Enhanced anti-melanoma efficacy through a combination of the armed oncolytic adenovirus ZD55-IL-24 and immune checkpoint blockade in B16-bearing immunocompetent mouse model.

Although the recent treatment in melanoma through the use of anti-PD-1 immunotherapy is successful, the efficacy of this approach remains to be improved. Here, we explore the feasibility of combination strategy with the armed oncolytic adenovirus ZD55-IL-24 and PD-1 blockade. We find that combination therapy with localized ZD55-IL-24 and systemic PD-1 blockade leads to synergistic inhibition of both local and distant established tumors in B16-bearing immunocompetent mouse model. Our further mechanism investigation reveals that synergistic therapeutic effect is associated with marked promotion of tumor immune infiltration and recognition in both local and distant tumors as well as spleens. PD-1 blockade has no obvious effect on promotion of tumor immune infiltration and recognition. Localized therapy with ZD55-IL-24, however, can help PD-1 blockade to overcome the limitation of relatively low tumor immune infiltration and recognition. This study provides a rationale for investigation of such combination therapy in the clinic.

Investigation and influencing factors about well-being level of elderly chronic patients during COVID-19 postpandemic period in Beijing.

ABSTRACT: The Corona Virus Disease 2019 (COVID-19) pandemic has huge impacts on the world, including human health and economic decline. The COVID-19 has severe infectivity, especially the elderly with chronic diseases will cause various complications after infection and accelerate the disease process. In addition, COVID-19 will also affect their mental health. Therefore, the mental health of elderly patients with chronic diseases cannot be ignored. The aim of this study was to investigate the well-being level of elderly people with chronic disease during COVID-19 postpandemic period in Beijing and analysis related influencing factors, so as to provide a basis for improving the well-being level of elderly chronic patients during the postpandemic period.Elderly patients with chronic diseases who met the inclusion criteria in 5 different administrative regions in Beijing were selected to carry out a questionnaire survey. The contents of the questionnaire included general data, the Memorial University of Newfoundland Happiness scale and the awareness situation of the COVID-19 pandemic. A total of 500 questionnaires were distributed by WeChat and 486 valid questionnaires were collected. The t test and one-way analysis of variance were used to compare Memorial University of Newfoundland Happiness scores between 2 or more groups, multiple linear regression analysis was used to conduct multiple factor analysis to explore the related factors about well-being level of elderly chronic patients.A total of 109 cases (22.43%) were evaluated high well-being level, 319 cases (65.64%) were evaluated moderate well-being level and 58 cases (11.93%) were evaluated low well-being according to the Memorial University of Newfoundland Happiness (MUNSH) scores rating. The multiple linear regression indicated that the education level, number of chronic diseases, medical expenses, frequency of children's visits, taking care of grandchildren or not, and group activity frequency significantly affected the well-being of patients with chronic diseases during COVID-19 postpandemic period in Beijing (P < .05).Most elderly patients with chronic diseases had moderate or above sense of well-being during postpandemic period, but we should still pay attention to the mental health of those elderly chronic patients with low education level, much comorbidity, more medical expenses, less visits by children, not take care of grandchildren and never participate in group activities.

HCCS1-armed, quadruple-regulated oncolytic adenovirus specific for liver cancer as a cancer targeting gene-viro-therapy strategy.

