Tumor-specific T cells in head and neck cancer have rescuable functionality and can be identified through single-cell co-culture.

BACKGROUND: Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) remains a treatment-resistance disease with limited response to immunotherapy. While T cells in HNSCC are known to display phenotypic dysfunction, whether they retain rescuable functional capacity and tumor-killing capability remains unclear. METHODS: To investigate the functionality and tumor-specificity of tumor-infiltrating lymphocytes (TILs) across HNSCCs, malignant cell lines and TILs were derived from 31 HPV-negative HNSCCs at the time of standard surgical resection. T cell functional capacity was evaluated through ex vivo expansion, immunophenotyping, and IsoLight single-cell proteomics. Tumor-specificity was investigated through both bulk and single-cell tumor-TIL co-culture. RESULTS: TILs could be successfully generated from 24 patients (77%), including both previously untreated and radiation recurrent HNSCCs. We demonstrate that across HNSCCs, TILs express multiple exhaustion markers but maintain a predominantly effector memory phenotype. After ex vivo expansion, TILs retain immunogenic functionality even from radiation-resistant, exhausted, and T cell-depleted disease. We further demonstrate tumor-specificity of T cells across HNSCC patients through patient-matched malignant cell-T cell co-culture. Finally, we use optofluidic technology to establish an autologous single tumor cell-single T cell co-culture platform for HNSCC. Cells derived from three HNSCC patients underwent single-cell co-culture which enabled identification and visualization of individual tumor-killing TILs in real-time in all patients. CONCLUSIONS: These studies show that cancer-specific T cells exist across HNSCC patients with rescuable immunogenicity and can be identified on a single-cell level. These data lay the foundation for development of patient-specific T cell immunotherapies in HNSCC.

Mechanism of ozone alleviation of malignant ascites in hepatocellular carcinoma through the inhibition of neutrophil extracellular traps.

Malignant ascites in hepatocellular carcinoma is usually a sign of advanced disease and poor prognosis and is also thought to be associated with chronic inflammation mediated by neutrophil extracellular trap (NET) networks. Although ozone, a strong oxidant, has significant antibacterial and anti-inflammatory effects, its effectiveness in treating malignant liver ascites is unclear. We first measured the levels of NETs in the peripheral blood of patients with liver cancer and healthy individuals. Next, we constructed the H22 tumor-bearing mouse model and observed the abdominal girth, body weight, survival rate, and survival time in each group; we marked the proteins associated with NETs in mouse intestinal tissues by immunofluorescence; cf-DNA and cytokines in ascites such as: tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), matrix metalloprotein 9 (MMP-9), and interferon gamma (IFN-γ) levels in ascites were measured by enzyme-linked immunosorbent assay. The expression levels of phosphorylated adenylate-activated protein kinase (P-AMPK) and scavenger receptor-A (SR-A) were detected by immunocytochemistry in the intestinal tissues of each group of mice. We further examined the expression of P-AMPK and SR-A proteins in ascites deposits by Western blotting. The results show, the plasma levels of NETs were higher in patients with hepatocellular carcinoma than in normal subjects (P < 0.01). Abdominal girth and body weight were significantly reduced in the ozone-treated group compared with the model group, while survival and survival time were dose dependently increased (both P < 0.05). NET-associated guanine histone H3 and myeloperoxidase were abundantly expressed at neutrophil aggregates in the intestinal tissues of the model mice, whereas their expression was significantly reduced in the ozone-treated group. The levels of cf-DNA, IL-6, IFN-γ, MMP-9, VEGF, and TNF-α were dose dependently increased in the ascites of H22 tumor-bearing mice in the ozone-treated group compared with the model group (all P < 0.01), while the expression of P-AMPK and SR-A proteins was increased in the ozone-treated group compared with the model group. Ozone showed significant antiperitoneal fluid production properties in H22 tumor-bearing mice, and ozone reduced peritoneal fluid production by activating AMPK and up-regulating SR-A phagocytosis damage-associated molecular patterns to reduce the production of NETs. This suggests that ozone could be used as a new drug for the treatment of malignant ascites in hepatocellular carcinoma.

Acinetobacter baumannii adaptation to the host pH microenvironment is mediated by allelic variation in a single residue of BauA protein.

