Therapeutic efficacy of cancer vaccine adjuvanted with nanoemulsion loaded with TLR7/8 agonist in lung cancer model.

Although immune checkpoint inhibitors have significantly improved clinical outcomes in various malignant cancers, only a small proportion of patients reap benefits, likely due to the low number of T cells and high number of immunosuppressive cells in the tumor microenvironment (TME) of patients with advanced disease. We developed a cancer vaccine adjuvanted with nanoemulsion (NE) loaded with TLR7/8 agonist (R848) and analyzed its therapeutic effect alone or in combination with immune checkpoint inhibitors, on antitumor immune responses and the reprogramming of suppressive immune cells in the TME. NE (R848) demonstrated robust local and systemic antitumor immune responses in both subcutaneous and orthotopic mouse lung cancer models, inducing tumor-specific T cell activation and mitigating T cell exhaustion. Combination with anti-PD-1 antibodies showed synergistic effects with respect to therapeutic efficacy and survival rate. Thus, NE (R848)-based cancer vaccines could prevent tumor recurrence and prolong survival by activating antitumor immunity and reprogramming immunosuppression.

Regulatory (FoxP3+) T cells and TGF-ß predict the response to anti-PD-1 immunotherapy in patients with non-small cell lung cancer.

Antitumor immune responses induced by immune checkpoint inhibitors anti-PD-1 or anti-PD-L1 have been used as therapeutic strategies in advanced non-small cell lung cancer (NSCLC) patients over the last decade. Favorable antitumor activity to immune checkpoint inhibitors is correlated with high PD-L1 expression, increased tumor-infiltrating lymphocytes, and decreased suppressive immune cells including Treg cells, myeloid-derived suppressor cells, or tumor-associated macrophages in various cancer types. In this study, we investigated the potential correlation between clinical outcomes and peripheral blood immune cell profiles, specifically focused on FoxP3+ Treg cells, collected at baseline and one week after anti-PD-1 therapy in two independent cohorts of patients with NSCLC: a discovery cohort of 83 patients and a validation cohort of 49 patients. High frequencies of circulating Treg cells one week after anti-PD-1 therapy were correlated with a high response rate, longer progression-free survival, and overall survival. Furthermore, high levels of TGF-ß and Treg cells were associated with favorable clinical outcomes. Our results suggest that higher levels of FoxP3+ Treg cells and TGF-ß can predict a favorable response to anti-PD-1 immunotherapy in patients with advanced NSCLC.

MDSC subtypes and CD39 expression on CD8+ T cells predict the efficacy of anti-PD-1 immunotherapy in patients with advanced NSCLC.

The major suppressive immune cells in tumor sites are myeloid derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and Treg cells, and the major roles of these suppressive immune cells include hindering T-cell activities and supporting tumor progression and survival. In this study, we analyzed the pattern of circulating MDSC subtypes in patients with non-small cell lung cancer (NSCLC) whether those suppressive immune cells hinder T-cell activities leading to poor clinical outcomes. First, we verified PMN-MDSCs, monocytic-MDSCs (M-MDSCs), and Treg cells increased according to the stages of NSCLC, and MDSCs effectively suppressed T-cell activities and induced T-cell exhaustion. The analysis of NSCLC patients treated with anti-PD-1 immunotherapy demonstrated that low PMN-MDSCs, M-MDSCs, and CD39+ CD8+ T cells as an individual and all together were associated with longer progression free survival and overall survival, suggesting PMN-MDSCs, M-MDSCs, and CD39+ CD8+ T cells frequencies in peripheral blood might be useful as potential predictive and prognostic biomarkers.

SB365 induces apoptosis and suppresses proliferation of glioblastoma cells.