BACKGROUND: In previously published studies, oncolytic adenovirus-mediated gene therapy has produced good results in targeting cancer cells. However, safety and efficacy, the two most important aspects in cancer therapy, remain serious challenges. The specific expression or deletion of replication related genes in an adenovirus has been frequently utilized to regulate the cancer cell specificity of a virus. Accordingly, in this study, we deleted 24 bp in E1A (bp924-bp947) and the entirety of E1B, including those genes encoding E1B 55kDa and E1B19kDa. We used the survivin promoter (SP) to control E1A in order to construct a new adenovirus vector named Ad.SP.E1A(Δ24).ΔE1B (briefly Ad.SPDD). HCCS1 (hepatocellular carcinoma suppressor 1) is a novel tumor suppressor gene that is able to specifically induce apoptosis in cancer cells. The expression cassette AFP-HCCS1-WPRE-SV40 was inserted into Ad.SPDD to form Ad.SPDD-HCCS1, enabling us to improve the safety and efficacy of oncolytic-mediated gene therapy for liver cancer. RESULTS: Ad.SPDD showed a decreased viral yield and less toxicity in normal cells but enhanced toxicity in liver cancer cells, compared with the cancer-specific adenovirus ZD55 (E1B55K deletion). Ad.SPDD-HCCS1 exhibited a potent anti-liver-cancer ability and decreased toxicity in vitro. Ad.SPDD-HCCS1 also showed a measurable capacity to inhibit Huh-7 xenograft tumor growth on nude mice. The underlying mechanism of Ad.SPDD-HCCS1-induced liver cancer cell death was found to be via the mitochondrial apoptosis pathway. CONCLUSIONS: These results demonstrate that Ad.SPDD-HCCS1 was able to elicit reduced toxicity and enhanced efficacy both in vitro and in vivo compared to a previously constructed oncolytic adenovirus. Ad.SPDD-HCCS1 could be a promising candidate for liver cancer therapy.

Optimization of the Administration Strategy for the Armed Oncolytic Adenovirus ZD55-IL-24 in Both Immunocompromised and Immunocompetent Mouse Models.

ZD55-IL-24 is an armed oncolytic adenovirus similar but superior to ONYX-015. Virotherapeutic strategies using ZD55-IL-24 have been demonstrated to be effective against several cancer types. However, it is unclear whether the traditional administration strategy is able to exert the maximal antitumor efficacy of ZD55-IL-24. In this study, we sought to optimize the administration strategy of ZD55-IL-24 in both A375-bearing immunocompromised mouse model and B16-bearing immunocompetent mouse model. Although the underlying antitumor mechanisms are quite different, the obtained results are similar in these two mouse tumor models. We find that the antitumor efficacy of ZD55-IL-24 increases as injection times increase in both of these two models. However, no obvious increase of efficacy is observed as the dose of each injection increases. Our further investigation reveals that the administration strategy of sustained ZD55-IL-24 therapy can achieve a better therapeutic effect than the traditional administration strategy of short-term ZD55-IL-24 therapy. Furthermore, there is no need to inject every day; every 2 or 3 days of injection achieves an equivalent therapeutic efficacy. Finally, we find that the sustained rather than the traditional short-term ZD55-IL-24 therapy can synergize with anti-PD-1 therapy to reject tumors in B16-bearing immunocompetent mouse model. These findings suggest that the past administration strategy of ZD55-IL-24 is in fact suboptimal and the antitumor efficacy can be further enhanced through administration strategy optimization. This study might shed some light on the development of clinically applicable administration regimens for ZD55-IL-24 therapy.

Synthesis and in vitro antibacterial activity of 7-(4-alkoxyimino-3-methyl-3-methylaminopiperidin-1-yl)quinolones.

To explore new agents of quinolone derivatives with high antibacterial activity, 7-(4-alkoxyimino-3-methyl-3-methylaminopiperidin-1-yl)quinolones were designed and synthesized, and their activity against gram-positive and gram-negative strains was tested in vitro. Sixteen target compounds were obtained. Their structures were established by 1H NMR, HRMS and X-ray crystallographic analysis. Compounds 14k and 14m-14o show good antibacterial activity against the tested five gram-positive strains and five gram-negative strains (MIC: 0.25-16 micromg x mL(-1)), of which the most active compound 14o is 8-fold more potent than levofloxacin against S. pneumoniae (MIC: 4 microg x mL(-1)), and comparable to levofloxacin against S. aureus, S. epidermidis, E. faecalis and E. coli (MIC: 0.25-1 microg x mL(-1)), but generally less potent than gemifloxacin.

The armed oncolytic adenovirus ZD55-IL-24 eradicates melanoma by turning the tumor cells from the self-state into the nonself-state besides direct killing.