Acinetobacter baumannii has been listed as one of the most critical pathogens in nosocomial infections; however, the key genes and mechanisms to adapt to the host microenvironment lack in-depth understanding. In this study, a total of 76 isolates (from 8 to 12 isolates per patient, spanning 128 to 188 days) were longitudinally collected from eight patients to investigate the within-host evolution of A. baumannii. A total of 70 within-host mutations were identified, 80% of which were nonsynonymous, indicating the important role of positive selection. Several evolutionary strategies of A. baumannii to increase its potential to adapt to the host microenvironment were identified, including hypermutation and recombination. Six genes were mutated in isolates from two or more patients, including two TonB-dependent receptor genes (bauA and BJAB07104_RS00665). In particular, the siderophore receptor gene bauA was mutated in multiple isolates from four patients with three MLST types, and all mutations were at amino acid 391 in ligand-binding sites. With 391T or 391A, BauA was more strongly bound to siderophores, which promoted the iron-absorption activity of A. baumannii at acidic or neutral pH, respectively. Through the A/T mutation at site 391 of BauA, A. baumannii displayed two reversible phases to adapt to distinct pH microenvironments. In conclusion, we demonstrated the comprehensive within-host evolutionary dynamics of A. baumannii, and discovered a key mutation of BauA site 391 as a genetic switch to adapt to different pH values, which may represent a model in the pathogen evolutionary adaption of the host microenvironment.

Pituitary adenylate cyclase-activating polypeptide receptor activation in the hypothalamus recruits unique signaling pathways involved in energy homeostasis.

Pituitary adenylate cyclase activating polypeptide (PACAP) exerts pleiotropic effects on ventromedial nuclei (VMN) of the hypothalamus and its control of feeding and energy expenditure through the type I PAC1 receptor (PAC1R). However, the endogenous role of PAC1Rs in the VMN and the downstream signaling responsible for PACAP's effects on energy balance are unknown. Numerous studies have revealed that PAC1Rs are coupled to both Gαs/adenylyl cyclase/protein kinase A (Gαs/AC/PKA) and Gαq/phospholipase C/protein kinase C (Gαq/PLC/PKC), while also undergoing trafficking following stimulation. To determine the endogenous role of PAC1Rs and downstream signaling that may explain PACAP's pleiotropic effects, we used RNA interference to knockdown VMN PAC1Rs and pharmacologically inhibited PKA, PKC, and PAC1R trafficking. Knocking down PAC1Rs increased meal sizes, reduced total number of meals, and induced body weight gain. Inhibition of either PKA or PKC alone in awake male Sprague-Dawley rats, attenuated PACAP's hypophagic and anorectic effects during the dark phase. However, PKA or PKC inhibition potentiated PACAP's thermogenic effects during the light phase. Analysis of locomotor activity revealed that PKA inhibition augmented PACAP's locomotor effects, whereas PKC inhibition had no effect. Finally, PACAP administration in the VMN induces surface PAC1R trafficking into the cytosol which was blocked by endocytosis inhibitors. Subsequently, inhibition of PAC1R trafficking into the cytosol attenuated PACAP-induced hypophagia. These results revealed that endogenous PAC1Rs uniquely engage PKA, PKC, and receptor trafficking to mediate PACAP's pleiotropic effects in VMN control of feeding and metabolism.NEW & NOTEWORTHY Endogenous PAC1 receptors, integral to VMN management of feeding behavior and body weight regulation, uniquely engage PKA, PKC, and receptor trafficking to mediate the hypothalamic ventromedial nuclei control of feeding and metabolism. PACAP appears to use different signaling mechanisms to regulate feeding behavior from its effects on metabolism.

Uterine tumor resembling an ovarian sex cord tumor: A case report and review of literature.

