First Report of Leaf Spot Caused by Alternaria alternata on Cyclocodon lancifolius in China.

Cyclocodon lancifolius Bunge in the family Campanulaceae, and commonly known as Hong Guo Ginseng, is found in the Indonesia, Philippines, Vietnam, Japan, and China. The leaves and roots of C. lancifolius are widely used as tonics by ethnic minorities in Guizhou and Hunan Provinces in China. In addition, the fruit is edible, and it is a new resource for both medicine and food. In June 2022, symptoms of leaf spot (Fig 1 A and B.) were observed on C. lancifolius plants in the medicinal plant greenhouse of Guizhou University of Traditional Chinese Medicine (106°61'E, 26°39'N), Guizhou Province. The incidence of leaf spot on C. lancifolius was approximately 40 to 70% of all leaves in canopy. Early symptoms on leaves were small circular or irregular brown spots. As the disease progressed the lesions gradually expanded, and multiple lesions coalesced to form large irregular brown spots. Eventually the seedlings died and leaves of mature plants wilted. In order to isolate the pathogen, ten leaf pieces (5×5 mm) were cut from the junction of the diseased and the healthy tissues, surface sterilized with 75% ethanol for 30 s, 0.1% mercuric chloride (HgCl2) solution for 60 s, rinsed in sterile water three times, finally dried and placed on potato dextrose agar (PDA) and cultured in the dark at 27°C for 4 days. Five purified fungal isolates were obtained by single spore isolation. The colonies were olivaceous to dark olive with white margins and abundant aerial mycelia. On potato carrot agar (PCA) medium, these fungi produced septate conidiophores. Conidia were obclavate or ellipsoid, brown, with one to four transverse septa and one to two longitudinal septa. Spores measured 7.64 to 14.20 × 3.32 to 6.38 µm (n=50). These morphological characteristics are consistent with Alternaria alternata (S. P. Wiltshire. 1933). To further confirm the identification, four genomic DNA regions including the ribosomal DNA internal transcribed spacer (ITS), translation elongation factor 1-a gene (TEF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), and Alternaria major allergen gene (Alt a1) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), TEF1-728F/TEF1-986R (Carbone and Kohn 1999), gpd1/gpd2 (Berbee et al. 1999), RPB2-5F/RPB2-7cR (Liu et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005), respectively. Sequences were deposited in GenBank with accession Nos. ITS: OQ128111, OQ690707, and OQ690708; TEF: OQ200380, OQ700996, and OQ700998; GAPDH: OQ200378, OQ700993, and OQ700995; RPB2:OQ200379, OQ701002, and OQ701004; Alt: OQ675614, OQ700999, and OQ701001. In a BLAST search, the sequences were 99-100% identical with corresponding sequences of A. alternata. A maximum likelihood phylogenetic tree was constructed with the combined sequence data sets of ITS, TEF, GAPDH, RPB2, and Alt a1 using MEGA 11. The isolate DHY0, DHY1, and DHY3 clustered with A. alternata (J. H. C. Woudenberg et al. 2015) (Fig. 2). To fulfill Koch's postulates, leaves on three healthy 3-month-old potted C. lancifolius seedlings were wounded with sterile needles and inoculated with 5 mm diameter mycelium, which was covered moist by sterile cotton for 24 h. Sterile water was used as the control. After inoculation, the plants were incubated at 27°C, 85% relative humidity, and a 12 h photoperiod. The experiment was repeated three times. Fifteen days after inoculation, all the leaves showed leaf spot symptoms that were similar to those observed in the greenhouse, while control leaves were asymptomatic (Fig. 1). A. alternata was successfully re-isolated from the symptomatic leaves and identified by morphology and the molecular methods described above. This pathogen has been reported to cause a leaf disease in a wide range of vegetables (Zhang et al. 2021), flowers (Zhang et al. 2022), and medicinal plants (Xing et al. 2020). To the best of our knowledge, this is the first report of A. alternata causing leaf spot on C. lancifolius in China. The accurate identification of this pathogen will provide a basis for the prevention and control of C. lancifolius leaf spot disease in the future.

Astragalus polysaccharide (APS) attenuated PD-L1-mediated immunosuppression via the miR-133a-3p/MSN axis in HCC.