CONTEXT: Glioblastoma is a malignant brain tumor with limited treatment modalities due to its nature. SB365, Pulsatilla saponin D, is known to induce apoptosis and inhibit the growth of many cancer cells. AIM: We elucidated the anticancer effects of SB365 in glioblastoma cells. METHODS: We examined the antiproliferative activity of SB365 in human glioblastoma cell lines. Apoptosis was evaluated using the Hoechst assay, TUNEL assay, DAPI nuclear staining, and Western blotting analysis. To test the antimetastatic capacity of SB365, cell migration assay was conducted, and hypoxia-inducible factor-1 alpha (HIF-1α) expression and vascular endothelial growth factor (VEGF) level were determined under hypoxic conditions. STATICAL ANALYSIS: Significance of the results was confirmed by a one-way analysis of variance analysis. RESULTS: SB365 treatment suppressed the growth of glioblastoma cells and resulted in apoptotic morphological features such as nuclear condensation and fragmentation, enhancing the expression of cleaved poly (ADP-ribose) polymerase and caspase-3. It also significantly delayed cell migration and decreased the HIF-1α expression and VEGF secretion. CONCLUSION: Our findings thus demonstrate that SB365 induced apoptosis and delayed the growth and migration of human glioblastoma cells. It is considered that SB365 would be a promising therapeutic option for glioblastoma.

Predictive Factors of Duration of Continuous Renal Replacement Therapy in Acute Kidney Injury Survivors.

The factors influencing continuous renal replacement therapy (CRRT) duration for critically ill patients with acute kidney injury (AKI) are unclear. Therefore, we investigated the clinical factors that could influence the duration of CRRT for AKI survivors. In this retrospective observational study, the medical records of all hospital survivors who required CRRT for AKI in intensive care units were analyzed. The CRRT duration (median, 6 days) was categorized as short-duration CRRT (≤ 6 days, n = 65) and long-duration CRRT (> 6 days, n = 59), according to the median CRRT duration. A urine output of less than 0.5 mL/kg/h (adjusted odds ratio [OR], 3.4; P = 0.010), mechanical ventilation use (adjusted OR, 7.9; P = 0.001), and extracorporeal membrane oxygenation (ECMO) use (adjusted OR, 6.5; P = 0.010) were independent predictors of long-duration CRRT, whereas serum creatinine and neutrophil gelatinase-associated lipocalin were not significant predictors. A clinical model demonstrated a good discriminatory ability to predict long-duration CRRT (area under the curve, 0.84; 95% confidence interval, 0.76-0.90). The urine output immediately before CRRT initiation and factors associated with disease severity significantly affected the duration of CRRT. Simultaneously considering the urine output, mechanical ventilation use, and ECMO use predicted CRRT duration in AKI survivors.

Change in alkaline phosphatase activity associated with intensive care unit and hospital length of stay in patients with septic acute kidney injury on continuous renal replacement therapy.

BACKGROUND: Evidence suggests that alkaline phosphatase attenuates inflammatory response in sepsis by lipopolysaccharide detoxification and adenosine triphosphate dephosphorylation. We sought to determine changes in alkaline phosphatase (AP) activity during septic acute kidney injury (AKI) and clinical parameters associated with AP activity. METHODS: In this retrospective study, we investigated baseline (when initiating CRRT) and follow-up AP activity on day 3, and associated outcomes in patients who underwent continuous renal replacement therapy (CRRT) due to septic AKI. RESULTS: We analyzed the baseline AP activity of 155 patients and day 3 AP activity in 123 patients. Baseline AP activity was not associated with renal or inflammatory biomarkers, or outcomes. It did not significantly differ between the 75 survivors and 80 non-survivors (p = 0.155). AP activity was higher on day 3 than at baseline (105 U/L [interquartile range, 79-156] vs 90 U/L [interquartile range, 59-133]). In particular, liver and bone isoforms increased significantly (p < 0.05), but intestine isoforms did not reach statistical significance (p = 0.367). In addition, day 3 AP activity showed a weak correlation with length of ICU stay (r = 0.213, p = 0.018) and length of hospital stay (r = 0.216, p = 0.017), but not with survival (r = - 0.035, p = 0.698). CONCLUSION: Endogenous AP activity significantly increased in patients with septic AKI. However, neither baseline nor follow-up AP activity was associated with survival.

