Anti-PD-1 cis-delivery of low-affinity IL-12 activates intratumoral CD8+T cells for systemic antitumor responses.

Immune checkpoint blockade (ICB) therapies function by alleviating immunosuppression on tumor-infiltrating lymphocytes (TILs) but are often insufficient to fully reactivate these dysfunctional TILs. Although interleukin 12 (IL-12) has been used in combination with ICB to improve efficacy, this remains limited by severe toxicity associated with systemic administration of this cytokine. Here, we engineer a fusion protein composed of an anti-PD-1 antibody and a mouse low-affinity IL-12 mutant-2 (αPD1-mIL12mut2). Systemic administration of αPD1-mIL12mut2 displays robust antitumor activities with undetectable toxicity. Mechanistically, αPD1-mIL12mut2 preferentially activates tumor-infiltrating PD-1+CD8+T cells via high-affinity αPD-1 mediated cis-binding of low-affinity IL-12. Additionally, αPD1-mIL12mut2 treatment exerts an abscopal effect to suppress distal tumors, as well as metastasis. Collectively, αPD1-mIL12mut2 treatment induces robust systemic antitumor responses with reduced side effects.

Hepatic portal venous gas initially manifesting as severe shock: a case series.

Hepatic portal venous gas is often referred to as the "sign of death" because it signifies a very poor prognosis if appropriate treatments are not promptly administered. The etiologies of hepatic portal venous gas are diverse and include severe complex abdominal infections, mesenteric ischemia, diving, and complications of endoscopic surgery, and the clinical manifestations are inconsistent among individual patients. Thus, whether emergency surgery should be performed remains controversial. In this report, we present three cases of hepatic portal venous gas. The patients initially exhibited symptoms consistent with severe shock of unknown etiology and were treated in the intensive care unit upon admission. We rapidly identified the cause of each individual patient's condition and selected problem-directed intervention measures based on active organ support, antishock support, and anti-infection treatments. Two patients recovered and were discharged without sequelae, whereas one patient died of refractory infection and multiple organ failure. We hope that this report will serve as a valuable reference for decision-making when critical care physicians encounter similar patients.

IL-2 delivery by engineered mesenchymal stem cells re-invigorates CD8+ T cells to overcome immunotherapy resistance in cancer.

Immune checkpoint blockade (ICB)-based immunotherapy depends on functional tumour-infiltrating lymphocytes (TILs), but essential cytokines are less understood. Here we uncover an essential role of endogenous IL-2 for ICB responsiveness and the correlation between insufficient IL-2 signalling and T-cell exhaustion as tumours progress. To determine if exogenous IL-2 in the tumour microenvironment can overcome ICB resistance, we engineered mesenchymal stem cells (MSCs) to successfully deliver IL-2 mutein dimer (SIL2-EMSC) to TILs. While MSCs have been used to suppress inflammation, SIL2-EMSCs elicit anti-tumour immunity and overcome ICB resistance without toxicity. Mechanistically, SIL2-EMSCs activate and expand pre-existing CD8+ TILs, sufficient for tumour control and induction of systemic anti-tumour effects. Furthermore, engineered MSCs create synergy of innate and adaptive immunity. The therapeutic benefits of SIL2-EMSCs were also observed in humanized mouse models. Overall, engineered MSCs rejuvenate CD8+ TILs and thus potentiate ICB and chemotherapy.

An engineered concealed IL-15-R elicits tumor-specific CD8+T cell responses through PD-1-cis delivery.