BACKGROUND: Endometrial stromal tumors originate from the endometrial stroma and account for < 2% of all uterine tumors. Uterine tumor resembling an ovarian sex cord tumor (UTROSCT) is a rare histological class of endometrial stromal and related tumors according to the latest World Health Organization classification of female genital tumors. Here, we report a case of UTROSCT in a 51-year-old woman. CASE SUMMARY: A 51-year-old woman had irregular menses for 6 mo. The patient visited a local hospital for vaginal bleeding. Pelvic computed tomography (CT) showed a mass in the pelvic cavity. Five days later, she came to our hospital for further diagnosis. The results of contrast-enhanced CT and pelvic ultrasound at our hospital suggested a malignant pelvic tumor. She then underwent total removal of the uterus with bilateral salpingectomy. Postoperative histological examination showed that the tumor cells had abundant cytoplasm, ovoid and spindle-shaped nuclei, fine chromatin, a high nucleoplasm ratio, and a lamellar distribution. The findings were consistent with UTROSCT, and the results of immunohistochemical analysis supported that diagnosis. The tumor was International Federation of Gynecology and Obstetrics stage IB. No adjuvant therapy was administered after radical surgery. The patient was followed up for 58 mo, and no recurrence was found. CONCLUSION: We report a case of UTROSCT with abnormal menstruation as a symptom, which is one of the most common symptoms. In patients with vaginal bleeding, ultrasonography can be used as a screening test because of its convenience, speed, and lack of radiation exposure. For patients with long-term tamoxifen use, routine monitoring of the endometrium is recommended. As UTROSCT may have low malignant potential, surgery remains the primary management strategy. Additionally, fertility preservation in patients of childbearing age is a vital consideration.

AtaT Improves the Stability of Pore-Forming Protein EspB by Acetylating Lysine 206 to Enhance Strain Virulence.

A novel type II toxin of toxin-antitoxin systems (TAs), Gcn5-related N-acetyltransferase (GNAT) family, was reported recently. GNAT toxins are mainly present in pathogenic species, but studies of their involvement in pathogenicity are rare. This study discovered that the GANT toxin AtaT in enterohemorrhagic Escherichia coli (EHEC) can significantly enhance strain pathogenicity. First, we detected the virulence of ΔataT and ΔataR in cell and animal models. In the absence of ataT, strains showed a lower adhesion number, and host cells presented weaker attaching and effacing lesions, inflammatory response, and pathological injury. Next, we screened the acetylation substrate of AtaT to understand the underlying mechanism. Results showed that E. coli pore-forming protein EspB, which acts as a translocon in type III secretion system (T3SS) in strains, can be acetylated specifically by AtaT. The acetylation of K206 in EspB increases protein stability and maintains the efficiency of effectors translocating into host cells to cause close adhesion and tissue damage.

Development and clinical application of deep learning model for lung nodules screening on CT images.

Lung cancer screening based on low-dose CT (LDCT) has now been widely applied because of its effectiveness and ease of performance. Radiologists who evaluate a large LDCT screening images face enormous challenges, including mechanical repetition and boring work, the easy omission of small nodules, lack of consistent criteria, etc. It requires an efficient method for helping radiologists improve nodule detection accuracy with efficiency and cost-effectiveness. Many novel deep neural network-based systems have demonstrated the potential for use in the proposed technique to detect lung nodules. However, the effectiveness of clinical practice has not been fully recognized or proven. Therefore, the aim of this study to develop and assess a deep learning (DL) algorithm in identifying pulmonary nodules (PNs) on LDCT and investigate the prevalence of the PNs in China. Radiologists and algorithm performance were assessed using the FROC score, ROC-AUC, and average time consumption. Agreement between the reference standard and the DL algorithm in detecting positive nodules was assessed per-study by Bland-Altman analysis. The Lung Nodule Analysis (LUNA) public database was used as the external test. The prevalence of NCPNs was investigated as well as other detailed information regarding the number of pulmonary nodules, their location, and characteristics, as interpreted by two radiologists.

Protective Immunity Elicited by VP1 Chimeric Antigens of Bacterial Ghosts against Hand-Foot-and-Mouth Disease Virus.

This study was designed to evaluate the immunogenicity and protective efficacy of two VP1 chimeric antigens of bacterial ghosts. Inoculation of the two VP1 chimeric antigens of bacterial ghosts into BALB/c mice markedly elicited humoral and mucosal immune responses. The specific antibodies induced by the chimeric ghosts protected mice not only against the virus that causes hand-foot-and-mouth disease but also against E. coli O157:H7 bacterial infection. In comparison with the negative control, immunization with the chimeric ghosts protected mice against two LD50 hand-foot-and-mouth disease viral infection. In addition, this specific immunity also protected the pups of pregnant mice immunized with the VP1 chimeric antigens of bacterial ghosts against 20 MLD E. coli O157:H7 infection. Taken together, the results of this study verify for the first time that the VP1 chimeric antigens of bacterial ghosts are target candidates for a new type of vaccine against hand-foot-and-mouth disease. Additionally, this vaccine strategy also elicited a stronger immune response against E. coli O157:H7.

