[Molecular characteristics of Japanese encephalitis virus carried by Culex tritaeniorhynchus in Dongchuan District, Kunming City, Yunnan Province].

OBJECTIVE: To isolate the Japanese encephalitis virus carried by Culex tritaeniorhynchus in Dongchuan District of Yunnan Province and analyze its molecular characteristics, so as to provide insights into the prevention and control of Japanese encephalitis in Yunnan Province. METHODS: Mosquito specimens were collected using mosquito-trapping lamps from pig farms in Batang Village and Xiaoxin Village, Dongchuan District, Kunming City, Yunnan Province in July 2016, and the mosquito species was identified according to the mosquito morphology. Then, 60 to 100 mosquitoes of each species served as a group and were ground. Baby hamster kidney-21 (BHK-21) cells and Aedes albopictus clone C6/36 cells were used for virus isolation, and positive isolates were identified using flavivirus primers. The positive isolates were amplified using reverse transcription polymerase chain reaction (RT-PCR) assay with 15 pairs of specific primers covering the full length of the genotype I Japanese encephalitis virus, and DNA sequence assembly was performed using the software SeqMan in the DNASTAR package. The obtained sequences were aligned with the complete sequences of 38 Japanese encephalitis virus downloaded from the GenBank with the software MegAlign, and the nucleotide and amino acid homology analyses of the obtained sequences were performed. The difference in amino acid sites was analyzed with the software GeneDoc, and phylogenetic trees were created based on the sequences of the coding region and E protein of the isolated Japanese encephalitis virus with the software Mega X. In addition, the secondary and tertiary structures of the E protein of the Japanese encephalitis virus were predicted using the online tool SOPMA and the software Swiss-Model. RESULTS: A total of 5 820 mosquitoes were collected and 3 843 Cx. tritaeniorhynchus (66.03%) were identified according to the mosquito morphology. A positive virus isolate, termed YNDC55-33, was isolated from Cx. tritaeniorhynchoides following batches of virus isolation from mosquito specimens, and cytopathic effect was observed following inoculation into BHK-21 and C6/36 cells. The YNDC55-33 virus isolate was successfully amplified with the flavivirus primes, and a long sequence containing 300 nucleotides was obtained. Following sequence alignment using the BLAST tool, the sequence of the YNDC55-33 virus isolate had high homology with that of the genotype I Japanese encephalitis virus. A long sequence with 10 845 nucleotides in length, which encoded 3 432 amino acids, was obtained by splicing the full sequence of the YNDC55-33 virus isolate. Phylogenetic analysis based on the whole-genome sequence and E gene sequence of the YNDC55-33 virus isolate showed that the new YNDC55-33 virus isolate was most closely related to the genotype I Guizhou isolate (GenBank accession number: HM366552), with nucleotide homology of 98.5% and amino acid homology of 99.4%, and the YNDC55-33 virus isolate shared 97.96% ± 0.33% nucleotide homology and 99.35% ± 0.08% amino acid homology with other genotype I Japanese encephalitis virus isolates, and < 90% nucleotide homology and < 98% amino acid homology with other genotypes of Japanese encephalitis virus. The YNDC55-33 virus isolate and the live attenuated virus vaccine candidate SA14-14-2 isolate differed at 16 amino acid sites on E gene, and 7 out of 8 key amino acid sites related to neurovirulence. The secondary and tertiary structures of the E protein of the YNDC55-33 virus isolate were predicted to be characterized by random coils. CONCLUSIONS: A genotype I Japanese encephalitis virus was isolated from Cx. tritaeniorhynchus in Dongchuan District, Kunming City. This virus isolate and the live attenuated virus vaccine candidate SA14-14-2 isolate does not differ at antigenic epitopes-related key amino acid sites, and the major protein structure of the virus isolate is random coils. This study adds new data for the epidemiological distribution of Japanese encephalitis virus in Yunnan Province, which may provide insights into the prevention and control of Japanese encephalitis in the province.

Potentiation of combined p19Arf and interferon-beta cancer gene therapy through its association with doxorubicin chemotherapy.