Checkpoint blockade immunotherapy releases the inhibition of tumor-infiltrating lymphocytes (TILs) but weakly induces TIL proliferation. Exogenous IL-15 could further expand TILs and thus synergize with αPD-L1 therapy. However, systemic delivery of IL-15 extensively expands peripheral NK cells, causing severe toxicity. To redirect IL-15 to intratumoral PD-1+CD8+T effector cells instead of NK cells for better tumor control and lower toxicity, we engineered an anti-PD-1 fusion with IL-15-IL-15Rα, whose activity was geographically concealed by immunoglobulin Fc region with an engineered linker (αPD-1-IL-15-R) to bypass systemic NK cells. Systematic administration of αPD-1-IL-15-R elicited extraordinary antitumor efficacy with undetectable toxicity. Mechanistically, cis-delivery of αPD-1-IL-15-R vastly expands tumor-specific CD8+T cells for tumor rejection. Additionally, αPD-1-IL-15-R upregulated PD-1 and IL-15Rß on T cells to create a feedforward activation loop, thus rejuvenating TILs, not only resulting in tumor control in situ, but also suppressing tumor metastasis. Collectively, renavigating IL-15 to tumor-specific PD-1+CD8+T cells, αPD-1-IL-15-R elicits effective systemic antitumor immunity.

Tumor-conditional IL-15 pro-cytokine reactivates anti-tumor immunity with limited toxicity.

IL-15 is a promising cytokine to expand NK and CD8+ T cells for cancer immunotherapy, but its application is limited by dose-limiting, on-target off-tumor toxicity. Here, we have developed a next-generation IL-15 that is activated inside the tumor microenvironment (TME). This pro-IL-15 has the extracellular domain of IL-15Rß fused to the N-terminus of sIL-15-Fc through a tumor-enriched Matrix Metalloproteinase (MMP) cleavable peptide linker to block its activity. Unlike sIL-15-Fc, pro-IL-15 does not activate the peripheral expansion of NK cells and T cells, thus reducing systemic toxicity, but it still preserves efficient anti-tumor abilities. In various mouse tumors, the anti-tumor effect of pro-IL-15 depends on intratumoral CD8+ T cells and IFN-γ. Pro-IL-15 increases the stem-like TCF1+Tim-3-CD8+ T cells within tumor tissue and helps overcome immune checkpoint blockade (ICB) resistance. Moreover, pro-IL-15 synergizes with current tyrosine kinase inhibitor (TKI) targeted-therapy in a poorly inflamed TUBO tumor model, suggesting that pro-IL-15 helps overcome targeted-therapy resistance. Our results demonstrate a next-generation IL-15 cytokine that can stimulate potent anti-tumor activity without severe toxicity.

Trichomonas vaginalis infection impairs anion secretion in vaginal epithelium.

Trichomonas vaginalis is a common protozoan parasite, which causes trichomoniasis associated with severe adverse reproductive outcomes. However, the underlying pathogenesis has not been fully understood. As the first line of defense against invading pathogens, the vaginal epithelial cells are highly responsive to environmental stimuli and contribute to the formation of the optimal luminal fluid microenvironment. The cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel widely distributed at the apical membrane of epithelial cells, plays a crucial role in mediating the secretion of Cl- and HCO3-. In this study, we investigated the effect of T. vaginalis on vaginal epithelial ion transport elicited by prostaglandin E2 (PGE2), a major prostaglandin in the semen. Luminal administration of PGE2 triggered a remarkable and sustained increase of short-circuit current (ISC) in rat vaginal epithelium, which was mainly due to Cl- and HCO3- secretion mediated by the cAMP-activated CFTR. However, T. vaginalis infection significantly abrogated the ISC response evoked by PGE2, indicating impaired transepithelial anion transport via CFTR. Using a primary cell culture system of rat vaginal epithelium and a human vaginal epithelial cell line, we demonstrated that the expression of CFTR was significantly down-regulated after T. vaginalis infection. In addition, defective Cl- transport function of CFTR was observed in T. vaginalis-infected cells by measuring intracellular Cl- signals. Conclusively, T. vaginalis restrained exogenous PGE2-induced anion secretion through down-regulation of CFTR in vaginal epithelium. These results provide novel insights into the intervention of reproductive complications associated with T. vaginalis infection such as infertility and disequilibrium in vaginal fluid microenvironment.

Increased intracellular Cl- concentration mediates Trichomonas vaginalis-induced inflammation in the human vaginal epithelium.