Gentamicin Induced Microbiome Adaptations Associate With Increased BCAA Levels and Enhance Severity of Influenza Infection.

Involvement of gut microbiota in pulmonary disease by the gut-lung axis has been widely observed. However, the cross-talk messengers between respiratory mucosal immunity and gut microbiota are largely unknown. Using selective pharmacologic destruction of gut microenvironment mouse models, we found gut microbiota displayed significantly lower alpha diversity and relative abundance of bacteria in Gentamicin treated mice. Metagenomic studies revealed functional differences in gut bacteria in altering metabolic profiles in mice blood. Branched-chain amino acids (BCAAs) are the essential factors linked between gut and lung. During this process, selective destruction of gut microbiota by Gentamicin induced high levels of BCAAs, and the high levels of BCAAs impacted the lung immunity against influenza virus. In vivo, Gentamicin-treated mice or mice fed with high BCAAs diets displayed reduced survival. At the sites of infection, the number of CD11b+Ly6G+ cells decreased, and CD8+ T cells increased accompanied by exuberant expression of pro-inflammatory cytokines could result in tissue damage. CD11b+Ly6G+ cells transplantation conferred remarkable protection from influenza virus infections. In vitro, BCAAs promoted bone marrow-derived cells differentiation to dendritic cells. Taken together, these findings demonstrate that Gentamicin induced disruption of the gut microbiota leads to increased BCAA levels that suppress CD11b+Ly6c+ cell development in association with overactive CD8+ T responses which may contribute to enhanced severity of the viral infection.

VP1 of Enterovirus 71 Protects Mice Against Enterovirus 71 and Coxsackievirus B3 in Lethal Challenge Experiment.

Enterovirus and Coxsackievirus are the major viruses that cause hand, foot, and mouth disease (HFMD) outbreaks worldwide. Several studies have shown the potential of viral envelope protein 1 (VP1) on providing protective effects from viral strains of different genotypes. However, whether VP1 has the cross-protection in Enteroviruses or Coxsackievirus has not been studied in-depth. In this study, the vp1 gene of Enterovirus 71 (EV71) and Coxsackievirus B3 (CB3) was inserted into the vector pET22b (+) to form the respective expression plasmids pEVP1 or pCVP1, and then transformed into Escherichia coli strain BL21 (DE3). The recombinant EVP1 or CVP1 protein was overexpressed successfully and effectively purified to homogeneity. Then, we identified that EVP1 and CVP1 protein could generate effectively specific humoral immunity and cellular immunity in mice, what's more, we determined the cross-protection of VP1 between EV71 and CB3 in a murine model. The results showed that immunization with EVP1 could effectively induce specific IgG and secretory IgA against CVP1 and the sera from EVP1-immunized mice could neutralize CB3 with mean titers 1:440. In contrast, no measurable neutralizing antibodies to EV71 were detected in CVP1-immunized mice. Then, newborn BALB/C mice, whose mother was immunized with EVP1 or CVP1, were administered with different lethal doses of EV71 or CB3. The EVP1 immunized group showed a 90% protective efficacy for a CB3 dosage of 120 LD50, but the CVP1 immunized group showed no significantly different protective efficacy against 15 LD50 of EV71 compared with the BSA immunized group. Hence, EVP1 is a promising subunit vaccine candidate against Enterovirus 71 and Coxsackievirus B3 caused HFMD.

Embelin Restores Carbapenem Efficacy against NDM-1-Positive Pathogens.

The emergence and spread of carbapenemase in Gram-negative pathogens poses an enormous threat to global public health. New Delhi metallo-ß-lactamase-1 (NDM-1) inactivates nearly every class of ß-lactam antibiotics, including carbapenem; however, there is no clinically useful NDM-1 inhibitor. Embelin, an important ingredient in traditional herbal medicine, has anti-tumor effects. The current study is the first to discover and examine the inhibitory activity of embelin against ß-lactamase NDM-1. The IC50 of embelin was 2.1 ± 0.2 µM when tested against NDM-1 carbapenemase. Most regions of the embelin molecule were buried within NDM-1's active site, and the hydroxyl group of embelin interacted directly with the metal ion Zn2+, as shown by molecular dynamic simulation. Systematic analysis of the antibacterial activities of embelin and antibiotics demonstrated that embelin restored meropenem activity against a panel of NDM-positive pathogens, such as Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. Based on these results, embelin could be a promising carbapenem adjuvant candidate against NDM-1-producing bacterial strains.