ZD55-IL-24 is similar but superior to the oncolytic adenovirus ONYX-015, yet the exact mechanism underlying the observed therapeutic effect is still not well understood. Here we sought to elucidate the underlying antitumor mechanism of ZD55-IL-24 in both immunocompetent and immunocompromised mouse model. We find that ZD55-IL-24 eradicates established melanoma in B16-bearing immunocompetent mouse model not through the classic direct killing pathway, but mainly through the indirect pathway of inducing systemic antitumor immunity. Inconsistent with the current prevailing view, our further results suggest that ZD55-IL-24 can induce antitumor immunity in B16-bearing immunocompetent mouse model in fact not due to its ability to lyse tumor cells and release the essential elements, such as tumor-associated antigens (TAAs), but due to its ability to put a "nonself" label in tumor cells and then turn the tumor cells from the "self" state into the "nonself" state without tumor cell death. The observed anti-melanoma efficacy of ZD55-IL-24 in B16-bearing immunocompetent mouse model was practically caused only by the viral vector. In addition, we also notice that ZD55-IL-24 can inhibit tumor growth in B16-bearing immunocompetent mouse model through inhibiting angiogenesis, despite it plays only a minor role. In contrast to B16-bearing immunocompetent mouse model, ZD55-IL-24 eliminates established melanoma in A375-bearing immunocompromised mouse model mainly through the classic direct killing pathway, but not through the antitumor immunity pathway and anti-angiogenesis pathway. These findings let us know ZD55-IL-24 more comprehensive and profound, and provide a sounder theoretical foundation for its future modification and drug development.

[Synthesis and antibacterial activity of 7-(3-amino-4-alkoxyimino-1 -piperidyl) -quinolones].

To explore new agents of quinolone derivatives with high activity against Gram-positive and Gram-negative microorganisms, 7-(3-amino-4-alkoxyimino-1-piperidyl) quinolones were designed and synthesized, and their activity against Gram-positive and Gram- negative microorganisms were tested in vivo and in vitro. Twenty one target compounds were obtained. Their structures were established by 1H NMR, HRMS and X-ray crystallographic analysis. The target compounds possess different antimicrobial activities against both Gram-negative and Gram-positive microorganisms. Compounds 14a and 14m have broad spectral antibacterial activities. They show better antibacterial activities against 12 strains Gram-positive bacteria than three references. In particular, their activities against S. aureus and S. epidermidis (including MRSA and MRSE) were 4 - 16 times than that of gemifloxacin and balofloxacin, and 8 - 64 times than that of levofloxacin. The MIC values to S. aureus strains of compounds 14a and 14m were 0.25 - 1 mg x L(-1) and 0.125 - 1 mg x L(-1), to S. epidermidis strains were 0.5 - 4 mg x L(-1) and 1 - 8 mg x L(-1) respectively. The in vivo results showed that they have as good internal protection as gemifloxacin and moxifloxacin against systemic infection model in mice (P > 0.05).

Veratric Acid Inhibits LPS-Induced IL-6 and IL-8 Production in Human Gingival Fibroblasts.

Veratric acid, one of the major benzoic acid isolated from vegetables and fruits, has been reported to have anti-inflammatory activity. The purpose of this study was to investigate the anti-inflammatory effects of veratric acid on lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (HGFs). HGFs were pretreated with veratric acid 1 h before LPS stimulation. The productions of IL-6 and IL-8 were detected by ELISA. The expression of iNOS, COX-2, PI3K, AKT, and NF-κB were detected by western blotting. The results showed that veratric acid inhibited LPS-induced IL-6 and IL-8 production, as well as iNOS and COX-2 expression. Veratric acid also inhibited LPS-induced NF-κB activation. In addition, veratric acid was found to suppress LPS-induced PI3K and AKT phosphorylation. In conclusion, the anti-inflammatory mechanism of veratric acid is due to its ability to inhibit PI3K/Akt/NF-κB signaling pathway.

Characteristics of participants' and caregivers' influence on non-response in a cross-sectional study of dementia in an older population.