Trichomonas vaginalis is a primary urogenital parasite that causes trichomoniasis, a common sexually transmitted disease. As the first line of host defense, vaginal epithelial cells play critical roles in orchestrating vaginal innate immunity and modulate intracellular Cl- homeostasis via the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that plays positive roles in regulating nuclear factor-κB (NF-κB) signalling. However, the association between T. vaginalis infection and intracellular Cl- disequilibrium remains elusive. This study showed that after T. vaginalis infection, CFTR was markedly down-regulated by cysteine proteases in vaginal epithelial cells. The intracellular Cl- concentration ([Cl-]i) was consequently elevated, leading to NF-κB signalling activation via serum- and glucocorticoid-inducible kinase-1. Moreover, heightened [Cl-]i and activated NF-κB signalling could be sustained in a positive feedback regulatory manner resulting from decreased intracellular cAMP through NF-κB-mediated up-regulation of phosphodiesterase 4. The results conclusively revealed that the intracellular Cl- of the human vaginal epithelium could be dynamically modulated by T. vaginalis, which contributed to mediation of epithelial inflammation in the human vagina.

Targeting IFNα to tumor by anti-PD-L1 creates feedforward antitumor responses to overcome checkpoint blockade resistance.

Many patients remain unresponsive to intensive PD-1/PD-L1 blockade therapy despite the presence of tumor-infiltrating lymphocytes. We propose that impaired innate sensing might limit the complete activation of tumor-specific T cells after PD-1/PD-L1 blockade. Local delivery of type I interferons (IFNs) restores antigen presentation, but upregulates PD-L1, dampening subsequent T-cell activation. Therefore, we armed anti-PD-L1 antibody with IFNα (IFNα-anti-PD-L1) to create feedforward responses. Here, we find that a synergistic effect is achieved to overcome both type I IFN and checkpoint blockade therapy resistance with the least side effects in advanced tumors. Intriguingly, PD-L1 expressed in either tumor cells or tumor-associated host cells is sufficient for fusion protein targeting. IFNα-anti-PD-L1 activates IFNAR signaling in host cells, but not in tumor cells to initiate T-cell reactivation. Our data suggest that a next-generation PD-L1 antibody armed with IFNα improves tumor targeting and antigen presentation, while countering innate or T-cell-driven PD-L1 upregulation within tumor.

Prognostic significance of CIP2A expression in solid tumors: A meta-analysis.

CIP2A, cancerous inhibitor of protein phosphatase 2A, was initially recognized as an oncoprotein. Recently several studies revealed that CIP2A could function as a prognosis biomarker, however, the result remained not comprehensive, partly due to small number of patients included individually. Here we carried out a meta-analysis of published studies to assess the prognostic significance of CIP2A in solid tumors. All eligible studies were identified through searching PubMed, Embase and Web of Science database. In this meta-analysis, 22 studies involving 4,579 participants were included, and we verified that CIP2A over-expression was significantly related with poor overall survival (pooled HR = 1.844, 95% CI = 1.528-2.225, P<0.001) and short disease free survival (pooled HR = 1.808, 95% CI = 1.591-2.055, P<0.001) in solid tumors. Additionally, subgroup analysis suggested that the trend of a poor overall survival with an increased CIP2A expression was present in East-Asian and European patients, as well as in lung cancer and colorectal cancer. To sum up, CIP2A over-expression was associated with poor survival in human solid tumors and might be a predictive factor of poor prognosis.

Development and Applications of Chromosome-Specific Cytogenetic BAC-FISH Probes in S. spontaneum.