Eukaryotic-like Kinase Expression in Enterohemorrhagic Escherichia coli: Potential for Enhancing Host Aggressive Inflammatory Response.

Enterohemorrhagic Escherichia coli (EHEC) or other attaching/effacing pathogen infections often cause host intestinal inflammation and pathology, which is thought to result in part from a host aggressive innate immune response. However, few effectors that play an important role in this pathology change have been reported. In this study, we discovered a previously unknown EHEC effector, Stk (putative serine/threonine kinase), which induces host aggressive inflammatory response during EHEC infection. Interestingly, homologous proteins of Stk are widely distributed in many pathogens. After translocating into the infected host cells, Stk efficiently phosphorylates IκBα and activates the NF-κB pathway. In EHEC-infected mice, Stk increases serum keratinocyte-derived cytokine (KC) levels and hyperactivates the inflammatory response of the colon, intensifying pathological injury of the colon. The virulence of Stk is based on its eukaryotic-like kinase activity. In conclusion, our data suggest that Stk is a new effector that induces the host aggressive inflammatory response during EHEC infection.

WITHDRAWN: LncRNA-HOTAIR Activates Tumor Cell Proliferation and Migration by Suppressing MiR-326 in Cervical Cancer.

Cervical cancer is the second most common malignant cancer in females. Recent findings indicate that LncRNA-HOTAIR played a crucial role in tumor progression. In our present study, we aimed to explore the regulating role of HOTAIR in the progression of cervical cancer. The expression of HOTAIR was found up-regulated in cervical cancer tissues and cell lines (Hela, CasKi, Me180 and C-33A) compared with the normal tissues and normal cervical cell line (Ect1/E6E7). To examine the function of HOTAIR, gene knockdown (KD) was performed using HOTAIR short hairpin RNAs (shRNAs). HOTAIR shRNA significantly suppressed cells proliferation and migration in Hela cells. Besides that, the targeting relationship between HOTAIR and miR-326 was firstly revealed by bioinformatics prediction. Simultaneously, suppressed expression of miR-326 was detected in tumor tissues and cell lines compared with the control. Then, suppressed expression level of miR-326 was elevated by adding miR-326 mimic in cervical cancer cells transfected with LncR-HOTAIR. Similarly, increased expression level of miR-326 was reduced by adding miR-326 inhibitor in cervical cancer cells transfected with HOTAIR shRNA. The targeting relationship between HOTAIR and miR-326 was further been confirmed through the luciferase report assay. Moreover, there existed a negative relationship between the expression of HOTAIR and miR-326. In addition, enhanced cell proliferation and migration abilities were suppressed by HOTAIR shRNA in cells transfected with miR-326 inhibitor. Finally, the in vivo experiment revealed that tumor growth and metastasis could also be inhibited by HOTAIR shRNA. Our present study elucidated the regulating role of HOTAIR/miR-326 axis in cervical cancer progression and provided a new potential therapeutic strategy for cervical cancer.

Molecular epidemiology of bla OXA-23 -producing carbapenem-resistant Acinetobacter baumannii in a single institution over a 65-month period in north China.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii poses a significant threat to hospitalized patients, as few therapeutic options remain. Thus, we investigated the molecular epidemiology and mechanism of resistance of carbapenem-resistant A.baumannii isolates in Beijing, China. METHODS: Carbapenem-resistant A.baumannii isolates (n = 101) obtained between June 2009 and November 2014 were used. Multilocus sequence typing (MLST) and PCR assays for class C and D ß-lactamase were performed on all isolates. S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization were performed to identify the resistance gene location. RESULTS: All 101 A.baumannii isolates were highly resistant to frequently used antimicrobials, and were considered multidrug resistant. A total of 12 sequence types (STs) were identified, including 10 reported STs and 2 novel STs. Eighty-seven isolates were classified to clonal complex 92 (CC92), among which ST191 and ST195 were the most common STs. The bla OXA-23 gene was positive in most (n = 95) of the A.baumannii isolates. Using S1-nuclease digestion PFGE and Southern blot hybridization, 3 patterns of plasmids carrying bla OXA-23 were confirmed. ST191 and ST195 (both harboring bla OXA-23 ) caused outbreaks during the study period, and this is the first report of outbreaks caused by ST191 and ST195 in north China. CONCLUSION: bla OXA-23 -producing A.baumannii ST191 and ST 195 isolates can disseminate in a hospital and are potential nosocomial outbreak strains. Surveillance of imipenem-resistant A.baumannii and antimicrobial stewardship should be strengthened.