Numerical expression of volume status using the bioimpedance ratio in continuous ambulatory peritoneal dialysis patients: A pilot study.

BACKGROUND: Volume overload results in higher mortality rates in patients on continuous ambulatory peritoneal dialysis (CAPD). The ratio of bioimpedance (RBI) might be a helpful parameter in adjusting dry body weight in CAPD patients. This study examined whether it is possible to distinguish between non-hypervolemic status and hypervolemic status in CAPD patients by using only RBI. METHODS: RBI was calculated as follows: RBI = impedance at 50 kHz/impedance at 500 kHz. Based on the experts' judgements, a total of 64 CAPD patients were divided into two groups, a non-hypervolemic group and a hypervolemic group. The RBI was measured from right wrist to right ankle (rw-raRBI) by bioimpedance spectroscopy (BCM®, Fresenius Medical Care) before and after the peritosol was emptied. Other RBIs were measured from the right side of the anterior superior iliac spine to the ipsilateral ankle (rasis-raRBI) to control for the electro-physiological effects of peritoneal dialysate. RESULTS: The mean rw-raRBI of non-hypervolemic patients was higher than that of hypervolemic patients in the presence (1.141 ± 0.022 vs. 1.121 ± 0.021, P < 0.001) of a peritosol. Likewise, the mean rasis-raRBI of non-hypervolemic patients was higher than that of hypervolemic patients (presence of peritosol: 1.136 ± 0.026 vs. 1.109 ± 0.022, P < 0.001; absence of peritosol: 1.131 ± 0.022 vs. 1.107 ± 0.022, P < 0.001). CONCLUSION: The volume status of CAPD patients was able to be simply expressed by RBI. Therefore, this study suggests that when patients cannot be analyzed using BCM, RBI could be an alternative.

Early continuous renal replacement therapy in septic acute kidney injury could be defined by its initiation within 24 hours of vasopressor infusion.

PURPOSE: The optimal timing for the initiation of early continuous renal replacement therapy (CRRT) is uncertain and requires a practically feasible definition with acceptable evidence. MATERIALS AND METHODS: We investigated the clinical impacts of 3-time interval parameters on the morbidity and mortality of 177 patients with septic shock-induced acute kidney injury: (1) time from vasopressor initiation to CRRT initiation (Tvaso-CRRT), (2) time from intensive care unit (ICU) admission to CRRT initation (TICU-CRRT), and (3) time from endotracheal intubation to CRRT initiation (Tendo-CRRT). RESULTS: The proportion of the patients with Tvaso-CRRT less than 24 h (median, 14 h, interquartile range [IQR], 5-30 h) was significantly higher in the survival group than in the non-survival group (84.3% vs. 58.5%, p < 0.001). Tvaso-CRRT less than 24 h and Sequential Organ Failure Assessment score were independent factors associated with 28-day mortality and 90-day mortality. TICU-CRRT (median, 17 h, IQR, 5-72 h) and Tendo-CRRT (median, 13 h, IQR, 4-48 h) were significantly correlated with both the length of ICU stay (p < 0.001) and mechanical ventilation duration (p < 0.001), but not mortality. CONCLUSIONS: Considering the possible therapeutic measurement by physician on the basis of the results in this study, early CRRT could be defined by a Tvaso-CRRT less than 24 h.

Design and synthesis of novel arylpiperazine derivatives containing the imidazole core targeting 5-HT(2A) receptor and 5-HT transporter.

Serotonin antagonist reuptake inhibitor (SARI) drugs that block both 5-HT(2) receptors and the serotonin transporters have been developed. The human 5-HT(2A/2C) receptor has been implicated in several neurological conditions, and potent selective 5-HT(2A/2C) ligands may have therapeutic potential for treatment of CNS diseases such as depression. An imidazole moiety usually provides good pharmacokinetic properties as a drug substance, and thus considerable efforts have been devoted to develop imidazole derivatives into drug candidates. The imidazole series of compounds was evaluated against 5-HT(2A/2C) and serotonin reuptake inhibition. A few of the compounds in the series showed promising IC(50) values and antidepressant-like effect in in vivo forced swimming test (FST). On the basis of these results, further lead optimization studies resulted in identifying promising compounds potentially for therapeutic use.