Balancing safety and efficacy is a major consideration for cancer treatments, especially when combining cancer immunotherapy with other treatment modalities such as chemotherapy. Approaches that induce immunogenic cell death (ICD) are expected to eliminate cancer cells by direct cell killing as well as activation of an antitumor immune response. We have developed a gene therapy approach based on p19Arf and interferon-ß gene transfer that, similar to conventional inducers of ICD, results in the release of DAMPS and immune activation. Here, aiming to potentiate this response, we explore whether association between our approach and treatment with doxorubicin (Dox), a known inducer of ICD, could further potentiate treatment efficacy without inducing cardiotoxicity, a critical side effect of Dox. Using central composite rotational design analysis, we show that cooperation between gene transfer and chemotherapy killed MCA205 and B16F10 cells and permitted the application of reduced viral and drug doses. The treatments also cooperated to induce elevated levels of ICD markers in MCA205, which correlated with improved efficacy of immunotherapy in vivo. Treatment of subcutaneous MCA205 tumors associating gene transfer and low dose (10 mg/kg) chemotherapy resulted in inhibition of tumor progression. Moreover, the reduced dose did not cause cardiotoxicity as compared to the therapeutic dose of Dox (20 mg/kg). The association of p19Arf/interferon-ß gene transfer and Dox chemotherapy potentiated antitumor response and minimized cardiotoxicity.

Response of human melanoma cell lines to interferon-beta gene transfer mediated by a modified adenoviral vector.

Since melanomas often retain wild type p53, we developed an adenoviral vector, AdRGD-PG, which provides robust transduction and transgene expression in response to p53. Previously, this vector was used for interferon-ß gene transfer in mouse models of melanoma, resulting in control of tumor progression, but limited cell killing. Here, the AdRGD-PG-hIFNß vector encoding the human interferon-ß cDNA (hIFNß) was used to transduce human melanoma cell lines SK-MEL-05 and SK-MEL-147 (both wild type p53). In vitro, cell death was induced in more than 80% of the cells and correlated with elevated annexinV staining and caspase 3/7 activity. Treatment with hIFNß promoted cell killing in neighboring, non-transduced cells, thus revealing a bystander effect. In situ gene therapy resulted in complete inhibition of tumor progression for SK-MEL-147 when using nude mice with no evidence of hepatotoxicity. However, the response in Nod-Scid mice was less robust. For SK-MEL-05, tumor inhibition was similar in nude and Nod-Scid mice and was less efficient than seen for SK-MEL-147, indicating both cell type and host specific responses. The AdRGD-PG-hIFNß vector provides extensive killing of human melanoma cells in vitro and a potent anti-tumor effect in vivo. This study provides a critical advance in the development of our melanoma gene therapy approach.

Seasonal variation in tap water δ2H and δ18O isotopes reveals two tap water worlds.

Stable isotope ratios of hydrogen and oxygen (δ2H and δ18O) in tap water provide important insights into the way that people interact with and manage the hydrological cycle. Understanding how these interactions vary through space and time allows for the management of these resources to be improved, and for isotope data to be useful in other disciplines. The seasonal variation of δ2H and δ18O in tap water within South Africa was assessed to identify municipalities that are supplied by seasonally invariant sources that have long residence periods, such as groundwater, and those supplied by sources that vary seasonally in a manner consistent with evapoconcentration, such as surface water-the proposed two tap water "worlds". Doing so allows for the cost-effective spatial interpolation of δ2H and δ18O values that likely reflect that of groundwater, removing the residual error introduced by other sources that are dependent on discrete, isolated factors that cannot be spatially generalised. Applying the proposed disaggregation may also allow for the efficient identification of municipalities that are dependent on highly variable or depleted surface water resources, which are more likely to be vulnerable to climate and demographic changes.

A case of systemic lupus erythematosus with rectal necrosis.

Lupus mesenteric vasculitis is one of the most devastating complications of systemic lupus erythematosus (SLE) and may lead to a spectrum of complications, including ulceration, haemorrhage, bowel necrosis, perforation, serositis and ascites. Among such complications, intestinal necrosis and intestinal perforation are the most serious. Rectal necrosis is a rare manifestation of SLE, with only two case reports in the English literature. Here, we report the case of a 59-year-old male patient with SLE complicated by rectal necrosis that was initially misdiagnosed as acne and rectal tumours. After two surgeries and the addition of immunosuppressive therapy, the patient was eventually cured and discharged.

