Preclinical safety assessment of the suction-assisted intradermal injection of the SARS-CoV-2 DNA vaccine candidate pGO-1002 in white rabbit.

pGO-1002, a non-viral DNA vaccine that expresses both spike and ORF3a antigens of SARS-CoV-2, is undergoing phase 1 and phase 2a clinical trials in Korea and the US. A preclinical repeated-dose toxicity study in New Zealand white rabbits in compliance with Good Laboratory Practice (GLP) was conducted to assess the potential toxicity, local tolerance, and immunogenicity of the vaccine and GeneDerm suction device. The dose rate was 1.2 mg/head pGO-1002, and this was administered intradermally to a group of animals (eight animals/sex/group) three times at 2-week intervals, followed by a 4-week recovery period. After each administration, suction was applied to the injection site using the GeneDerm device. Mortality, clinical signs, body weight, food consumption, skin irritation, ophthalmology, body temperature, urinalysis, and clinical pathology were also monitored. Gross observations and histopathological evaluation were performed. Overall, pGO-1002 administration-related changes were confined to minor damage or changes at the injection site, increased spleen weight and minimal increased cellularity in white pulp. All changes of injection site were considered local inflammatory changes or pharmacological actions due to the vaccine with the changes in spleen considered consistent with vaccine-induced immune activation. All findings showed reversibility during the 4-week recovery period. Animals vaccinated with pGO-1002, administered by intradermal injection and followed by application of suction with GeneDerm, developed humoral and cellular responses against the SARS-CoV-2 antigens consistent with prior studies in rats. Collectively, it was concluded that the pGO-1002 vaccine was safe and effective under these experimental conditions and these data supported future human study of the vaccine, now known as GLS-5310, for clinical trial use.

Accelerating Global Deletion of the Abnormal Toxicity Test for vaccines and biologicals. Planning common next steps. A workshop Report.

This online workshop Accelerating Global Deletion of the Abnormal Toxicity Test for vaccines and biologicals. Planning common next steps was organized on October 14th, 2021, by the Animal Free Safety Assessment Collaboration (AFSA), the Humane Society International (HSI), the European Federation of Pharmaceutical Industries and Associations (EFPIA), in collaboration with the International Alliance of Biological Standardization (IABS). The workshop saw a participation of over a hundred representatives from international organizations, pharmaceutical industries and associations, and regulatory authorities of 28 countries. Participants reported on country- and region-specific regulatory requirements and, where present, on the perspectives on the waiving and elimination of the Abnormal Toxicity Test. With AFSA, HSI, EFPIA and IABS representatives as facilitators, the participants also discussed specific country/global actions to further secure the deletion of ATT from all regulatory requirements worldwide.

Disruption of Membrane Integrity as a Molecular Initiating Event Determines the Toxicity of Polyhexamethylene Guanidine Phosphate Depending on the Routes of Exposure.

Polyhexamethylene guanidine phosphate (PHMG-P), a cationic biocide, is widely used in household products due to its strong bactericidal activity and low toxicity. However, it causes fatal lung damage when inhaled. In this study, we investigated why PHMG-P causes fatal lung injury when inhaled, and demonstrated that the disruption of membrane integrity through ionic interaction-a molecular initiating event of PHMG-P-determines toxicity. Mice were injected intravenously with 0.9 or 7.2 mg/kg PHMG-P (IV group), or instilled intratracheally with 0.9 mg/kg PHMG-P (ITI group); they were euthanatized at 4 h and on days 1 and 7 after treatment. Increased total BAL cell count and proinflammatory cytokine production, along with fibrotic changes in the lungs, were detected in the ITI group only. Levels of hepatic enzymes and hepatic serum amyloid A mRNA expression were markedly upregulated in the 7.2 mg/kg IV and ITI groups at 4 h or day 1 after treatment, but returned to baseline. No pathological findings were detected in the heart, liver, or kidneys. To simulate the IV injection, A549, THP-1, and HepG2 cells were treated with PHMG-P in cell culture media supplemented with different serum concentrations. Increased serum concentration was associated with an increase in cell viability. These results support the idea that direct contact between PHMG-P and cell membranes is necessary for PHMG-induced toxicity.

Preclinical safety assessment of a therapeutic human papillomavirus DNA vaccine combined with intravaginal interleukin-7 fused with hybrid Fc in female rats.