OBJECTIVE: The issue of non-response in dementia epidemiological studies, which may result in the underestimation of the prevalence of dementia, has attracted little attention. We aimed to explore the causes and related factors of non-response in a dementia survey among Chinese veterans. METHODS: A two-phase, cross-sectional study investigated the prevalence of dementia and mild cognitive impairment in Chinese veterans aged ≥ 60 years. We collected the socio-demographic data and prior medical history, evaluated the health status of veterans and their caregivers, assessed the cognitive status of veterans, and evaluated the care burden of caregivers by Caregiver Burden Inventory (CBI). RESULTS: Of 9676 eligible participants, 525 (5.4%) veterans in phase 1 and 1706 (35.0%) veterans among 4875 veterans in phase 2 did not respond. Illness, hospitalization and death accounted for 63.0% and 75.5% non-response in phases 1 and 2, respectively. Non-participation in social activities, self-perceived poor health status, worsened health changes, self-reported need for life care, and history of hearing loss or glaucoma independently predicted non-response in phase 1 or 2. The heavy care burden, suggested by the higher CBI scores and self-reported health deterioration of the primary caregivers, predicted non-response in phase 1 or 2. CONCLUSIONS: The negative factors from both the participants and their caregivers independently predicted the non-response in the dementia study in an older population. Preventative strategies from the perspectives of the participants and caregivers should be developed to improve the response rates in both phases in a cross-sectional study.

An armed oncolytic adenovirus system, ZD55-gene, demonstrating potent antitumoral efficacy.

ONYX-015 is an attractive therapeutic adenovirus for cancer because it can selectively replicate in tumor cells and kill them. To date, clinical trials of this adenovirus have demonstrated marked safety but not potent enough when it was used alone. In this paper, we put forward a novel concept of Gene-ViroTherapy strategy and in this way, we constructed an armed therapeutic oncolytic adenovirus system, ZD55-gene, which is not only deleted of E1B 55-kD gene similar to ONYX-015, but also armed with foreign antitumor gene. ZD55-gene exhibited similar cytopathic effects and replication kinetics to that of ONYX-015 in vitro. Importantly, the carried gene is expressed and the expression level can increase with the replication of virus. Consequently, a significant antitumoral efficacy was observed when ZD55-CD/5-FU was used as an example in nude mice with subcutaneous human SW620 colon cancer. Our data demonstrated that ZD55-gene, which utilizing the Gene-ViroTherapy strategy, is more efficacious than each individual component in vivo.

Cancer-specific killing by the CD suicide gene using the human telomerase reverse transcriptase promoter.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase, is transcriptionally upregulated in more than 90% of tumor cells. It may be used as a tool for driving a gene to kill tumors specifically. To test this idea, luciferase reporter gene was used and the results showed that hTERT promoter could restrict the gene expression in the telomerase-positive tumor cells. A tumor-specific expression plasmid phTERT-CD was constructed, in which the E. coli cytosine deaminase (CD) gene was controlled by the hTERT promoter. A colorectal cancer cell line (LoVo) and a normal amnion cell line (WISH) were transfected by this plasmid. It was shown that the expression of the CD gene increased the sensitivity of LoVo cells to the prodrug, 5-fluorocytosine (5FC), over 800-fold, while the sensitivity of WISH cells to 5FC was increased only 6-fold. Mixed cell experiments showed a strong "bystander effect" on CD-negative cells. Furthermore, a significant anti-tumor effect of the phTERT-CD/5FC system was observed in nude mice bearing mammalian carcinoma induced by s.c. inoculation of LoVo cells when the mice were given 250 mg/kg 5FC twice a day for 10 consecutive days. These results indicated that hTERT promoter could target the suicidal effect of CD gene to tumor cells, and therefore, may be a novel and promising targeting approach to the treatment of cancer.

Interleukin-2 gene has superior antinociceptive effects when delivered intrathecally.