MHC-II neoantigens shape tumour immunity and response to immunotherapy.

The ability of the immune system to eliminate and shape the immunogenicity of tumours defines the process of cancer immunoediting1. Immunotherapies such as those that target immune checkpoint molecules can be used to augment immune-mediated elimination of tumours and have resulted in durable responses in patients with cancer that did not respond to previous treatments. However, only a subset of patients benefit from immunotherapy and more knowledge about what is required for successful treatment is needed2-4. Although the role of tumour neoantigen-specific CD8+ T cells in tumour rejection is well established5-9, the roles of other subsets of T cells have received less attention. Here we show that spontaneous and immunotherapy-induced anti-tumour responses require the activity of both tumour-antigen-specific CD8+ and CD4+ T cells, even in tumours that do not express major histocompatibility complex (MHC) class II molecules. In addition, the expression of MHC class II-restricted antigens by tumour cells is required at the site of successful rejection, indicating that activation of CD4+ T cells must also occur in the tumour microenvironment. These findings suggest that MHC class II-restricted neoantigens have a key function in the anti-tumour response that is nonoverlapping with that of MHC class I-restricted neoantigens and therefore needs to be considered when identifying patients who will most benefit from immunotherapy.

[Emerging strategies for the treatment of Meibomian gland dysfunction].

Meibomian gland dysfunction (MGD), as a chronic,diffuse Meibomian gland disease,is one of the most common ophthalmological clinical diseases. Symptoms can be mild,such as ocular discomfort, but severe cases resulting in ocular surface damage could affect patients' visual function. Moreover,with the absence of a thorough examination of eyelid status and Meibomian gland prior to ocular surface surgery, it could cause severe postoperative complications. As a usual but easily overlooked disease, MGD and its associated ocular surface diseases have drawn greater attention,on the other hand,some emerging therapies,in addition to the clinically recognized treatments, provided doctors with more effective treatments at their disposal,and plenty of research achievements have been published. This article emphasizes on new physical approaches in the treatments of MGD and its associated ocular surface diseases. (Chin J Ophthalmol, 2019,55:465-468).

High-Dimensional Analysis Delineates Myeloid and Lymphoid Compartment Remodeling during Successful Immune-Checkpoint Cancer Therapy.

Although current immune-checkpoint therapy (ICT) mainly targets lymphoid cells, it is associated with a broader remodeling of the tumor micro-environment. Here, using complementary forms of high-dimensional profiling, we define differences across all hematopoietic cells from syngeneic mouse tumors during unrestrained tumor growth or effective ICT. Unbiased assessment of gene expression of tumor-infiltrating cells by single-cell RNA sequencing (scRNAseq) and longitudinal assessment of cellular protein expression by mass cytometry (CyTOF) revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. Surprisingly, we observed multiple subpopulations of monocytes/macrophages, distinguishable by the markers CD206, CX3CR1, CD1d, and iNOS, that change over time during ICT in a manner partially dependent on IFNγ. Our data support the hypothesis that this macrophage polarization/activation results from effects on circulatory monocytes and early macrophages entering tumors, rather than on pre-polarized mature intratumoral macrophages.

Immunomodulatory and antitumor effects of type I interferons and their application in cancer therapy.

During the last decades, the pleiotropic antitumor functions exerted by type I interferons (IFNs) have become universally acknowledged, especially their role in mediating interactions between the tumor and the immune system. Indeed, type I IFNs are now appreciated as a critical component of dendritic cell (DC) driven T cell responses to cancer. Here we focus on IFN-α and IFN-ß, and their antitumor effects, impact on immune responses and their use as therapeutic agents. IFN-α/ß share many properties, including activation of the JAK-STAT signaling pathway and induction of a variety of cellular phenotypes. For example, type I IFNs drive not only the high maturation status of DCs, but also have a direct impact in cytotoxic T lymphocytes, NK cell activation, induction of tumor cell death and inhibition of angiogenesis. A variety of stimuli, including some standard cancer treatments, promote the expression of endogenous IFN-α/ß, which then participates as a fundamental component of immunogenic cell death. Systemic treatment with recombinant protein has been used for the treatment of melanoma. The induction of endogenous IFN-α/ß has been tested, including stimulation through pattern recognition receptors. Gene therapies involving IFN-α/ß have also been described. Thus, harnessing type I IFNs as an effective tool for cancer therapy continues to be studied.