This study was conducted to establish the toxicological profile of combination treatment with therapeutic HPV DNA vaccines (GX-188E) and the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (IL-7hyFc). GX-188E was administered intramuscularly by electroporation with or without IL-7hyFc intravaginally once per 2 weeks for 8 weeks (five times) in female Sprague-Dawley rats. Because up-regulation of immune responses and migration of antigen-specific T cells in cervicoviginal tissue were predicted as therapeutic effects, we distinguished adverse effects from therapeutic effects based on the severity of the systemic immune response, reversibility of lymphoid tissue changes, target tissue damage, and off-target immune responses. We observed that the number of neutrophils was increased, and the number of lymphocytes was decreased in the blood. Further, myofiber degeneration, necrosis, fibroplasia, and cell infiltration were observed at the GX-188E administration site. These changes were fully or partially recovered over a 4-week period. Analysis of lymphocytes in spleen revealed that CD4+ T cells and total T cells decreased in rats treated with GX-188E in combination with a high dose of IL-7hyFc (1.25 mg/animal). However, these changes were not considered adverse because they were transient and may have been related to electroporation-mediated DNA delivery or the local migration of lymphocytes induced by IL-7. Therefore, the potential toxicity of the combination of GX-188E and IL-7hyFc treatment was comparable to that of GX-188E treatment alone, and the no observed adverse effect level for GX-188E with IL-7hyFc was considered as 320 µg/animal for GX-188E and 1.25 mg/animal for IL-7hyFc.

Inhibitory Effects of AF-343, a Mixture of Cassia tora L., Ulmus pumila L., and Taraxacum officinale, on Compound 48/80-Mediated Allergic Responses in RBL-2H3 Cells.

The purpose of this study was to determine the antiallergic effects of AF-343, a mixture of natural plant extracts from Cassia tora L., Ulmus pumila L., and Taraxacum officinale, on rat basophilic leukemia (RBL-2H3) cells. The inhibitory effects on cell degranulation, proinflammatory cytokine secretion, and reactive oxygen species (ROS) production were studied in compound 48/80-treated RBL-2H3 cells. The bioactive compounds in AF-343 were also identified by HPLC-UV. AF-343 was found to effectively suppress compound 48/80-induced b-hexosaminidase release, and interleukin (IL)-4 and tumor necrosis factor-a (TNF-a) production in RBL-2H3 cells. In addition, AF-343 exhibited DPPH free radical scavenging effects in vitro (half-maximal inhibitory concentration (IC50) = 105 µg/mL) and potently inhibited compound 48/80-induced cellular ROS generation in a 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay. Specifically, treatment with AF-343 exerted stronger antioxidant effects in vitro and antiallergic effects in cells than treatment with three single natural plant extracts. Furthermore, AF-343 was observed to contain bioactive compounds, including catechin, aurantio-obtusin, and chicoric acid, which have been reported to elicit antiallergic responses. This study reveals that AF-343 attenuates allergic responses via suppression of b-hexosaminidase release, IL-4 and TNF-a secretion, and ROS generation, perhaps through mechanisms related to catechin, aurantio-obtusin, and chicoric acid. The results indicate that AF-343 can be considered a treatment for various allergic diseases.

Safety assessment of freeze-dried powdered Tenebrio molitor larvae (yellow mealworm) as novel food source: Evaluation of 90-day toxicity in Sprague-Dawley rats.

Worldwide demand for novel food source has grown and edible insects are a promising food sources for humans. Tenebrio molitor, as known as yellow mealworm, has advantages of being rich in protein, and easy to raise as a novel food source. The objective of this study was to evaluate subchronic toxicity, including potential hypersensitivity, of freeze-dried powdered T. molitor larvae (fdTML) in male and female Sprague-Dawley rats. The fdTML was administered orally once daily at dose levels of 0, 300, 1000 and 3000 mg/kg/day for 90 days. A toxicological assessment was performed, which included mortality, clinical signs, body and organ weights, food consumption, ophthalmology, urinalysis, hematology, serum chemistry, gross findings, histopathologic examination and allergic reaction. There were no fdTML- related findings in clinical signs, urinalysis, hematology and serum chemistry, gross examination, histopathologic examination or allergic reaction. In conclusion, the No Observed Adverse Effect Level (NOAEL) for fdTML was determined to be in excess of 3000 mg/kg/day in both sexes of rats under the experimental conditions of this study.