CONTEXT: Astragalus polysaccharide (APS) is a new tumour therapeutic drug, that has an inhibitory effect on a variety of solid tumours. Tumour cell immunosuppression is related to the up-regulation of programmed death ligand 1 (PD-L1). However, whether APS exerts its antitumor effect by regulating PD-L1 remains unclear. OBJECTIVE: To explore whether APS exerts its antineoplastic effect via regulating PD-L1-mediated immunosuppression in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: SMMC-7721 cells were subcutaneous injected into BALB/C mice for HCC model establishment. Mice were intraperitoneally injected with 100, 200 and 400 mg/kg APS for 12 days. Immunohistochemistry (IHC) was performed to assess CD8+ T cells' rate and PD-L1 level in HCC tissues. HCC cells were pre-treated with 0.1, 0.5 and 1 mg/mL APS for 4 h, then were treated with 10 ng/mL IFN-γ 24 h. PD-L1 level and cell apoptosis was detected by flow cytometry. PD-L1 and Moesin (MSN) proteins were measured by western blot. MiR-133a-3p and MSN mRNA levels were assessed by qRT-PCR. The targets of miR-133a-3p were predicted by starBase, and which was verified by dual-luciferase reporter assay. RESULTS: Our findings illustrated that APS dose-dependently inhibited HCC growth tested with IC50 values of 4.2 mg/mL, and IFN-γ-induced PD-L1 expression and attenuated PD-L1-mediated immunosuppression in HCC cells. APS attenuated PD-L1-mediated immunosuppression via miR-133a-3p in HCC cells. Besides, miR-133a-3p targeted to MSN, and MSN inhibited the antitumor effect of APS by maintaining the stability of PD-L1. Moreover, APS attenuated PD-L1-mediated immunosuppression via the miR-133a-3p/MSN axis. CONCLUSIONS: APS attenuated PD-L1-mediated immunosuppression via miR-133a-3p/MSN axis to develop an antitumor effect. APS may be an effective drug for HCC treatment.

Modeling MEN1 with Patient-Origin iPSCs Reveals GLP-1R Mediated Hypersecretion of Insulin.

Multiple endocrine neoplasia type 1 (MEN1) is an inherited disease caused by mutations in the MEN1 gene encoding a nuclear protein menin. Among those different endocrine tumors of MEN1, the pancreatic neuroendocrine tumors (PNETs) are life-threatening and frequently implicated. Since there are uncertainties in genotype and phenotype relationship and there are species differences between humans and mice, it is worth it to replenish the mice model with human cell resources. Here, we tested whether the patient-origin induced pluripotent stem cell (iPSC) lines could phenocopy some defects of MEN1. In vitro ß-cell differentiation revealed that the percentage of insulin-positive cells and insulin secretion were increased by at least two-fold in MEN1-iPSC derived cells, which was mainly resulted from significantly higher proliferative activities in the pancreatic progenitor stage (Day 7-13). This scenario was paralleled with increased expressions of prohormone convertase1/3 (PC1/3), glucagon-like peptide-1 (GLP-1), GLP-1R, and factors in the phosphatidylinositol 3-kinase (PI3K)/AKT signal pathway, and the GLP-1R was mainly expressed in ß-like cells. Blockages of either GLP-1R or PI3K significantly reduced the percentages of insulin-positive cells and hypersecretion of insulin in MEN1-derived cells. Furthermore, in transplantation of different stages of MEN1-derived cells into immune-deficient mice, only those ß-like cells produced tumors that mimicked the features of the PNETs from the original patient. To the best of our knowledge, this was the first case using patient-origin iPSCs modeling most phenotypes of MEN1, and the results suggested that GLP-1R may be a potential therapeutic target for MEN1-related hyperinsulinemia.

Effect of Hydrogen Inhalation Therapy on Hearing Loss of Patients With Nasopharyngeal Carcinoma After Radiotherapy.

Objective: To evaluate the clinical efficacy and safety of hydrogen inhalation in improving hearing loss in patients with long-term survival of nasopharyngeal carcinoma after radiotherapy. Methods: The eustachian tube dysfunction score, pure tone air conduction threshold, bone conduction threshold, the score of tympanogram and otoscope were prospectively observed in patients with deafness after radiotherapy only or combined radiotherapy and chemotherapy for nasopharyngeal carcinoma. Paired t test and one-way analysis of variance were used to analyze the data before and after treatment. Results: A total of 17 patients were observed. The median time from radiotherapy to now was 228 months, and the median time from the diagnose of deafness to now was 92 months. After 4 weeks of hydrogen inhalation, the score of eustachian tube dysfunction, air conduction and bone conduction hearing thresholds were significantly reduced, P values were 0.0293, 0.0027, 0.0404, respectively. The mean air-bone gap, the score of otoendoscopy and tympanogram were also decreased, but the differences were not significant (P = 0.2079, P = 0.0536, P = 0.1056). Patients with radiotherapy alone and concurrent chemo-radiotherapy had significantly lower air conduction hearing threshold after hydrogen absorption (P = 0.0142, P = 0.0495). The results of air and bone hearing thresholds before, 4 and 12 weeks after hydrogen inhalation showed a descending trend. The air and bone hearing thresholds before hydrogen inhalation were 74.69 ± 27.03 dB and 45.70 ± 21.58 dB, respectively. At the 12th week, the mean values of air and bone hearing thresholds were the lowest, which were 66.88 ± 20.88 dB and 40.94 ± 18.93 dB, respectively, but there was no significant difference in air and bone hearing thresholds among all groups (P = 0.6755, P = 0.7712). After hydrogen inhalation treatment, no adverse reactions such as nosebleed, chest pain, dyspnea, nausea, vomiting, dizziness, earache and allergic reaction were observed. Conclusion: This is the first prospective study on the effect of hydrogen inhalation on hearing improvement in patients with deafness after radiotherapy/chemotherapy for nasopharyngeal carcinoma, suggesting that continuous hydrogen inhalation may be an alternative rehabilitation therapy for these patients.