Saccharum spontaneum is a major Saccharum species that contributed to the origin of modern sugarcane cultivars, and due to a high degree of polyploidy is considered to be a plant species with one of the most complex genetics. Fluorescence in situ hybridization (FISH) is a powerful and widely used tool in genome studies. Here, we demonstrated that FISH based on bacterial artificial chromosome (BAC) clones can be used as a specific cytological marker to identify S. spontaneum individual chromosomes and study the relationship between S. spontaneum and other related species. We screened low-copy BACs as probes from the sequences of a high coverage of S. spontaneum BAC library based on BLAST search of the sorghum genome. In total, we isolated 49 positive BAC clones, and identified 27 BAC clones that can give specific signals on the S. spontaneum chromosomes. Of the 27 BAC probes, 18 were confirmed to be able to discriminate the eight basic chromosomes of S. spontaneum. Moreover, BAC-24, BAC-66, BAC-78, BAC-69, BAC-71, BAC-73, and BAC-77 probes were used to construct physical maps of chromosome 1 and chromosome 2 of S. spontaneum, which indicated synteny in Sb01 between S. spontaneum and sorghum. Furthermore, we found that BAC-14 and BAC-19 probes, corresponding to the sorghum chromosomes 2 and 8, respectively, localized to different arms of the same S. spontaneum chromosome, suggesting that there was an inter-chromosomal rearrangement event between S. spontaneum and sorghum. Our study provides the first set of chromosome-specific cytogenetic markers in Saccharum and is critical for future advances in cytogenetics and genome sequencing studies in Saccharum.

Neuroprotection of (+)-2-(1-Hydroxyl-4-Oxocyclohexyl) Ethyl Caffeate Against Hydrogen Peroxide and Lipopolysaccharide Induced Injury via Modulating Arachidonic Acid Network and p38-MAPK Signaling.

Oxidative stress and neuroinflammation are highly relevant to the pathological processes of various neurodegenerative diseases including Alzheimer's disease (AD). (+)-2-(1-hydroxyl-4-oxocyclohexyl) ethyl caffeate (HOEC), a novel 5-lipoxygenase inhibitor, was isolated from the whole plant of Incarvillea mairei var granditlora (Wehrhahn) Grierson. In this study, we investigated the protective effect of HOEC on hydrogen peroxide (H2O2) and lipopolysaccharide (LPS) -induced cytotoxicity and neuroinflammation in vitro and in vivo. MTT assay, LDH release assay, morphological observation and Hoechst 33342/PI dual staining followed by EIA, immunofluorescence staining and Western Blotting analysis were performed to elucidate the neuroprotective effect of HOEC. Treatment with HOEC at various concentrations prior to H2O2 exposure significantly enhanced cell viability, decreased LDH release, prevented cell morphologic changes and apoptosis. Instead of PGE2 reduction, HOEC markedly inhibited the production of LTB4 and suppressed the macrophage-mediated neurotoxicity. Western blotting and immunofluorescence staining showed that HOEC inhibited H2O2-induced p38 phosphorylation and NF-κB activation. Neuroprotective effect of HOEC was abolished by a p38 inhibitor. Further in vivo studies of LPS-induced neuroinflammation confirmed the anti-inflammatory effects of HOEC. These findings that HOEC protects SH-SY5Y cells from H2O2 and LPS-induced injury via arachidonic acid network modulation followed by p38 MAPK and NF-κB signaling, might make HOEC be considered as a therapeutic candidate for prevention and treatment of neurodegenerative diseases involving oxidative stress or/and inflammation.

The protection of acetylcholinesterase inhibitor on ß-amyloid-induced the injury of neurite outgrowth via regulating axon guidance related genes expression in neuronal cells.