The antinociceptive effect of interleukin-2 gene on rat carrageenan-induced pain was explored using different delivery methods. Intrathecal (i.t.) or plantar s.c. delivery of plasmid harbouring the interleukin-2 gene produced a marked antinociceptive effect, which was maintained up to 6 days; the administration of recombinant human interleukin-2 only had a transitory effect. The antinociceptive effect lasted longer and was more potent when the interleukin-2 gene was administered i.t. than when delivered s.c. The effect of the interleukin-2 gene was related to its protein expression, was dose dependent, and could be potentiated by liposome. The results suggest that the interleukin-2 gene has a good prospect for clinical use.

A Novel Type of Apoptosis Induced by Cadmium in BA/F3beta Cells.

Apoptosis is usually accompanied by DNA fragmentation and up-regulation of reactive oxygen species, and it can be inhibited by overexpression of Bcl-2. Here, cadmium was found to induce apoptosis in BA/F3beta cells. MTT assay, Hochest 33258 staining, and transmission electron microscopy analysis were used to detect the apoptosis, however, neither DNA fragmentation nor up-regulation of reactive oxygen species were observed in this type of apoptosis. Furthermore, Bcl-2 overexpression had no effect on this type of apoptosis. In conclusion, these data suggested that cadmium induced a novel type of apoptosis in BA/F3beta cells.

Expression of Brain-derived Neurotrophic Factor (BDNF) Gene in Rat Myoblast Cells.

Brain-derived neurotrophic factor (BDNF) gene was cloned into retrovirous vector plasmid pLNCX to form the pLNC/BDNF. After packaging in PA317 cell line, the infectious particles were used to infect rat myoblast cell line L-6TG. After selection with G418, the L-6TG/BDNF cells were collected. Results from Southern blot show that bdnf gene has successfully integrated into the L-6TG genomic DNA. The expression of bdnf gene has been proved by Northern blot and Dot blot. The content of BDNF in the culture medium of L-6TG/BDNF was about 25 ng.10(-6) cells per 24 h per ml.

Use of Carcinoembryonic Antigen Gene Promoter in Colorectal Carcinoma-specific Suicidal Gene Therapy.

The possibility of tumor-specific suicidal gene therapy of colorectalcarcinoma was investigated using carcinoembryonic antigen (CEA) -positive human colorectal carcinoma cell line LoVo and CEA-negative HeLa cell line as a model. After confirming cellular specificity of cea promoter by CAT assay, eukaryotic expression plasmid pCEACD was constructed in which the expresstion of E. coli cytosine deaminase (CD) gene was under the control of cea promoter. The expression of CD gene increased the sensitivity of LoVo cells to 5-fluorocytosine (5FC) by 750 fold, while the sensitivity of HeLa cells to 5FC was increased by only 7.5 fold. These results suggest that the expression of CD gene driven by cea promoter specifically killed CEA-positive colorectal carcinoma cells. Transmission electron microscopy and DNA fragmentation assay demonstrate that CD/5FC system induced apoptosis in LoVo cells.

Expression of Cytosine Deaminase Gene in Human Colon Carcinoma Cells by Recombinant Retroviral Vector.

The recombinant retroviral vector pLCDSN containing E. coli cytosine deaminase gene was constructed. After packaging with PA317 cell line, the infectious particles were used to infect human colon carcinoma cell line LoVo. A single clone harbouring EC-CD gene was picked after G418 selection. There was no significant difference in cell growth curve or morphology between the LoVo/LCDSN and LoVo cells. Both of them were very sensitive to 5-FU in vitro (IC(50), approximately 0.5 &mgr;mol/L). However, the expression of the CD gene did increase the sensitivity of these cells to the nontoxic prodrug, 5-FC, decreasing the IC(50) for 5-FC from 22 000 &mgr;mol/L for parental LoVo cells to 13 &mgr;mol/L for LoVo/LCDSN cells. Obvious by side effect was also observed. When cells transduced with CD gene were mixed with wild type cells at a ratio of 30:70, above 80% of the cancer cells could be killed after treatment with a nontoxic concentration of 5-FC.

Interaction between Jak3 and Nucleosome Assembly Protein 1.