Uncovering the immunotherapeutic cycle initiated by p19Arf and interferon-ß gene transfer to cancer cells: An inducer of immunogenic cell death.

Simultaneous reestablishment of p53/p19Arf and interferon-ß pathways in melanoma cells culminates in a cell death process that displays features of necroptosis along with the release of immunogenic cell death molecules and unleashes an antitumor immune response mediated by natural killer cells, neutrophils as well as CD4+ and CD8+ T lymphocytes.

Intratumoral Immunization by p19Arf and Interferon-ß Gene Transfer in a Heterotopic Mouse Model of Lung Carcinoma.

Therapeutic strategies that act by eliciting and enhancing antitumor immunity have been clinically validated as an effective treatment modality but may benefit from the induction of both cell death and immune activation as primary stimuli. Using our AdRGD-PG adenovector platform, we show here for the first time that in situ gene transfer of p19Arf and interferon-ß (IFNß) in the LLC1 mouse model of lung carcinoma acts as an immunotherapy. Although p19Arf is sufficient to induce cell death, only its pairing with IFNß significantly induced markers of immunogenic cell death. In situ gene therapy with IFNß, either alone or in combination with p19Arf, could retard tumor progression, but only the combined treatment was associated with a protective immune response. Specifically in the case of combined intratumoral gene transfer, we identified 167 differentially expressed genes when using microarray to evaluate tumors that were treated in vivo and confirmed the activation of CCL3, CXCL3, IL1α, IL1ß, CD274, and OSM, involved in immune response and chemotaxis. Histologic evaluation revealed significant tumor infiltration by neutrophils, whereas functional depletion of granulocytes ablated the antitumor effect of our approach. The association of in situ gene therapy with cisplatin resulted in synergistic elimination of tumor progression. In all, in situ gene transfer with p19Arf and IFNß acts as an immunotherapy involving recruitment of neutrophils, a desirable but previously untested outcome, and this approach may be allied with chemotherapy, thus providing significant antitumor activity and warranting further development for the treatment of lung carcinoma.

AAVPG: a vigilant vector where transgene expression is induced by p53.

Using p53 to drive transgene expression from viral vectors may provide on demand expression in response to physiologic stress, such as hypoxia or DNA damage. Here we introduce AAVPG, an adeno-associated viral (AAV) vector where a p53-responsive promoter, termed PG, is used to control transgene expression. In vitro assays show that expression from the AAVPG-luc vector was induced specifically in the presence of functional p53 (1038±202 fold increase, p<0.001). The AAVPG-luc vector was an effective biosensor of p53 activation in response to hypoxia (4.48±0.6 fold increase in the presence of 250µM CoCl2, p<0.001) and biomechanical stress (2.53±0.4 fold increase with stretching, p<0.05). In vivo, the vigilant nature of the AAVPG-luc vector was revealed after treatment of tumor-bearing mice with doxorubicin (pre-treatment, 3.4×10(5)±0.43×10(5)photons/s; post-treatment, 6.6×10(5)±2.1×10(5)photons/s, p<0.05). These results indicate that the AAVPG vector is an interesting option for detecting p53 activity both in vitro and in vivo.

Hydrogen-rich saline alleviates experimental noise-induced hearing loss in guinea pigs.