Allogeneic Vγ9Vδ2 T-cell immunotherapy exhibits promising clinical safety and prolongs the survival of patients with late-stage lung or liver cancer.

Vγ9Vδ2 T cells are promising candidates for cellular tumor immunotherapy. Due to their HLA-independent mode of action, allogeneic Vγ9Vδ2 T cells can be considered for clinical application. To apply allogeneic Vγ9Vδ2 T cells in adoptive immunotherapy, the methodology used to obtain adequate cell numbers with optimal effector function in vitro needs to be optimized, and clinical safety and efficacy also need to be proven. Therefore, we developed a novel formula to improve the expansion of peripheral γδ T cells from healthy donors. Then, we used a humanized mouse model to validate the therapeutic efficacy of expanded γδ T cells in vivo; furthermore, the expanded γδ T cells were adoptively transferred into late-stage liver and lung cancer patients. We found that the expanded cells possessed significantly improved immune effector functions, including proliferation, differentiation, and cancer cell killing, both in vitro and in the humanized mouse model. Furthermore, a phase I clinical trial in 132 late-stage cancer patients with a total of 414 cell infusions unequivocally validated the clinical safety of allogeneic Vγ9Vδ2 T cells. Among these 132 patients, 8 liver cancer patients and 10 lung cancer patients who received ≥5 cell infusions showed greatly prolonged survival, which preliminarily verified the efficacy of allogeneic Vγ9Vδ2 T-cell therapy. Our clinical studies underscore the safety and efficacy of allogeneic Vγ9Vδ2 T-cell immunotherapy, which will inspire further clinical investigations and eventually benefit cancer patients.

Two weeks of hydrogen inhalation can significantly reverse adaptive and innate immune system senescence patients with advanced non-small cell lung cancer: a self-controlled study.

Following standard treatments, the traditional model for enhancing anti-tumor immunity involves performing immune reconstitution (e.g., adoptive immune cell therapies or immunoenhancing drugs) to prevent recurrence. For patients with advanced non-small cell lung cancer, we report here on two objectives, the immunosenescence for advanced non-small cell lung cancer and hydrogen gas inhalation for immune reconstitution. From July 1st to September 25th, 2019, 20 non-small cell lung cancer patients were enrolled to evaluate the immunosenescence of peripheral blood lymphocyte subsets, including T cell, natural killer/natural killer T cell and gamma delta T cell. Two weeks of hydrogen inhalation was performed during the waiting period for treatment-related examination. All patients inhaled a mixture of hydrogen (66.7%) and oxygen (33.3%) with a gas flow rate of 3 L/min for 4 hours each day. None of the patients received any standard treatment during the hydrogen inhalation period. After pretreatment testing, major indexes of immunosenescence were observed. The abnormally higher indexes included exhausted cytotoxic T cells, senescent cytotoxic T cells, and killer Vδ1 cells. After 2 weeks of hydrogen therapy, the number of exhausted and senescent cytotoxic T cells decreased to within the normal range, and there was an increase in killer Vδ1 cells. The abnormally lower indexes included functional helper and cytotoxic T cells, Th1, total natural killer T cells, natural killer, and Vδ2 cells. After 2 weeks of hydrogen therapy, all six cell subsets increased to within the normal range. The current data indicate that the immunosenescence of advanced non-small cell lung cancer involves nearly all lymphocyte subsets, and 2 weeks of hydrogen treatment can significantly improve most of these indexes. The study was approved by the Ethics Committee of Fuda Cancer Hospital, Jinan University in China (approval No. Fuda20181207) on December 7th, 2018, and was registered on ClinicalTrials.gov (ID: NCT03818347) on January 24th, 2019.

First Report of Ceratocystis changhui Causing Postharvest Fruit Rot of Peach in Kunming, China.

