheral blood samples from healthy donors and analyzed the percentages of monocyte-derived dendritic cells with CD206 (CD206+ DCs) before expansion, the proportion of Tregs, and the subpopulations after expansion treated with bvPLA2 or PBS using flow cytometry and the correlations among them. The percentage of Tregs tended to be higher in the bvPLA2 group than in the control group. There were significant positive correlations between the CD206 population in hPBMC and the proportions of Tregs treated with bvPLA2, especially in the Treg fold change comparing the increase ratio of Tregs in bvPLA2 and in PBS. These findings indicate that bvPLA2 increased the proportion of Tregs in healthy human peripheral blood and the number of CD206+ DCs could be a predictor of the bvPLA2 response of different individuals.

Inhibition of lysophosphatidic acid receptor 1-3 deteriorates experimental autoimmune encephalomyelitis by inducing oxidative stress.

BACKGROUND: Lysophosphatidic acid receptors (LPARs) are G-protein-coupled receptors involved in many physiological functions in the central nervous system. However, the role of the LPARs in multiple sclerosis (MS) has not been clearly defined yet. METHODS: Here, we investigated the roles of LPARs in myelin oligodendrocyte glycoprotein peptides-induced experimental autoimmune encephalomyelitis (EAE), an animal model of MS. RESULTS: Pre-inhibition with LPAR1-3 antagonist Ki16425 deteriorated motor disability of EAElow. Specifically, LPAR1-3 antagonist (intraperitoneal) deteriorated symptoms of EAElow associated with increased demyelination, chemokine expression, cellular infiltration, and immune cell activation (microglia and macrophage) in spinal cords of mice compared to the sham group. This LPAR1-3 antagonist also increased the infiltration of CD4+/IFN-γ+ (Th1) and CD4+/IL-17+ (Th17) cells into spinal cords of EAElow mice along with upregulated mRNA expression of IFN-γ and IL-17 and impaired blood-brain barrier (BBB) in the spinal cord. The underlying mechanism for negative effects of LPAR1-3 antagonist was associated with the overproduction of reactive oxygen species (ROS)-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) 2 and NOX3. Interestingly, LPAR1/2 agonist 1-oleoyl-LPA (LPA 18:1) (intraperitoneal) ameliorated symptoms of EAEhigh and improved representative pathological features of spinal cords of EAEhigh mice. CONCLUSIONS: Our findings strongly suggest that some agents that can stimulate LPARs might have potential therapeutic implications for autoimmune demyelinating diseases such as MS.