OBJECTIVE: To examine the efficiency of hydrogen-rich saline in the treatment of intensive noise-induced cochlear injury. MATERIALS AND METHODS: Forty guinea pigs were assigned to one of four groups: HS+NOISE (i.p. injection hydrogen-rich saline), NS+NOISE (i.p. injection normal saline), NOISE ALONE (noise control), and NO TREATMENT (normal control) groups. The HS+NOISE, NS+NOISE, and NOISE ALONE groups were exposed to intensive noise (4 h at 115 dB SPL noise of 4000±100 Hz). The auditory brainstem response (ABR) was used to examine the hearing threshold in each group. Distortion product otoacoustic emission (DPOAE) was used to examine outer hair cell function. We also examined cochlear morphology to evaluate inner and outer hair cell trauma induced by noise exposure. Hydrogen-rich saline was administered twice daily for 6 days (2.5 ml/kg, i.p.) 24 h after noise exposure. RESULTS: Baseline ABR thresholds and DPOAE values were normal in all groups at the measured frequencies (2, 4, 8, and 16 kHz) before noise exposure. The ABR threshold shift was 50-55 dB across the frequencies tested, and average DPOAE declined in the NOISE ALONE, NS+NOISE, and HS+NOISE groups 24 h after noise exposure. However, the changes in cochlear parameters were different between groups. The HS+NOISE group showed a significantly decreased ABR threshold value as compared with the NS+NOISE or NOISE ALONE group (P<0.01) on day 7. The mean DPOAE recovered to some extent in the three noise exposure groups, but at most frequencies the HS+NOISE group showed significantly increased DPOAE on day 7 as compared with the NS+NOISE group or NOISE ALONE group (P<0.01). Surface Corti organ preparations stained with succinate dehydrogenase (SDH) showed that most outer hair cells (OHCs) were still dropsical and a few were missing 7 days after noise exposure in the NS+NOISE group. Only a few OHCs were slightly dropsical in the HS+NOISE group. The numbers of missing hair cells 7 days after noise exposure were significantly greater in the NOISE ONLY and NS+NOISE groups than the HS+NOISE group (P<0.01). CONCLUSIONS: Hydrogen-rich saline can alleviate experimental noise-induced hearing loss in guinea pigs, partially by preventing the death of cochlear hair cells after intensive noise exposure.

A one pot synthesis of mono- and di-lactosyl sphingosines.

The importance of analogues of lactosyl ceramides as basic structures of many natural glycosphingolipids provided a rationale for developing an efficient synthetic route to these compounds. We report herein a novel approach to synthesize several members of this family. Glycosylation of N-diphenylmethylene-spingosine, which exists in an imine-oxazolidine tautomeric mixture, with acetobromolactose under a modified Koenigs-Knorr condition yielded lactosyl beta-(1 --> 1) sphingosine, lactosyl beta-(1 --> 3) sphingosine and dilactosyl sphingosine in good yields. A similar glycosylation could be applicable to the synthesis of other glycosphingolipids.

[Amelioration of nerve growth factor against noise-induced threshold shift: a transmission electron microscope observation].

OBJECTIVE: To study the protective effects of nerve growth factor(NGF) on noise-induced hearing damage in guinea pigs. METHODS: NGF injected Guinea pigs were consecutively exposed to white noise of 115 dB(A) for 6 days continually (45 min.d-1). Auditory thresholds were measured using auditory cortex evoked response to tone bursts in different post-exposure intervals (1 h, 1 d, 2 d, 3 d and 6 d). The ultrastructural changes within hair cells were also observed by a transmission electron microscope(TEM). RESULTS: The auditory threshold shifts in test group A(NGF:1,000 U.kg-1.d-1, i.m.), B(NGF:2,000 U.kg-1.d-1, i.m.) and C(NGF:3,000 U.kg-1.d-1, i.m.) were significantly fewer than that in the control group(Saline: 1 ml.kg-1.d-1, i.m.). Threshold shifts almost recovered in test group B and C 3 days after the exposure; while a threshold shift of (16.43 +/- 6.91) dB was present 6 days after the exposure in the control group. TEM showed that all three rows of the outer hair cells(OHCs) of the basal turn in the control group displayed significant pathological changes. Depolymerization of actin filiaments within stereocilia, swelling of submembraneous cistern and the efferent nerve-ending and slight edema of hair cells were evident. In test group A, the hair cells display slight pathological changes, which are confined in the third row of OHCs in a local position of the basal turn. In group B and C hair cells have nearly normal appearance. CONCLUSION: NGF is able to reduce threshold shift, and promote the recovery of auditory threshold in acoustic trauma. This factor can, to some extent, protect against noise-induced hearing damage.