The peach (Prunus persica (L) Batsch) is a predominant commercially grown stone fruit in China (Lee et al. 1990). Ceratocystis changhui is an aggressive pathogen causing typical black rot symptoms on corms of taro (Colocasia esculenta) (Liu et al. 2018), it has not been reported on other hosts. During the summer and autumn of 2013, a postharvest fruit rot disease was observed on several peaches at a farmer's market (N 25°02'; E 102°42') in Kunming City, Yunnan Province, China. The incidence of the disease varied from 5 to 20%. Necrotic spots were first observed on the infected peach fruit (Prunus persica cv. shuimitao). The spots enlarged gradually and developed into a brown, water-soaked and rotted lesion. Eventually, the whole fruit became soft, rotted and covered with a gray-brown mycelium (Fig. 1 A, B). The isolates were obtained from the symptomatic tissues incubated on slices of fresh carrot root (Moller et al. 1968). After 5 to 10 days of incubation, perithecia and mycelium were observed growing on carrot slices. Spore masses were removed from the apices of perithecia, transferred to potato dextrose agar medium (PDA) and incubated at 25°C for 5 to 10 days, followed by single-spore isolation. All eight single-spore isolates from peach fruits obtained in this study were deposited in the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, China. In culture, mycelium was initially white, gradually turned to greyish-green or brown (Fig. 1E, F). Measurements were made 7 days after the formation of perithecia. Perithecia (Fig. 1G) were black, globose, 185.71 to 305.56 µm × 142.86 to 264.29 µm and showed a long black neck, 600 to 957.14 µm (Fig. 1H). Ascospores (Fig. 1I) were helmet-hat shaped and 2.86 to 6.67 µm ×3.81 to 4.76 µm. Cylindrical conidia (Fig. 1J) 6.67 to 38. 95 µm × 2.86 to7.62 µm were observed. Chlamydospore (Fig. 1K), 8.57 to 13.33 µm × 5.71 to 9.52 µm, were ovoid or obpyriform, smooth. To further verify pathogen identity the internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS1F and ITS4 (Thorpe et al. 2005), and the total genomic DNA from the mycelia of five isolates was extracted using a CTAB method (Lee &Taylor 1990). The nucleotide sequences have been blasted and deposited in the GenBank database. Analysis of the ITS sequences from the isolates T1-1yp, T1-2yp, T2-1yp (GenBank accession no. KY580895-KY580897) showed 99% to 100% similarity with isolates C. changhui CMW43272 (KY643886), CMW43281 (KY643884), CMW46112 (KY643891) and CMW46113 (KY643892) from taro in China. Phylogenetic trees based on the maximum-likelihood (ML) method were constructed using MEGA 7. ITS sequences of other Ceratocystis spp. were attained from NCBI for comparative analysis (Liu et al. 2018), and Davidsoniella virescens (CMW11164) served as outgroup. The robustness of ML tree was evaluated with 1,000 bootstrap (BS) values. The pathogen was identified as C. changhui based on the phylogenetic analysis (Fig. 2). Three isolates (T1-1yp, T1-2yp, T2-1yp) were used for pathogenicity. Nine Prunus persica cv. yingzuitao fruits at early maturity (8 points out of 10) were wound inoculated with 200µL conidia suspension of the fungus (approximately 2.0 × 106 conidia / mL). Degreasing cotton dipped in sterile water was used to raise the humidity in preservation boxes. Boxes were incubated for 10 days at 25°C. Three peaches as controls were treated only with sterile distilled water in the same way. Symptoms of sunken lesions and fruit rot were observed two days after inoculation, and measured at 1.8 to 3.2 cm from the inoculation point within 5 days (Fig. 1C: right, D). The same pathogen was re-isolated from them confirming Koch's postulates. Control peaches remained symptomless. This fungus was morphologically and phylogenetically identified as C. changhui. To our knowledge, this is the first report of C. changhui on postharvest peach in Yunnan, China. The disease will affect quality and taste of peach, so it is critical to deploy appropriate management strategies to limit the fungus spread.

Ginger wilt and rot disease caused by Ceratocystis fimbriata in China.