The Dose-Response Relationship of bacillus Calmette-Guérin and Urothelial Carcinoma Cell Biology.

PURPOSE: Attenuated mycobacterium bacillus Calmette-Guérin is widely used as intravesical immunotherapy of nonmuscle invasive urothelial carcinoma. Currently there are limited data on the relationship between bacillus Calmette-Guérin dose intensity and tumor response. We evaluated the dose-response relationship of bacillus Calmette-Guérin to nonmuscle invasive bladder cancer in vitro using urothelial carcinoma cell lines and in vivo using an orthotopic mouse model. MATERIALS AND METHODS: Two human urothelial carcinoma cell lines were used to study the effect of bacillus Calmette-Guérin dose on the tumor cell response. Internalization, activation of signaling pathways, gene transactivation, cell viability, lactate dehydrogenase and HMGB1 release were study end points. An orthotopic tumor model was used to compare the effect of different doses on the antitumor efficacy of bacillus Calmette-Guérin. RESULTS: Bacillus Calmette-Guérin internalization by urothelial carcinoma cells increased as a function of time and dose with a plateau at higher doses and/or long exposure times. Intracellular signaling demonstrated a similar direct, dose dependent increase. Cytokine expression by urothelial carcinoma cells as a function of dose was variable. Some genes increased progressively but others showed a decrease at the highest dose. While nonviable cell number increased in proportion to dose, the number of cells undergoing necrotic cell death decreased at higher doses. A higher dose of bacillus Calmette-Guérin (1:200) showed a better antitumor effect than a standard dose (1:50) (p <0.01). CONCLUSIONS: Bacillus Calmette-Guérin dose has a direct impact on urothelial carcinoma cell biology. Increased dose intensity, particularly in nonresponders, may represent a strategy to increase bacillus Calmette-Guérin treatment efficacy.

H2O2 generation by bacillus Calmette-Guérin induces the cellular oxidative stress response required for bacillus Calmette-Guérin direct effects on urothelial carcinoma biology.

PURPOSE: Exposure of urothelial carcinoma cells to bacillus Calmette-Guérin affects cellular redox status and tumor cell biology but the mechanism(s) remain unclear. We examined free radical production by bacillus Calmette-Guérin in tumor cells in response to the bacillus using global profiling of reactive oxygen species/reactive nitrogen species. The relationship between free radical generation and downstream cellular events was evaluated. MATERIALS AND METHODS: Using fluorescent probes we performed global profiling of reactive oxygen species/reactive nitrogen species in heat killed and viable bacillus Calmette-Guérin, and in the 253J and T24 urothelial carcinoma cell lines after exposure to the bacillus. Inhibition of bacillus Calmette-Guérin internalization and H2O2 pharmacological scavenging were studied for their effect on cellular reactive oxygen species/reactive nitrogen species generation and various physiological end points. RESULTS: Viable bacillus Calmette-Guérin produced H2O2 and O2(-) but nitric oxide was not generated. Loss of viability decreased H2O2 production by 50% compared to viable bacillus. Bacillus Calmette-Guérin internalization was necessary for the bacillus to induce reactive oxygen species/reactive nitrogen species generation in urothelial carcinoma cells. Pharmacological H2O2 scavenging reversed reactive oxygen species/reactive nitrogen species mediated signaling in urothelial carcinoma cells. Bacillus Calmette-Guérin dependent alterations in tumor biology, including intracellular signaling, gene expression and cytotoxicity, depended on free radical generation. CONCLUSIONS: This study demonstrates the importance of free radical generation by bacillus Calmette-Guérin and intracellular generation of cellular oxidative stress on the urothelial carcinoma cell response to the bacillus. Manipulating the cellular oxidative stress induced by bacillus Calmette-Guérin represents a potential target to increase the efficacy of the bacillus.

Loss of bacillus Calmette-Guérin viability adversely affects the direct response of urothelial carcinoma cells to bacillus Calmette-Guérin exposure.