Characterization of the autophosphorylating kinase, PkaF, in Streptomyces coelicolor A3(2) M130.

Streptomyces coelicolor, the model species for morphologically complex actinomycete bacteria, has unique characteristics such as morphological and physiological differentiation, which are controlled by various factors and several protein kinases. From the whole genomic sequence of S. coelicolor A3(2), 44 putative serine/threonine (Ser/Thr) protein kinases were identified, and the pkaF gene was chosen as the best-conserved protein for typical Ser/Thr protein kinases. pkaF encodes a 667-amino acid protein with a predicted N-terminal Ser/Thr kinase domain and four repeated C-terminal penicillin-binding domains and Ser/Thr kinase-associated (PASTA) domains. Based on PCR, a pkaF gene was cloned and heterologously expressed. PkaF expressed in Escherichia coli had the bigger molecular size than the expected value (75 kDa) and was further purified by Ni2+-NTA agarose affinity column chromatography to homogeneity. The purified PkaF was autophosphorylated through the transfer of the γ-phosphate group of ATP. The extent of phosphorylation was proportional to the amount of PkaF, and the phospho-PkaF was dephosphorylated by the addition of the cell lysate of S. coelicolor A3(2). Although no change was observed in the pkaF disruptant, overexpression of pkaF induced severe repression of morphogenesis and actinorhodin production, but not undecylprodigiosin production, implying that PkaF specifically regulates morphogenesis and actinorhodin production in S. coelicolor.

Discovery of 2-(4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-bromophenyl)-1-(2-chlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-1,3,4-thiadiazole (GCC2680) as a potent, selective and orally efficacious cannabinoid-1 receptor antagonist.

Structure-activity relationship studies in a series of diarylpyrazolyl thiadiazoles identified cannabinoid-1 receptor antagonists with excellent potency and selectivity. Based on its exceptional in vivo efficacy in animal models and its favorable pharmacokinetic and toxicological profiles, 2-(4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-bromophenyl)-1-(2-chlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-1,3,4-thiadiazole (GCC2680) was selected as a preclinical candidate for the treatment of obesity.

Synthesis and structure-activity relationship of 1,2,4-triazole-containing diarylpyrazolyl carboxamide as CB1 cannabinoid receptor-ligand.

Numerous research groups have been engaged in searching for novel CB1 receptor antagonists, since SR141716A (rimonabant), a CB1 receptor antagonist, proved to be efficacious in human for the treatment of obesity. In the present study, a series of 1,2,4-triazole-containing diarylpyrazolyl carboxamides based on the 1,5-diarylpyrazole template of rimonabant, was synthesized and tested for CB1 receptor binding affinity. The structure-activity relationship studies demonstrated that incorporation of 1,2,4-triazole ring onto the pyrazole scaffold via a methylene linker led to a significant improvement for CB1 receptor binding affinity. Importantly, these analogues also exhibited excellent selectivity for CB1 receptor over CB2 receptor.

Oral administration of 1,4-aryl-2-mercaptoimidazole inhibits T-cell proliferation and reduces clinical severity in the murine experimental autoimmune encephalomyelitis model.

T cells play a pivotal role in the initiation and progression of multiple sclerosis. We have found that 1,4-aryl-2-mercaptoimidazole (KRM-III) inhibited T-cell antigen receptor- and phorbol myristate acetate/ionomycin-induced activation of nuclear factor of activated T cells (NFAT) and T-cell proliferation with an IC(50) of 5 microM. The KRM-III-mediated inhibitory effect was specific for NFAT activation but not for nuclear factor kappaB. Oral administration of 90 mg/kg KRM-III resulted in complete abrogation of anti-CD3 antibody-induced T-cell activation and a 45.8% reduction in footpad swelling in bovine serum albumin-induced delayed-type hypersensitivity. In the murine experimental autoimmune encephalomyelitis (EAE) model, oral administration of KRM-III significantly attenuated the severity of disease when given before or after disease onset. Draining lymph node cells from KRM-III-treated mice showed markedly reduced proliferation in response to myelin oligodendrocyte glycoprotein peptide. Histological analysis indicated that KRM-III reduced the infiltration of inflammatory cells to the white matter of spinal lumbar cords. These results demonstrate that KRM-III efficiently inhibits T-cell activation and inflammatory responses and lessens EAE clinical signs, which suggest KRM-III as a potential lead compound for the treatment of T-cell-driven autoimmune diseases.