Ginger (Zingiber officinale Rosc.) is an herb that has been grown in China for more than 2500 years. It can be used as both a spice and a therapeutic drug. In July 2013, ginger plants were found to have wilting symptoms and yellowing leaves with netrotics leaf tips in a farm in Kunming city of Yunnan province (25. 02 N; 102.42 E), southwest China, and we also found gray-black lesion on the surface of the harvest gingers in a market in Kunming. Initial symptoms on harvest gingers appeared as gray-black mycelia growth on the surface of the harvested ginger, which enlarged and extended internally. Carrot baiting was used to isolate the pathogen from rotted gingers and diseased ginger leaves (Moller and Devay. 1968). After two weeks, spores developing from perithecia on the carrot pieces were transferred to malt extract agar (MEA) and incubate at 25°C constant-temperature incubator. Six single-spore isolates (ZOR-1 to ZOR-6) were obtained, the isolates were stored in 15% glycerol at -80°C refrigerator in State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan Agricultural University. Cultures varied in color from white to brownish green to brown. N = 50 for all measurements. Blackish brown, globose perithecia (131.9 to 186.0 µm × 138.5 to 188.3 µm) with a long black neck (400.2 to 644.7 µm) were immersed, partially embedded or superficial on the substrate. Ascospores were globose or had a "hat-like" morphology typical of Ceratocystis fimbriata, and were 4.0 to 5.3 µm × 4.8 to 6.2 µm. Endoconidia were cylindrical and clavate (2.9 to 7.4 µm × 7.5 to 32.8 µm), conidia were barrel-shaped (4.4 to 10.4 µm× 6.2 to 12.9 µm), and chlamydospores were smooth, blackish brown, ovoid or obpyriform (8.42 to 12.21 µm × 10.47 to 17.65 µm) (Webster and Butler. 1967; Engelbrecht and Harrington. 2005). Genomic DNA was extracted from two isolates (ZOR-1, ZOR-2) using the CTAB method (Lee and Taylor 1990). The internal transcribed spacers (ITS) region of rDNA was amplified using primers ITS1F/ITS4 (Thorpe et al. 2005). The nucleotide sequences of ZOR-1 and ZOR-2 (GenBank accessions KJ511490 and KJ511491) were 100% homologous to those of the isolates of C. fimbriata from diseased Cucumis sativus L. and Punica granatum L. in China (GenBank accessions MH535909 and KT963159). Thus, the pathogen was identified as C. fimbriata. Pathogenicity tests were made on fresh ginger rhizomes in laboratory, the pathogen was cultured for 14 days on MEA (ZOR-1, ZOR-2), which were washed with sterilized water and the resulting spore suspensions diluted to 1.0 × 106 spores/ml . Wounds (0.5 × 0.5 cm) were made on the surface of healthy mature ginger rhizomes by scraping with a sterile scalpel, then treated with a 100 ul spore suspension. Control ginger rhizomes were coated sterile water. Ginger rhizomes were stored at room temperature. Each treatment was performed in triplicate. After 5 days, grey-black mycelia developed on the rhizome surface, becoming a visible black mould after 1 week. We reisolated the pathogen from infected tissues, but not from the controls. In the greenhouse, 20ml of 1.0 × 106 spores/ml suspensions from isolates ZOR-1 and ZOR-2, or sterile water were injected into two-month- old ginger seedlings in triplicate. The inoculated site on the stem turned black in 5 days. 6 weeks after inoculation, the inoculated plants developed yellowing leaves and wilting symptoms. The same fungus was re-isolated from inoculated plants, but not from the controls. According to Koch's Postulation, the inoculated strains of ZOR-1 and ZOR-2 were the pathogens causing ginger wilt and rot disease. To the best of our knowledge, ginger is a new host plant of Ceratocystis fimbriata from China. In recent years, we have found that this disease incidence was approxmiatelt 5 to 10% of the farmland and 5 to 15% of the stored condition respectively in Yunnan Province. If not prevented ginger production in China will be affected.

First Report of Wilt of Rubber Tree Caused by Chalaropsis thielavioides in China.

Rubber tree (Hevea brasiliensis (Willd. ex Adr. Juss) Müll. Arg.) is used for the extraction of natural rubber and is an economically and socially important estate crop commodity in many Asian countries such as Indonesia, Malaysia, Thailand, India, Sri Lanka, China and several countries in Africa (Pu et al, 2007). Xishuangbanna City and Wenshan City are the main rubber cultivation areas in Yunnan Province, China. In November 2012, rubber tree showing typical wilt symptoms (Fig. 1 A) and vascular stains (Fig. 1 B) were found in Mengla County, Xishuangbanna City. This disease was destructive in these trees and plant wilt death rate reached 5%. The diseased wood pieces (0.5cm long) from trunk of rubber was surface disinfected with 75% ethanol for 30s and 0.1% mercuric chloride (HgCl2) for 2min, rinsed three times with sterile distilled water, plated onto malt extract agar medium (MEA), and incubated at 28℃. After 7 days, fungal-like filaments were growing from the diseased trunk. Six cultures from 6 rubber trunk were obtained and incubated on MEA at 28℃, after 7 days to observe the cultural features. The mycelium of each culture was white initially on MEA, and then became dark green. Cylindrical endoconidia apices rounded, non-septate, smooth, single or borne in chains (8.9 to 23.6 × 3.81 to 6.3µm) (Fig. 1 C). Chlamydospores (Fig. 1 D) were abundant, thick walled, smooth, forming singly or in chains (11.1 to 19.2 × 9.4 to 12.0µm). The mould fungus was identifed as Chalaropsis based on morphology (Paulin-Mahady et al. 2002). PCR amplification was carried out for 3 isolates, using rDNA internal transcribed spacer (ITS) primer pairs ITS1F and ITS4 (Thorpe et al. 2005). The nucleotide sequences were deposited in the GenBank data base and used in a Blast search of GenBank. Blast analysis of sequenced isolates XJm8-2-6, XJm8-2 and XJm10-2-6 (accessions KJ511486, KJ511487, KJ511489 respectively) had 99% identity to Ch. thielavioides strains hy (KF356186) and C1630 (AF275491). Thus the pathogen was identified as Ch. thielavioides based on morphological characteristics and rDNA-ITS sequence analysis. Pathogenicity test of the isolate (XJm8-2) was conducted on five 1-year-old rubber seedlings. The soil of 5 rubber seedlings was inoculated by drenching with 40 ml spore suspension (106 spores / ml). Five control seedlings were inoculated with 40 ml of sterile distilled water. All the seedlings were maintained in a controlled greenhouse at 25°C and watered weekly. After inoculated 6 weeks, all the seedlings with spore suspension produced wilt symptoms, as disease progressed, inoculated leaves withered (Fig. 1 E) and vascular stains (Fig. 1 F) by 4 months. While control seedlings inoculated with sterile distilled water remained healthy. The pathogen re-isolated from all inoculated symptomatic trunk was identical to the isolates by morphology and ITS analysis. But no pathogen was isolated from the control seedlings. The pathogenicity assay showed that Ch. thielavioides was pathogenic to rubber trees. Blight caused on rubber tree by Ceratocystis fimbriata previously in Brazil (Valdetaro et al. 2015), and wilt by Ch. thielavioides was not reported. The asexual states of most species in Ceratocystis are "chalara" or "thielaviopsis" (de Beer et al. 2014). To our knowledge, this is the first report of this fungus causing wilt of rubber in China. The spread of this disease may pose a threat to rubber production in China.