Tyrosine kinase Jak3 plays a critical role in the interleukin 2 IL-2 signaling because it not only participates the Jak-Stat pathway, but also interacts with unidentified signal transducers and regulates expression of some oncogenes such as c-fos and c-myc. Abundant evidence demonstrated that phosphorylated tyrosine was necessary for the interaction between two proteins. Therefore, in order to clarify the role of Jak3 in IL-2 signal transduction, the tyrosine-phosphorylation-involved yeast two-hybrid system was constructed and the N-terminal region JH3-JH7 of Jak3 was used as a bait to screen a peripheral blood cDNA library. About 50 double-positive colonies were obtained. Sequence analysis indicated that one of them was from nucleosome assembly protein 1 gene (Nap1), and encoded a protein of 392 amino acid residues. Two-hybrid system results demonstrated that interaction between Jak3 and Nap1 depended on the level of tyrosine phosphorylation. Furthermore, immunoprecipitation and Western blot experiments confirmed that Jak3 really interacted with Nap1 in murine pro-B lymphocyte BAF/BO3beta cells.

Ubiquitin Conjugating Enzyme Ubc9 is Involved in Protein Degradation of Redox Factor-1 (Ref-1).

Redox factor-1 (Ref-1) is a bifunctional protein playing an important role in both cellular redox regulation and DNA apurinic/apyrimidinic sites' repair. To find Ref-1interacting proteins (Rips), a yeast two-hybrid screening was performed by using Ref-1 redox domain as the 'bait', and five positive clones were obtained. One of them (Rip3) was identified to be the ubiquitin-conjugating enzyme Ubc9. Simultaneous overexpression of Ubc9 in Hela cells dramatically inhibited the enhancement of AP-1 reporter gene by Ref-1. Western blot indicated that the protein level of Ref-1 dropped down as the result of simultaneous overexpression of Ubc9. These results suggest that Ubc9 is involved in the protein degradation of Ref-1, resulting in the downregulation of Ref-1 physiological function.

Cloning of Glial Cell Line-derived Neurotrophic Factor Gene and Its Expression in Eukaryotic Cells.

Glial cell line-derived neurotrophic factor gene (gdnf) was obtained by RT-PCR method. Its pro-duct in COS7 cells showed neurotrophic and protective effect on the dopaminergic neurons. Then gdnf gene was cloned into retrovirus vector pLNCX and packaged with PA317. Infectious particles thus obtained were used to infect rat myoblast cell line L-6TG. A cell clone L-6TG/gdnf was obtained after G418 selection. It will be applied to the ex vivo gene therapy study of Parkinson's disease.

[Synthesis and antibacterial activity of 7-(7-aminomethyl-5-azaspiro [2,4] hept-5-yl)-1-cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid and its analogues].

AIM: To find new antibacterial agents of quinolone with high activity and low toxicity. METHODS: To design and synthesize 7-(7-aminomethyl-5-azaspiro [2,4] hept-5-yl)-1-cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid and its analogues, and to study their antibacterial activity in vitro and in vivo. RESULTS: Twenty new compounds (2 - 11, 17 - 26) were obtained including five targeted compounds (22 - 26). The structures of the compounds were confirmed by 1HNMR, MS and HRMS. Compounds 22 - 26 showed broad spectrum of antibacterial activity against Gram-positive and Gram-negative organisms. Especially for compound 24, the relevant MIC values for 13 strains of Gram-positive organisms were < 0.001 - 0.03 mg(-1), including 4 strains of S. pneumoniae, 2 strains of S. pyogenes, 3 strains of S. aureus and 2 strains of Enterococci which exhibited more potent activity than contrast agents (clinafloxacin and gatifloxacin). The MIC values of 24 for 6 strains Gram-positive organisms were 0.01 - 1 mg x L(-1), which exhibited equal or lower activity than contrast agents. They were more effective than ciprofloxacin and gatifloxacin against intraperitoneal infections caused by S. pneumoniae and S. aureus in mice. CONCLUSION: Compounds (23, 24 and 26) showed excellent antibacterial activity in vitro and in vivo and should be worth further investigation.