Exposure to polyhexamethyleneguanidine phosphate in early life dampens pulmonary damage compared to adult mice.

Polyhexamethyleneguanidine phosphate (PHMG-P) is a biocide of guanidine family that can cause a fatal lung damage if exposed directly to the lungs. No reports exist regarding the toxicity of PHMG-P in neonatal animals. Therefore, this study aimed to determine PHMG-P toxicity in neonatal and 8-week-old mice after they were intranasally instilled with 1.5 mg/kg, 3 mg/kg, and 4.5 mg/kg PHMG-P. PHMG-P lung exposure resulted in more severe pulmonary toxicity in adult mice than in newborn mice. In the high-dose group of newborn mice, a minimal degree of inflammatory cell infiltration and fibrosis in the lung were detected, whereas more severe pathological lesions including granulomatous inflammation, fibrosis, and degeneration of the bronchiolar epithelium were observed in adult mice. At day 4, C-C motif chemokine ligand 2 (CCL2), a potent chemokine for monocytes, was upregulated but recovered to normal levels at day 15 in newborn mice. However, increased CCL2 and IL-6 levels were sustained at day 15 in adult mice. When comparing the differentially expressed genes of newborn and adult mice through RNA-seq analysis, there were expression changes in several genes associated with inflammation in neonates that were similar or different from those in adults. Although no significant lung damage occurred in newborns, growth inhibition was observed which was not reversed until the end of the experiment. Further research is needed to determine how growth inhibition from neonatal exposure to PHMG-P affects adolescent and young adult health.

Ferulate protects the epithelial barrier by maintaining tight junction protein expression and preventing apoptosis in tert-butyl hydroperoxide-induced Caco-2 cells.

Epithelial barrier function is determined by both transcellular and paracellular permeability, the latter of which is mainly influenced by tight junctions (TJs) and apoptotic leaks within the epithelium. We investigated the protective effects of ferulate on epithelial barrier integrity by examining permeability, TJ protein expression, and apoptosis in Caco-2 cells treated with tert-butyl hydroperoxide (t-BHP), a strong reactive species inducer. Caco-2 cells pretreated with ferulate (5 or 15 µM) were exposed to t-BHP (100 µM), and ferulate suppressed the t-BHP-mediated increases in reactive species and epithelial permeability in Caco-2 cells. Moreover, ferulate inhibited epithelial cell leakage induced by t-BHP, which was accompanied by decreased expression of the TJ proteins zonula occludens-1 and occludin. In addition, pretreatment with ferulate markedly protected cells against t-BHP-induced apoptosis, as evidenced by decreased nuclear condensation, cytochrome c release, and caspase-3 cleavage and an increased Bax/Bcl-2 ratio. These results suggest that ferulate protects the epithelial barrier of Caco-2 cells against oxidative stress, which results in increased epithelial permeability, decreased TJ protein expression, and increased apoptosis. The most significant finding of our study is the demonstration of protective, ferulate-mediated antioxidant effects on barrier integrity, with a particular focus on intracellular molecular mechanisms.

Implementation of Systematic Bioanalysis of Antibody-Drug Conjugates for Preclinical Pharmacokinetic Study of Ado-Trastuzumab Emtansine (T-DM1) in Rats.

Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies covalently bound to cytotoxic drugs by a linker. They are designed to selectively bind target antigens and present a promising cancer treatment without the debilitating side effects of conventional chemotherapies. Ado-trastuzumab emtansine (T-DM1) is an ADC that received US FDA approval for the treatment of HER2-positive breast cancer. The purpose of this study was to optimize methods for the quantification of T-DM1 in rats. We optimized four analytical methods: (1) an enzyme-linked immunosorbent assay (ELISA) to quantify the total trastuzumab levels in all drug-to-antibody ratios (DARs), including DAR 0; (2) an ELISA to quantify the conjugated trastuzumab levels in all DARs except DAR 0; (3) an LC-MS/MS analysis to quantify the levels of released DM1; and (4) a bridging ELISA to quantify the level of anti-drug antibodies (ADAs) of T-DM1. We analyzed serum and plasma samples from rats injected intravenously with T-DM1 (20 mg/kg, single dose) using these optimized methods. Based on these applied analytical methods, we evaluated the quantification, pharmacokinetics, and immunogenicity of T-DM1. This study establishes the systematic bioanalysis of ADCs with validated assays, including drug stability in matrix and ADA assay, for future investigation on the efficacy and safety of ADC development.