A narrative review of hydrogen oncology: from real world survey to real world evidence.

The use of hydrogen for cancer control has made great progress in cytology and animal experiments. With the increasing number of hydrogen products on the market, larger numbers of advanced cancer patients have participated in clinical trials or received treatment at home after purchase. Our study reported a real-world survey from 82 patients with good cancer control using hydrogen products, including real world evidence from patients who received ineffective traditional treatment, patients who received traditional treatment that failed, or patients who refused traditional treatment. Two typical cases were reported herein. Subsequently, we included studies on the mechanism of hydrogen oncology. The mechanism of cancer control using hydrogen includes the inhibition of tumor cells and the activation of exhausted lymphocytes. Large-scale real world evidence has shown clinical value, and yet remains to be further developed and researched.

Hydrogen therapy can be used to control tumor progression and alleviate the adverse events of medications in patients with advanced non-small cell lung cancer.

Chemotherapy, targeted therapy, and immunotherapy are used against advanced non-small cell lung cancer. A clinically efficacious method for relieving the adverse events associated of such therapies is lacking. Fifty-eight adult patients were enrolled in our trial to relieve pulmonary symptoms or the adverse events of drugs. Twenty patients who refused drug treatment were assigned equally and randomly to a hydrogen (H2)-only group and a control group. According to the results of tumor-gene mutations and drug-sensitivity tests, 10, 18, and 10 patients were enrolled into chemotherapy, targeted therapy, and immunotherapy groups in which these therapies were combined with H2-therapy, respectively. Patients underwent H2 inhalation for 4-5 hours per day for 5 months or stopped when cancer recurrence. Before study initiation, the demographics (except for tumor-mutation genes) and pulmonary symptoms (except for moderate cough) of the five groups showed no significant difference. During the first 5 months of treatment, the prevalence of symptoms of the control group increased gradually, whereas that of the four treatment groups decreased gradually. After 16 months of follow-up, progression-free survival of the control group was lower than that of the H2-only group, and significantly lower than that of H2 + chemotherapy, H2 + targeted therapy, and H2 + immunotherapy groups. In the combined-therapy groups, most drug-associated adverse events decreased gradually or even disappeared. H2 inhalation was first discovered in the clinic that can be used to control tumor progression and alleviate the adverse events of medications for patients with advanced non-small cell lung cancer. This study was approved by the Ethics Committee of Fuda Cancer Hospital of Jinan University on December 7, 2018 (approval No. Fuda20181207), and was registered at ClinicalTrials.gov (Identifier: NCT03818347) on January 28, 2019.

The future of cryoablation: An abscopal effect.

Cryoablation has become a popular modality to treat a variety of malignant tumors in solid organs and soft tissues. In the future, the use of cryoablation should focus on its abscopal effect. The present review discusses the increased immune response triggered by cryoablation alone or by cryoablation combined with immunotherapies, which can improve the immune response and limit immunosuppression. First, cryoablative techniques should be improved to increase the area of necrosis and reduce the area of apoptosis. Second, cryoablation should be combined with immunotherapies, for example, cyclophosphamide, natural killer cells, granulocyte monocyte colony stimulating factor (GM-CSF), cytotoxic T lymphocyte-associated antigen (CTLA)-4, and programmed death receptor 1 (PD)-1 inhibitors. Cryoablation could also be combined with Hydrogen gas molecules, which were shown recently to stimulate peroxisome proliferator activated receptor gamma coactivator (PGC)-1α, thereby promoting mitochondrial function, which might rescue exhausted CD8+ T cells, leading to prolonged progression-free survival and overall survival of patients with advanced colorectal cancer.

Hydrogen-oxygen therapy can alleviate radiotherapy-induced hearing loss in patients with nasopharyngeal cancer.