Substituted pyrimidines as cannabinoid CB1 receptor ligands.

Cannabinoid CB1 receptors have been the avenue of extensive studies since the first clinical results of rimonabant (SR141716) for the treatment of obesity and obesity-related metabolic disorders were reported in 2001. To further evaluate the properties of CB receptors, we have designed and efficiently prepared a series of substituted pyrimidines based on chemical structure of Merck's taranabant, a cannabinoid CB1 receptor inverse agonist. Noticeably, N4-((2S,3S)-3-(3-bromophenyl)-4-(4-chlorophenyl)butan-2-yl)-N6-butylpyrimidine-4,6-diamine (13b) demonstrated good binding affinity and decent selectivity for CB1 receptor (IC(50)=16.3nM, CB2/CB1=181.6).

Helicobacter pylori genotyping findings from multiple cultured isolates and mucosal biopsy specimens: strain diversities of Helicobacter pylori isolates in individual hosts.

OBJECTIVES: To determine whether the genotypes of virulent genes in Helicobacter pylori isolates and mucosal biopsy specimens differ in individuals, and to investigate whether different isolates from single hosts show strain differences. METHODS: Sixty-one Korean patients with H. pylori infection were enrolled. PCR and DNA sequencing for cagA, vacA, iceA, and oipA were performed using DNA extracted from H. pylori isolates cultured (2.6 H. pylori isolates per host) directly from antral mucosal biopsy specimens. Strain diversities were analyzed in 234 H. pylori isolates obtained from 43 hosts with at least two H. pylori isolates from antrum and body, respectively, and random amplified polymorphic DNA fingerprinting was carried out on isolates obtained from patients who showed genotype diversity. RESULTS: The patients with inconsistent genotyping results between H. pylori isolates and mucosal biopsies were as follows: 16.4% for cagA, 19.7% for vacA m, 47.5% for vacA s1, 6.6% for vacA i-region, 34.4% for iceA, and 21.3% for oipA. Genotyping of H. pylori isolates from same hosts showed diversity in 58.1% (25/43 patients). When random amplified polymorphic DNA -PCR fingerprinting was carried out on 104 H. pylori isolates from 19 patients who showed genotype diversity among their isolates, 68.4% (13 of 19 patients) of patients were found to be colonized by multiple H. pylori strains. CONCLUSION: This study shows that the genotypes of virulent genes from biopsy samples produced different results when compared with those obtained from H. pylori isolates, especially for vacA s1, and iceA. In addition, about 60% of our patients were infected by multiple H. pylori strains.

Methylsulfonylpyrazolyl oxadiazoles and thiadiazoles as potent, orally bioavailable cannabinoid-1 receptor antagonists for the treatment of obesity.

BACKGROUND: Since the cannabinoid receptor 1 (CB1) antagonist SR141716 (rimonabant) was previously reported to modulate food intake, CB1 antagonism has been considered as a new therapeutic target for the treatment of obesity. DISCUSSION: In the present study, biarylpyrazole analogues based on a sulfur-containing pyrazole core coupled with 1,3,4-oxadiazole and 1,3,4-thiadiazole were synthesized and assayed for rat CB1 receptor binding affinity. RESULTS: The structure-activity relationship studies to optimize pyrazole substituents as well as 1,3,4-oxadiazole or 1,3,4-thiadiazole rings led to four novel CB1 antagonists with IC(50) values of approximately 1 nM for the rat CB1 receptor binding. Among these derivatives, we identified trifluoromethylcyclobutyl analogues 19e and 19l as promising precandidates for the development as anti-obesity agents.