Immunomodulatory Effect of Hispolon on LPS-Induced RAW264.7 Cells and Mitogen/Alloantigen-Stimulated Spleen Lymphocytes of Mice.

Hispolon is a potent anticancer, anti-inflammatory, antioxidant, and antidiabetic agent isolated from Phellinus linteus, an oriental medicinal mushroom. However, the immunomodulatory mechanisms by which hispolon affects macrophages and lymphocytes remain poorly characterized. We investigated the immunomodulatory effects of hispolon on oxidative stress, inflammatory responses, and lymphocyte proliferation using lipopolysaccharide (LPS)-treated RAW264.7 macrophages or mitogen/alloantigen-treated mouse splenocytes. Hispolon inhibited LPS-induced reactive oxygen and nitrogen species (ROS/RNS) generation and decreased total sulfhydryl (SH) levels in a cell-free system and RAW264.7 cells. Hispolon exerted significant anti-inflammatory effects by inhibiting production of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) and activation of nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) in LPS-treated RAW264.7 cells. Hispolon also modulated NF-κB and STAT3 activation by suppressing the NF-κB p65 interaction with phospho-IκBα and the STAT3 interaction with JAK1, as determined via coimmunoprecipitation analysis. Additionally, hispolon significantly decreased lymphocyte proliferation, T cell responses and T helper type 1 (Th1)/type 2 (Th2) cytokines production in mitogen/alloantigen-treated splenocytes. We conclude that hispolon exerts immunomodulatory effects on LPS-treated macrophages or mitogen/alloantigen-treated splenocytes through antioxidant, anti-inflammatory, and antiproliferative activities. Thus, hispolon may be a therapeutic agent for treating immune-mediated inflammatory diseases.

The role of endothelin receptor A during myelination of developing oligodendrocytes.

Endothelin (ET)-1 and its receptors (ETA and ETB receptor) are present in the central nervous system. ET exerts biological effects on gliogenesis and glial cell functions. In order to define a possible mechanism of ETA receptor signaling, the distribution of the ETA receptor in developing oligodendrocytes and the effects of ET-1 on the myelination of oligodendrocytes were examined. ETA receptor immunoreactivity was confined to the perivascular elements of the blood vessels during early postnatal development. However later in development, ETA receptor immunoreactivity was no longer observed in the vessels but became localized to the myelinating oligodendrocytes of the primitive corpus callosum of the white matter, apart from the vessels. ET-1 induced myelin basic protein (MBP) in primary oligodendrocyte precursor cell culture though the ETA receptor and was blocked by an ETA receptor antagonist. In addition, ET-1 evoked the release of Ca(2+) which is a central regulator of oligodendrocyte differentiation. Our results provide a link between ET-1 and its ETA receptor and myelination during oligodendrocyte differentiation.

Transcriptomic Analysis of Polyhexamethyleneguanidine-Induced Lung Injury in Mice after a Long-Term Recovery.

Polyhexamethyleneguanidine phosphate (PHMG-P) is one of the causative agents of humidifier disinfectant-induced lung injury. Direct exposure of the lungs to PHMG-P causes interstitial pneumonia with fibrosis. Epidemiological studies showed that patients with humidifier disinfectant-associated lung injuries have suffered from restrictive lung function five years after the onset of the lung injuries. We investigated whether lung damage was sustained after repeated exposure to PHMG-P followed by a long-term recovery and evaluated the adverse effects of PHMG-P on mice lungs. Mice were intranasally instilled with 0.3 mg/kg PHMG-P six times at two weeks intervals, followed by a recovery period of 292 days. Histopathological examination of the lungs showed the infiltration of inflammatory cells, the accumulation of extracellular matrix in the lung parenchyma, proteinaceous substances in the alveoli and bronchiolar-alveolar hyperplasia. From RNA-seq, the gene expression levels associated with the inflammatory response, leukocyte chemotaxis and fibrosis were significantly upregulated, whereas genes associated with epithelial/endothelial cells development, angiogenesis and smooth muscle contraction were markedly decreased. These results imply that persistent inflammation and fibrotic changes caused by repeated exposure to PHMG-P led to the downregulation of muscle and vascular development and lung dysfunction. Most importantly, this pathological structural remodeling induced by PHMG-P was not reversed even after long-term recovery.