Cognitive deficits in AD correlate with progressive synaptic dysfunction and loss. The Rho family of small GTPases, including Rho, Rac, and Cdc42, has a central role in cellular motility and cytokinesis. Acetylcholinesterase inhibitor has been found to protect cells against a broad range of reagents-induced injuries. Present studies examined if the effect of HupA on neurite outgrowth in Aß-treated neuronal cells executed via regulating Rho-GTPase mediated axon guidance relative gene expression. Affymetrix cDNA microarray assay followed by real-time RT-PCR and Western Blotting analysis were used to elucidate and analyze the signaling pathway involved in Aß and HupA's effects. The effects of Aß and HupA on the neurite outgrowth were further confirmed via immunofluorescence staining. Aß up-regulated the mRNA expressions of NFAT5, LIMK1, EPHA1, NTN4 and RAC2 markedly in SH-SY5Y cells. Co-incubation of Aß and HupA reversed or decreased the changes of NFAT5, NTN4, RAC2, CDC42 and SEMA4F. HupA treated alone increased NFAT5, LIMK1, NTN4 significantly. Following qRT-PCR validation showed that the correlation of the gene expression ratio between microarray and qRT-PCR is significant. Western blot result showed that the change of CDC42 protein is consistent with the mRNA level while RAC2 is not. The morphological results confirmed that HupA improved, or partly reversed, the Aß-induced damage of neurite outgrowth. The protective effect of HupA from Aß induced morphological injury might be correlative to, at least partially, regulating the network of neurite outgrowth related genes.

Effects of L-carnosine on splenic sympathetic nerve activity and tumor proliferation.

l-Carnosine (ß-alanyl-l-histidine), a dipeptide of the amino acids ß-alanine and histidine, is found in mammalian tissues including those in the central nervous system and in skeletal muscles. In the present study, we examined the effects of intraduodenal (ID) injection of l-carnosine on splenic sympathetic nerve activity (splenic-SNA) in urethane-anesthetized rats and found that ID injection of 3.3mg/kg of body weight of l-carnosine significantly suppressed splenic-SNA. Since it has been suggested that splenic-SNA reduction increases natural killer (NK) activity of splenic cells, which in turn elevates tumor immunity, we then investigated the effect of l-carnosine on the proliferation of human colon cancer cells transplanted into athymic nude mice. The findings of this study revealed that 1mg/mL of l-carnosine solution given as the only drinking water inhibited tumor proliferation. These results suggest that l-carnosine suppresses splenic-SNA and inhibits cancer cell proliferation, probably by elevating NK activity.

[Determination of biphenyl ether herbicides in water using HPLC with cloud-point extraction].

OBJECTIVE: To determine residues of multiple biphenyl ether herbicides simultaneously in water using high performance liquid chromatography (HPLC) with cloud-point extraction. METHODS: The residues of eight biphenyl ether herbicides (including bentazone, fomesafen, acifluorfen, aclonifen, bifenox, fluoroglycofenethy, nitrofen, oxyfluorfen) in water samples were extracted with cloud-point extraction of Triton X-114. The analytes were separated and determined using reverse phase HPLC with ultraviolet detector at 300 nm. Optimized conditions for the pretreatment of water samples and the parameters of chromatographic separation applied. RESULTS: There was a good linear correlation between the concentration and the peak area of the analytes in the range of 0.05-2.00 mg/L (r = 0.9991-0.9998). Except bentazone, the spiked recoveries of the biphenyl ether herbicides in the water samples ranged from 80.1% to 100.9%, with relative standard deviations ranging from 2.70% to 6.40%. The detection limit of the method ranged from 0.10 microg/L to 0.50 microg/L. CONCLUSION: The proposed method is simple, rapid and sensitive, and can meet the requirements of determination of multiple biphenyl ether herbicides simultaneously in natural waters.

Effects of L-arginine and L-lysine mixtures on splenic sympathetic nerve activity and tumor proliferation.

Oral supplementations of L-arginine and L-lysine show tumor inhibition abilities. The splenic sympathetic nerve is involved in central modulation of cellular immunity and suppresses splenic natural killer cell activity in rats. An intravenous administration of a mixture of 10 mM L-arginine and L-lysine decreased splenic sympathetic nerve activity (splenic-SNA). We examined the effect of L-arginine and L-lysine mixtures on splenic-SNA in urethane-anesthetized rats by administration of 1 ml mixtures of 2 mM, 10 mM, and 50 mM L-arginine and L-lysine. We also studied the effect of the above mixtures on human colon cancer cell proliferation in athymic nude mice. An increase in splenic-SNA and tumor volume (2 mM), no effect (10 mM), and a decrease in both values (50 mM) were seen. Bivariate correlation analysis revealed a positive correlation between changes in splenic-SNA and tumor volume, indicating the tumor suppressing ability of weakened splenic-SNA.