Highly efficient and versatile synthesis of libraries of constrained beta-strand mimetics.

The general approach of using a bicyclic template to produce inhibitors of the protease superfamily of enzymes has been investigated. The Diels Alder cycloaddition reaction on solid support has been found to be highly efficient for the synthesis of libraries of compounds that mimic the beta-strand secondary structure of proteins. Several potent and selective inhibitors of proteases have been discovered.

N,N-dimethylsphingosine 1-phosphate activates human platelets.

We recently reported that N,N-dimethylsphingosine 1-phosphate (DMS-1-P) can be formed from N,N-dimethylsphingosine (DMS) in activated platelets [Y. Yatomi et al., Biochem. Biophys. Res. Commun. 231 (1997) 848-851]. In this study, we synthesized, for the first time, DMS-1-P and examined the functional effects of DMS-1-P and its related sphingolipids on platelets. Although exogenous DMS was inactive, its phosphorylated derivative, DMS-1-P, induced platelet intracellular Ca2+ mobilization and shape change, but not aggregation or release reactions. Since sphingosine 1-phosphate (Sph-1-P) is structurally related to DMS-1-P and activates platelets more strongly than DMS-1-P, a competitive binding experiment for [3H]Sph-1-P was performed using DMS-1-P. DMS-1-P reduced the binding of [3H]Sph-1-P to platelets almost as much as unlabeled Sph-1-P did. These results suggest that DMS-1-P activates platelets via an interaction with a platelet surface receptor for Sph-1-P.

Sphingosine 1-phosphate regulates melanoma cell motility through a receptor-coupled extracellular action and in a pertussis toxin-insensitive manner.

Our previous work showed that sphingosine 1-phosphate (Sph-1-P) inhibits the cell motility of mouse melanoma B16/F10, and other types of cells at 10-100 nM concentrations. In the present paper, we have identified and characterized specific cell surface binding sites for Sph-1-P in F10 cells. Sph-1-P immobilized on controlled pore glass beads inhibited the motility of F10 cells, suggesting that Sph-1-P acts on the cells from the outside. Binding assays with [3H]Sph-1-P revealed the presence of specific cell surface binding sites for Sph-1-P in F10 cells. Scatchard analysis demonstrated a single class of binding sites for Sph-1-P. The binding of [3H]Sph-1-P to F10 cells was inhibited by the addition of excess unlabeled Sph-1-P but not other natural sphingolipids. The specific binding was also sensitive to treatment with a protease. Using Sph-1-P-immobilized affinity chromatography, we, for the first time, identified 41-kDa and 79-kDa Sph-1-P binding proteins on the melanoma cell surface, although the 41-kDa protein was less specific to Sph-1-P. We demonstrated that pertussis toxin (PTX) treatment did not abolish the motility inhibition by Sph-1-P, suggesting that no PTX-sensitive G-protein is involved in the signaling. Furthermore, Sph-1-P was found to be specifically released from mouse BALB/3T3 clone A31 cells and F10 cells. Collectively, these results strongly suggest that Sph-1-P regulates melanoma cell motility through an extracellular action by specific binding to cell surface receptor protein(s), which is independent of PTX-sensitive G-protein.

N,N-dimethylsphingosine phosphorylation in human platelets.

Metabolism of sphingosine (Sph) derivatives in human platelets was examined. [3H]Sph was rapidly and heavily phosphorylated into sphingosine 1-phosphate, similarly in resting and stimulated platelets. [14C]N,N-dimethylsphingosine was stable in resting platelets, while it was converted into N,N-dimethylsphingosine 1-phosphate (DMS-1-P), although weakly, in platelets stimulated with thrombin or 12-O-tetradecanoylphorbol 13-acetate. This DMS-1-P formation was inhibited by staurosporine, a potent protein kinase inhibitor. [3H]C2-ceramide was unchanged both in resting and stimulated platelets. Our report is the first to describe production of DMS-1-P in a biological system.