Assessment of acute and repeated pulmonary toxicities of oligo(2-(2-ethoxy)ethoxyethyl guanidium chloride in mice.

Oligo(2-(2-ethoxy)ethoxyethyl guanidinium chloride (PGH) and polyhexamethyleneguanidine phosphate (PHMG-P) are cationic biocides containing a guanidine group. Direct exposure of the lungs to PHMG-P is known to induce pulmonary inflammation and fibrotic changes. Few studies have assessed the pulmonary toxicity of PGH, another member of the guanidine family. In this study, we assessed the acute and repeated toxicity of PGH and PHMG-P to compare the pathological progression induced by both chemicals. PGH (1.5 mg/kg) or PHMG (0.6 mg/kg) was instilled intratracheally to mice once or three times every 4 days; subsequently, cytokine levels were quantified and a histopathological examination was performed. To verify the toxic mechanism of PGH, we quantified cell viability and cytokine production induced by PGH or PHMG-P in the presence or absence of anionic material in cells. Instillation of PGH and PHMG-P into the mouse lung increased cytokine production, immune cell infiltration, and pulmonary fibrotic changes. These pathological changes were exacerbated over time in the single- and the repeated-dose PHMG-P groups, but were resolved over time in the PGH groups. PGH or PHMG-P showed cytotoxic effects, IL-1ß secretion, and ROS production in a dose-dependent manner in human cell lines. However, the co-treatment of anionic materials with PGH or PHMG-P significantly reduced these toxic responses, which confirmed that the cation of PGH disrupted the plasma membrane via ionic interaction, as observed for PHMG-P. In addition, we suggest the disruption of plasma membrane as a molecular initiating event of cationic chemicals-induced adverse outcomes when exposed directly to the lungs.

Preclinical immunogenicity testing using anti-drug antibody analysis of GX-G3, Fc-fused recombinant human granulocyte colony-stimulating factor, in rat and monkey models.

Human granulocyte colony-stimulating factor (G-CSF, this study used Fc-fused recombinant G-CSF; GX-G3) is an important glycoprotein that stimulates the proliferation of granulocytes and white blood cells. Thus, G-CSF treatment has been considered as a crucial regimen to accelerate recovery from chemotherapy-induced neutropenia in cancer patients suffering from non-myeloid malignancy or acute myeloid leukemia. Despite the therapeutic advantages of G-CSF treatment, an assessment of its immunogenicity must be performed to determine whether the production of anti-G-CSF antibodies causes immune-related disorders. We optimized and validated analytical tools by adopting validation parameters for immunogenicity assessment. Using these validated tools, we analyzed serum samples from rats and monkeys injected subcutaneously with GX-G3 (1, 3 or 10 mg/kg once a week for 4 weeks followed by a 4-week recovery period) to determine immunogenicity response and toxicokinetic parameters with serum concentration of GX-G3. Several rats and monkeys were determined to be positive for anti-GX-G3 antibodies. Moreover, the immunogenicity response of GX-G3 was lower in monkeys than in rats, which was relevant to show less inhibition of toxicokinetic profiles in monkeys, at least 1 mg/kg administrated group, compared to rats. These results suggested the establishment and validation for analyzing anti-GX-G3 antibodies and measurement of serum levels of GX-G3 and anti-GX-G3 antibodies, which was related with toxicokinetic profiles. Taken together, this study provides immunogenicity assessment which is closely implicated with toxicokinetic study of GX-G3 in 4-week repeated administrated toxicological studies.

Frequency of and risk factors for reversion of QuantiFERON test in healthcare workers in an intermediate-tuberculosis-burden country.