PURPOSE: The attenuated mycobacterium bacillus Calmette-Guérin is widely used as intravesical immunotherapy for nonmuscle invasive urothelial carcinoma. Previous studies demonstrated that in the laboratory and clinical settings bacillus Calmette-Guérin viability is a variable that correlates with antitumor efficacy. We evaluated how loss of viability impacted a number of molecular and phenotypic intermediate end points that characterize and/or contribute to the direct effect of bacillus Calmette-Guérin on urothelial carcinoma cells. MATERIALS AND METHODS: We studied the effect of loss of bacillus Calmette-Guérin viability on the tumor cell response to the treatment in 2 human urothelial carcinoma cell lines. The cellular response to bacillus Calmette-Guérin rendered nonviable by heat killing was compared to the response to viable bacillus. Response end points included the induction of oxidative stress, activation of intracellular signaling pathways, gene transactivation and phenotypic changes. RESULTS: Loss of viability resulted in a quantitative decrease in the tumor cell response relative to that of viable bacillus Calmette-Guérin for all measured end points. The decrease in response varied by cell line, ranging from 15% to 100% of the response to viable bacillus. While responses were quantitatively different, nonviable bacillus continued to induce responses that were qualitatively similar to those of bacillus Calmette-Guérin relative to that in untreated controls. CONCLUSIONS: Bacillus Calmette-Guérin viability is an important variable influencing the direct tumor cell response to the treatment. Although the magnitude of its effects are attenuated, heat killed bacillus Calmette-Guérin remains active for the induction of bacillus Calmette-Guérin responsive biological end points.

iNOS expression and NO production contribute to the direct effects of BCG on urothelial carcinoma cell biology.

OBJECTIVES: Evidence suggests that oxidative stress occurring as a consequence of inducible nitric oxide synthase/nitric oxide (iNOS/NO) contributes to the biologic effects of bacille Calmette-Guérin (BCG). Objective of this study is to examine iNOS expression, NO production, and the biologic effect of NO on established intermediate end points for the human urothelial carcinoma cell response to BCG. MATERIALS AND METHODS: Quantitative reverse transcriptase-polymerase chain reaction and real-time measurement of NO was used to assess iNOS and NO production, respectively, in 2 human urothelial carcinoma (UC) cell lines, in response to BCG. The effect of blocking NO production using the specific iNOS inhibitor 1400W was determined for multiple intermediate end points characterizing BCG's direct effects on tumor cell biology. Activation of nuclear factor kappa B and nuclear factor (erythroid-derived 2)-like 2 signaling pathways, transactivation of genes, including p21, CD54, IL6, IL8, CXCL1, CXCL3, CCL20, and cytotoxicity, as measured by vital dye exclusion, lactate dehydrogenase release, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were measured in response to BCG with and without iNOS inhibition. RESULTS: Exposure of UC cells to BCG significantly increased both iNOS expression and NO production. Inhibition of iNOS activity with 1400W significantly inhibited BCG's direct biologic effect on UC cells for all of the end points evaluated. CONCLUSIONS: iNOS expression, NO production, and the associated oxidative stress play a central role in the response of UC cells to BCG exposure. Manipulation of oxidative stress may afford an opportunity to enhance the antitumor effects of BCG.

Development of a fusion protein SNVP as substrate for assaying multi-serotype botulinum neurotoxins.

The SNARE super family has three core members, namely SNAP-25, VAMP-2, and syntaxin. SNAP-25 is cleaved by botulinum toxins (BoNTs)/A, /C, and /E, whereas VAMP-2 is the substrate for proteolytic BoNTs/B, /D, /F, and /G. In this study, we constructed a hybrid gene encoding the fusion protein SNVP that encompasses SNAP-25 residues Met1 to Gly206 and VAMP-2 residues Met1 to Lys94. The hybrid gene was cloned in a prokaryotic vector carrying an N-terminal pelB signal sequence and overexpressed in Escherichia coli BL21(DE3) Rosetta. To easily purify the protein, 6× His double-affinity tags were designed as the linker and C terminus of the fusion protein. SNVP was purified to homogeneity by affinity chromatography on a HisTrap FF column and determined to be more than 97% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal sequencing of the purified protein showed that signal peptide was successfully removed. The fusion protein SNVP contained the protease cleavage sites of all seven serotypes of BoNTs. SNVP was also proved to be recognized and cleaved by the endopeptidase of BoNTs (BoNT/A-LC, BoNT/B-LC, BoNT/E-LC, and BoNT/G-LC). The novel fusion substrate SNVP exhibited high biological activity under the optimal conditions, suggesting its potential use as a reagent for BoNT assay.