Three patients with nasopharyngeal carcinoma developed binaural secretory otitis media 12, 2, and 0.5 years after radiotherapy, respectively. The secretions subsided after conventional drug and drainage treatments, but hearing continued to deteriorate until severe loss was documented in both ears. After examination of the eardrum and tympanum, patients were enrolled in a clinical trial in the first half of 2019 (ClinicalTrials.gov: NCT03818347). After 0.5, 1 and 2 months of continuous hydrogen-oxygen therapy, our first three patients reported different levels of improvement in binaural hearing. This is the first report to show that, after treatment for nasopharyngeal carcinoma, hearing loss can be alleviated using hydrogen-oxygen therapy.

A Gallbladder Carcinoma Patient With Pseudo-Progressive Remission After Hydrogen Inhalation.

BACKGROUND: Hydrogen therapy has been reported to convert exhausted programmed cell death receptor (PD-1)+CD8+ T cells to PD-1-CD8+ T cells, in advanced colorectal cancer patients, which is associated with significantly prolonged survival. CASE PRESENTATION: A 72-year-old female patient presented with metastatic gallbladder cancer and underwent symptomatic treatment combined with hydrogen therapy. The tumors were initially enlarged and displayed increased tumor marker expression following hydrogen inhalation therapy, after which they continued to remit, similar to the pseudo-progression that occurs after anti-PD-1 treatment. During one month of hydrogen therapy, the patient's gallbladder and liver tumors continued to progress, and intestinal obstruction occurred. The intestinal obstruction was gradually relieved after symptomatic treatment, and the metastases in the abdominal cavity gradually decreased in size, anemia and hypoalbuminemia were corrected, and both the lymphocyte and tumor marker levels returned to normal. The patient was able to resume normal life two and a half months after hydrogen inhalation and survived over 10 months. CONCLUSION: This is the first report of pseudo-progression followed by sustained remission after hydrogen inhalation. This phenomenon is similar to the pseudo-progression-remission pattern that occurs following PD-1 antibody treatment. These findings suggest that hydrogen may have an inhibitory effect on PD-1 expression.

"Real world survey" of hydrogen-controlled cancer: a follow-up report of 82 advanced cancer patients.

Advanced cancer treatment is a huge challenge and new ideas and strategies are required. Hydrogen exerts antioxidant and anti-inflammatory effects that may be exploited to control cancer, the occurrence and progression of which is closely related to peroxidation and inflammation. We conducted a prospective follow-up study of 82 patients with stage III and IV cancer treated with hydrogen inhalation using the "real world evidence" method. After 3-46 months of follow-up, 12 patients died in stage IV. After 4 weeks of hydrogen inhalation, patients reported significant improvements in fatigue, insomnia, anorexia and pain. Furthermore, 41.5% of patients had improved physical status, with the best effect achieved in lung cancer patients and the poorest in patients with pancreatic and gynecologic cancers. Of the 58 cases with one or more abnormal tumor markers elevated, the markers were decreased at 13-45 days (median 23 days) after hydrogen inhalation in 36.2%. The greatest marker decrease was in achieved lung cancer and the lowest in pancreatic and hepatic malignancies. Of the 80 cases with tumors visible in imaging, the total disease control rate was 57.5%, with complete and partial remission appearing at 21-80 days (median 55 days) after hydrogen inhalation. The disease control rate was significantly higher in stage III patients than in stage IV patients (83.0% and 47.7%, respectively), with the lowest disease control rate in pancreatic cancer patients. No hematological toxicity was observed although minor adverse reactions that resolved spontaneously were seen in individual cases. In patients with advanced cancer, inhaled hydrogen can improve patients' quality-of-life and control cancer progression. Hydrogen inhalation is a simple, low-cost treatment with few adverse reactions that warrants further investigation as a strategy for clinical rehabilitation of patients with advanced cancer. The study protocol received ethical approval from the Ethics Committee of Fuda Cancer Hospital of Jinan University on December 7, 2018 (approval number: Fuda20181207).

Hydrogen gas therapy induced shrinkage of metastatic gallbladder cancer: A case report.

BACKGROUND: We present the case of a 72-year-old female patient with gallbladder cancer (GBC) who developed in situ recurrence and liver metastases 9 mo after irreversible electroporation ablation and oral tegafur (a fluoropyrimidine derivative) chemotherapy, which failed to control the progression of the disease. The patient further developed metastases in the lymph nodes around the head of the pancreas. The patient had severe anemia, requiring weekly blood transfusions. The gallbladder tumor invaded the descending part of the duodenum, causing intestinal leakage and hepatic colonic adhesion. CASE SUMMARY: The patient refused other treatments and began daily hydrogen inhalation therapy. After 1 mo of treatment, the gallbladder and liver tumors continued to progress, and intestinal obstruction occurred. After continuous hydrogen therapy and symptomatic treatments including gastrointestinal decompression and intravenous nutrition support, the intestinal obstruction was gradually relieved. Three months after hydrogen therapy, the metastases in the abdominal cavity gradually reduced in size, her anemia and hypoalbuminemia were corrected, lymphocyte and tumor marker levels returned to normal, and the patient was able to resume normal life. CONCLUSION: This is the first report of an efficacy and safety study about hydrogen therapy in patient with metastatic GBC and a critical general condition, who has remained stable for more than 4 months.