OBJECTIVES: High-risk healthcare workers (HCWs) are often screened for latent tuberculosis infection (LTBI) using QuantiFERON tests (QFTs), with annual serial tests often showing reversion from positive to negative results. We assessed the frequency of and risk factors for reversion of QFTs in HCWs in an intermediate-tuberculosis burden country. METHODS: We enrolled high-risk HCWs at a tertiary-care hospital in South Korea, who were assessed by QFTs at least twice between 2017 and 2019. RESULTS: Of the 1870 HCWs screened, 1542 (82%) had persistent negative results, 229 (12%) had persistent positive results, 53 (3%) showed reversion, and 46 (2%) showed conversion from negative to positive. Multivariate analysis comparing the characteristics of the 229 HCWs with persistent positive results and the 53 who experienced reversion showed that older age (adjusted odds ratio (aOR): 0.96; 95% confidence interval (CI): 0.92-0.99), male sex (aOR: 0.29; 95% CI: 0.11-0.78) and high (≥0.70 IU/mL) baseline QFT results (aOR: 0.15; 95% CI: 0.07-0.31) were inversely associated with reversion. Using an ROC curve-derived cut-off of <0.738 IU/mL, the area under the curve was 0.79. Of 53 HCWs with reversion, 36 (78%) had below 0.738 IU/mL of baseline QFT, while 181 (79%) of 229 HCWs without reversion had above 0.738 IU/mL of baseline QFT. CONCLUSION: Reversion during serial testing is unlikely in HCWs who are male, older in age, and have higher baseline QFT results. Serial testing without LTBI treatment may be indicated in HCWs who are female, younger and, especially, have lower QFT results.

Bromodomain protein Brd4 regulates human immunodeficiency virus transcription through phosphorylation of CDK9 at threonine 29.

Positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 (CDK9) and cyclin T, is a global transcription factor for eukaryotic gene expression, as well as a key factor for human immunodeficiency virus (HIV) transcription elongation. P-TEFb phosphorylates the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II), facilitating the transition from nonprocessive to processive transcription elongation. Recently, the bromodomain protein Brd4 has been shown to interact with the low-molecular-weight, active P-TEFb complex and recruit P-TEFb to the HIV type 1 long terminal repeat (LTR) promoter. However, the subsequent events through which Brd4 regulates CDK9 kinase activity and RNAP II-dependent transcription are not clearly understood. Here we provide evidence that Brd4 regulates P-TEFb kinase activity by inducing a negative pathway. Moreover, by analyzing stepwise initiation and elongation complexes, we demonstrate that P-TEFb activity is regulated in the transcription complex. Brd4 induces phosphorylation of CDK9 at threonine 29 (T29) in the HIV transcription initiation complex, inhibiting CDK9 kinase activity. P-TEFb inhibition is transient, as Brd4 is released from the transcription complex between positions +14 and +36. Removal of the phosphate group at T29 by an incoming phosphatase released P-TEFb activity, resulting in increased RNAP II CTD phosphorylation and transcription. Finally, we present chromatin immunoprecipitation studies showing that CDK9 with phosphorylated T29 is associated with the HIV promoter region in the integrated and transcriptionally silent HIV genome.

Inhibition of methyltransferases results in induction of g2/m checkpoint and programmed cell death in human T-lymphotropic virus type 1-transformed cells.

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia. The HTLV-1-encoded protein Tax transactivates the viral long terminal repeat and plays a critical role in virus replication and transformation. Previous work from our laboratory demonstrated that coactivator-associated arginine methytransferase 1, a protein arginine methytransferase, was important for Tax-mediated transactivation. To further investigate the role of methyltransferases in viral transcription, we utilized adenosine-2,3-dialdehyde (AdOx), an adenosine analog and S-adenosylmethionine-dependent methyltransferase inhibitor. The addition of AdOx decreased Tax transactivation in C81, Hut102, and MT-2 cells. Unexpectedly, we found that AdOx potently inhibited the growth of HTLV-1-transformed cells. Further investigation revealed that AdOx inhibited the Tax-activated NF-kappaB pathway, resulting in reactivation of p53 and induction of p53 target genes. Analysis of the NF-kappaB pathway demonstrated that AdOx treatment resulted in degradation of the IkappaB kinase complex and inhibition of NF-kappaB through stabilization of the NF-kappaB inhibitor IkappaBalpha. Our data further demonstrated that AdOx induced G(2)/M cell cycle arrest and cell death in HTLV-1-transformed but not control lymphocytes. These studies demonstrate that protein methylation plays an important role in NF-kappaB activation and survival of HTLV-1-transformed cells.