Effect of the culture extract of Lentinus edodes mycelia on splenic sympathetic activity and cancer cell proliferation.

The spleen is an important organ for tumor immunity, and the splenic sympathetic nerve has a suppressive effect on splenic natural killer (NK) cytotoxicity. On the basis of this and reports that Lentinus edodes (Shiitake mushroom) has tumor-inhibitory effects, the authors hypothesized that an extract of a mycelial culture of L. edodes grown in a solid medium of sugar-cane bagasse and defatted rice bran-L.E.M-might affect the sympathetic splenic sympathetic nerve activity (Splenic-SNA) and thus inhibit tumor proliferation. Thus, the effect of L.E.M on Splenic-SNA and human cancer cell proliferation was examined. Splenic-SNA was found to be suppressed by an intraduodenal L.E.M injection in urethane-anesthetized rats, which significantly inhibited increases in the tumor volume of human colon and breast cancer cells implanted in athymic nude mice. These findings suggest that L.E.M has an inhibitory effect on tumor proliferation possibly via a reduction in NK cytotoxicity through the suppression of Splenic-SNA.

Effects of olfactory stimulations with scents of grapefruit and lavender oils on renal sympathetic nerve and blood pressure in Clock mutant mice.

Previously, we observed that in mice, olfactory stimulation with scent of grapefruit oil elevates renal sympathetic nerve activity and blood pressure. In contrast, olfactory stimulation with scent of lavender oil has opposite effects in mice. Moreover, electrolytic lesions of the mouse hypothalamic suprachiasmatic nucleus eliminated changes in renal sympathetic nerve activity and blood pressure induced by either scent of grapefruit oil or scent of lavender oil. Here, we show that grapefruit oil-induced elevations in renal sympathetic nerve activity and blood pressure were not observed in Clock mutant mice, which harbor mutations in Clock and lack normal circadian rhythms, whereas lavender oil-suppressions were preserved in Clock mutant mice. In addition, responses of c-Fos inductions in the suprachiasmatic nucleus and paraventricular nucleus of the hypothalamus to scent of grapefruit oil observed in wild-type mice were not observed in Clock mutant mice. These findings suggest that the Clock gene might be implicated in elevating responses of autonomic and cardiovascular functions to olfactory stimulation with scent of grapefruit oil.

Involvement of the histaminergic system in renal sympathetic and cardiovascular responses to leptin and ghrelin.

Previous studies have demonstrated that histamine affects blood pressure (BP) in anesthetized rats. Here, we examined the effects of lateral cerebral ventricular (LCV) injection of various doses of histamine on renal sympathetic nerve activity (RSNA) and BP in anesthetized rats. LCV injection of a low dose of histamine (0.0001nmol) suppressed RSNA and BP. Conversely, a high dose of histamine (100nmol) elevated both RSNA and BP. Moreover, inhibiting effects of a low dose of histamine were eliminated by LCV pre-injection of thioperamide, an antagonist of histaminergic H3-receptor, and accelerating effects of a high dose of histamine were abolished by LCV pre-injection of diphenhydramine, an antagonist of histaminergic H1-receptor. Thus, these evidences suggest that central histamine affects RSNA and BP via histaminergic receptors. In addition, we examined a role for histaminergic system in cardiovascular modulators such as leptin and ghrelin. The LCV pre-injection of thioperamide clearly blocked suppressing effects of ghrelin on RSNA and BP. The LCV pre-injection of diphenhydramine also blocked elevating effects of leptin. Therefore, these results suggest that leptin and ghrelin might affect RSNA and BP by mediating central histaminegic H1- and H3-receptors, respectively.