Long-term response of metastatic renal clear cell carcinoma following a subcutaneous injection of mixed bacterial vaccine: a case report.

In this study, we present the case of a 56-year-old patient with renal clear cell carcinoma who developed lung metastases 13 months after nephrectomy and subsequently received tyrosine kinase inhibitor (sunitinib) and PD-1 antibody (nivolumab) immunotherapy, which failed to control the progression of the disease. The patient further developed metastases to the left pleura, bilateral hilar lymph nodes, liver, right lower kidney, scapula, left sixth rib, right tonsil, and other organs. There was severe anemia, requiring weekly blood transfusions. Karnofsky score was 30. After receiving mixed bacterial vaccine (MBV) consisting of 6 kinds of heat-inactivated bacteria plus Poly I:C, the patient's condition rapidly improved, systemic metastases gradually reduced in size or disappeared, anemia was corrected, and the patient was able to resume normal life and work. MBV treatment in the setting of failure of previous immunotherapy treatment appears to have achieved objective response for this patient with metastatic renal clear cell carcinoma, which has lasted more than 20 months.

Allogenic Vγ9Vδ2 T cell as new potential immunotherapy drug for solid tumor: a case study for cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly aggressive and fatal tumor. CCA occurs in the epithelial cells of bile ducts. Due to increasing incidences, CCA accounts for 3% of all gastrointestinal malignancies. In addition to comprehensive treatments for cancer, such as surgery, chemotherapy, and radiotherapy, during the past few years, cellular immunotherapy has played an increasingly important role. As a result of our research, we have discovered the γδ T cell-based immunotherapy for CCA. CASE PRESENTATION: A 30-year-old male ( https://www.clinicaltrials.gov/ ID: NCT02425735) was diagnosed with recurrent mediastinal lymph node metastasis after liver transplantation because of Cholangiocarcinoma (stage IV). In the course of his therapy sessions, he only received allogenic γδ T cell immunotherapy from August, 2017 through February, 2018 (8 infusions in total). γδ T cells were expanded from peripheral blood mononuclear cells (PBMCs) of healthy donor, and ~ 4 × 108 cells were adoptive transferred to the patient. CONCLUSION: In the above case report of the Cholangiocarcinoma (stage IV) patient who had received liver transplantation and afterward was diagnosed with recurrent mediastinal lymph node metastasis, we clinically proved that allogenic γδ T cell treatment had no adverse effects. We observed that allogenic γδ T cell treatments positively regulated peripheral immune functions of the patient, depleted tumor activity, improved quality of life, and prolonged his life span. After 8 γδ T cell treatments, the size of lymph nodes was remarkably reduced with activity depletion. This clinical work suggested that allogenic γδ T cell immunotherapy could be developed into a promising therapy drug for CCA.

Brain Metastases Completely Disappear in Non-Small Cell Lung Cancer Using Hydrogen Gas Inhalation: A Case Report.

Lung cancer is the most common type of tumor, prone to contralateral lung, bone and brain metastasis. We report a 44-year-old woman diagnosed with lung cancer with multiple metastases in November 2015. Oral targeted drugs were initiated after the removal of brain metastases, and most lesions remained stable for 28 months. In March 2018, intracranial multiple metastases, as well as hydrocephalus accumulation in the third ventricle and lateral ventricles, and metastases in bone, adrenal gland, liver were noted. Hydrogen-gas monotherapy was started to control the tumor a month later. After 4 months, the size of multiple brain tumors was reduced significantly, and the amount of hydrocephalus in the third ventricle and lateral ventricles reduced significantly. After 1 year, all brain tumors had disappeared, and there were no significant changes in metastases in the liver and lung. These data show that, after standard treatments had failed, hydrogen-gas monotherapy elicited significant effective control of tumors (especially those in the brain), and survival time was lengthened.

Iodine-125 seed implantation and allogenic natural killer cell immunotherapy for hepatocellular carcinoma after liver transplantation: a case report.

For advanced hepatocellular carcinoma (HCC) patients, liver transplantation (LT) is an optimal treatment with limitation of high risk of tumor recurrence related to the immunosuppressive chemotherapy as usually recommended. In this study, a 29-year-old man suffered from HCC recurrence after LT. He underwent radiotherapy (total dose: 45 Gy) but had no significant response. Then, he received iodine-125 seed implantation combined with allogenic natural killer (NK) cell immunotherapy. Liver function, immune function, circulating tumor cell counts and computed tomography scans were evaluated to determine the clinical effect. We found that this combined treatment produced enhanced immune function of the patient and reduction in tumor size. This is the first report of an efficacy and safety study about clinical regimen comprising allogenic NK cell immunotherapy combined with iodine-125 seed implantation for the treatment of HCC recurrence after LT.