Small-molecule inhibitor which reactivates p53 in human T-cell leukemia virus type 1-transformed cells.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of the aggressive and fatal disease adult T-cell leukemia. Previous studies have demonstrated that the HTLV-1-encoded Tax protein inhibits the function of tumor suppressor p53 through a Tax-induced NF-kappaB pathway. Given these attributes, we were interested in the activity of small-molecule inhibitor 9-aminoacridine (9AA), an anticancer drug that targets two important stress response pathways, NF-kappaB and p53. In the present study, we have examined the effects of 9AA on HTLV-1-transformed cells. Treatment of HTLV-1-transformed cells with 9AA resulted in a dramatic decrease in cell viability. Consistent with these results, we observed an increase in the percentage of cells in sub-G(1) and an increase in the number of cells positive by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay following treatment of HTLV-1-transformed cells with 9AA. In each assay, HTLV-1-transformed cells C8166, Hut102, and MT2 were more sensitive to treatment with 9AA than control CEM and peripheral blood mononuclear cells. Analyzing p53 function, we demonstrate that treatment of HTLV-1-transformed cells with 9AA resulted in an increase in p53 protein and activation of p53 transcription activity. Of significance, 9AA-induced cell death could be blocked by introduction of a p53 small interfering RNA, linking p53 activity and cell death. These results suggest that Tax-repressed p53 function in HTLV-1-transformed cells is "druggable" and can be restored by treatment with 9AA. The fact that 9AA induces p53 and inhibits NF-kappaB suggests a promising strategy for the treatment of HTLV-1-transformed cells.

Lysophosphatidylcholine enhances oxidative stress via the 5-lipoxygenase pathway in rat aorta during aging.

Lysophosphatidylcholine (LPC) is a lysolipid, acting as a potent cellular mediator of various biological processes. The purpose of this study was to define the role of LPC as a possible causative factor of disrupted redox balance in aged aorta from rats. In this study, we found elevated serum LPC levels in 24-month-old rats that were correlated with the age-related increase in cytosolic phospholipase A(2) (PLA(2)) activity. We also found that aortas from old rats showed increased 5-lipoxygenase (5-LO) activity. With the LPC-treated endothelial cells (YPEN-1 cells), we observed a rapid generation of reactive species, leading to enhanced oxidative stress. Our further investigations using specific 5-LO inhibitors led to the identification of a 5-LO pathway as the reactive species production source in the LPC-treated cells. Additional validation of this 5-LO pathway was made by the detection of increased leukotriene B4 generation in the LPC-treated cells. These in vitro data supported findings of increased expression and activation of aortic 5-LO in old rats by LPC. Together, our data strongly suggested that LPC caused the enhancement of oxidative stress in aged aorta through reactive species generation by an activated 5-LO pathway. LPC may well be an important contributor to age-related oxidative stress in aging aorta.

Polyhexamethyleneguanidine phosphate induces cytotoxicity through disruption of membrane integrity.

Polyhexamethyleneguanidine phosphate (PHMG-P) is a polymeric biocide with a guanidine group. It has multiple positive charges in physiological conditions due to nitrogen atom in the guanidine and this cationic property contributes antimicrobial effect by disrupting cell membranes. To determine whether the cationic nature of PHMG-P results in cytotoxicity in human cell lines, anionic compounds were treated with PHMG-P. The cytotoxic effect was evaluated with ROS production and HMGB1 release into media. To verify the protection effect of anion against PHMG-P-induced cell death in vivo, a zebrafish assay was adopted. In addition, membrane disruption by PHMG-P was evaluated using fluorescein diacetate and propidium iodine staining. As a result, anionic substances such as DNA and poly-l-glutamic acids, decreased PHMG-P induced cell death in a dose-dependent manner. While HMGB1 and ROS production increased with PHMG-P concentration, the addition of anionic compounds with PHMG-P reduced the ROS production and HMGB1 release. The mortality of the zebrafish increased with PHMG-P concentration and co-treatment of anionic compounds with PHMG-P decreased mortality in a dose-dependent manner. In addition, FDA and PI staining confirmed that PHMG-P disrupts plasma membrane. Taken together, a cationic property is considered to be one of the main causes of PHMG-P-induced mammalian cell toxicity.