Characterization of the sgtR1 and sgtR2 genes and their role in regulating expression of the sprT gene encoding Streptomyces griseus trypsin.

The sgtR1 and sgtR2 genes encoding putative regulators similar to the Aha1 and ArsR families, respectively, were identified downstream from the sprT gene. To investigate their function, expression vectors containing various combinations of sprT, sgtR1, and sgtR2 were transformed into Streptomyces lividans and Streptomyces griseus. The trypsin activity levels produced by S. lividans harboring pWHM3-TR2 (sprT and sgtR2) or pWHM3-TR1R2 (sprT, sgtR2, and sgtR2) were, respectively, 6.6 or 8.9 times that of S. lividans transformed with pWHM3-T (sprT). In the pWHM3-TR1R2 transformant, the transcription of sprT consistently occurred during the earlier stages of growth and was maintained at a higher level throughout the 6 days of cultivation. Streptomyces griseus IFO13350 harboring pWHM3-TR1R2 also produced trypsin activity 2.1 times that of the pWHM3-T transformant. However, all S. griseus Delta adpA transformants produced lower SGT activity than the wild-type strain, and none could overcome the deficiency in AdpA transcriptional activator, suggesting that AdpA is an absolute prerequisite for sprT expression. The sprT transcript was detected at a high level only in the wild-type strain, but the sgtR1 and sgtR2 transcript levels were very similar between the S. griseus IFO13350 and Delta adpA strains. This clearly demonstrates that the transcription of the sgtR1 and sgtR2 genes is not dependent on AdpA and that they are therefore not members of the AdpA regulon.

The inhibitory mechanism of methylmercury on differentiation of human neuroblastoma cells.

Methylmercury (MeHg) is a ubiquitous environmental toxicant and shows neurotoxicity to central nerve system (CNS) or neuronal cells. It has been known that MeHg has more influence to developing or differentiating CNS/neuronal cells than adult or differentiated CNS/neuronal cells. This study examined the effect of MeHg on differentiation of human neuroblastoma SH-SY5Y cells induced by all-trans-retinoic acid (RA). MeHg caused the impairment of the RA-induced G(1/0) phase arrest; it was induced the reduction of G(1/0) phase and S phase arrest. Extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase C (PKC) are involved in the RA-mediated differentiation and cell cycle progression. Activation of ERK1/2 by RA was increased more in MeHg-treated differentiating cells, comparing with only RA-treated groups. Furthermore, in both cases of inhibition of ERK1/2 with PD98059 or inhibition of PKC with GF109203X, RA/MeHg-induced ERK1/2 phosphorylation was reduced and G(1/0) phase arrest was induced. Thus, it indicates that the neuronal differentiation with RA was mediated by the ERK1/2 and PKC related pathway and MeHg resulted in neurotoxic influences through the disturbance in steps of differentiation by this pathway. These results suggest that MeHg inhibits RA-induced differentiation in SH-SY5Y cells by a pathway dependent ERK1/2 and PKC.

Genetic and phenotypic diversity of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy)propionic acid]-degrading bacteria isolated from soils.

Twelve mecoprop-degrading bacteria were isolated from soil samples, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genus Sphingomonas. Ten different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 12 isolates. The isolates were found to be able to utilize the chiral herbicide mecoprop as a sole source of carbon and energy. While seven of the isolates were able to degrade both (R)- and (S)-mecoprop, four isolates exhibited enantioselective degradation of the (S)-type and one isolate could degrade only the (R)-enantiomer. All of the isolates were observed to possess plasmid DNAs. When certain plasmids were removed from isolates MP11, MP15, and MP23, those strains could no longer degrade mecoprop. This compelling result suggests that plasmid DNAs, in this case, conferred the ability to degrade the herbicide. The isolates MP13, MP15, and MP24 were identified as the same strain; however, they exhibited different plasmid profiles. This indicates that these isolates acquired different mecoprop-degradative plasmids in different soils through natural gene transfer.