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.

Cetuximab combined with natural killer cells therapy: an alternative to chemoradiotherapy for patients with advanced non-small cell lung cancer (NSCLC).

Natural killer (NK) cells therapy has the potential to prolong survival in patients with advanced non-small cell lung cancer (NSCLC). We conducted a clinical trial to investigate the safety and efficacy of cetuximab plus NK cells therapy in patients with advanced NSCLC. Between June 2015 and August 2016, 54 patients with advanced EGFR-expressing NSCLC were assigned randomly to the cetuximab plus NK cells therapy group (A; n = 27) or cetuximab alone group (B; n = 27). Patients in group A received two courses of NK cells therapy continuously. Cetuximab was administered intravenously and the weekly maintenance dose was continued until tumor progression. All adverse effects were manageable and no significant difference was noted between the two groups (P > 0.05). Levels of CEA, NSE and circulating tumor cells (CTCs) in group A were significantly lower than those before treatment (P < 0.05). Patients in group A had a significant improvement in immune function and quality of life (QOL) (P < 0.05). Patients in group A survived longer than those in group B (median PFS: 6 months vs 4.5 months; median OS: 9.5 months vs 7.5 months; P < 0.05). Combination therapy could be an alternative to chemoradiotherapy for patients with advanced NSCLC.

Advances in cryoablation for pancreatic cancer.

Pancreatic carcinoma is a common cancer of the digestive system with a poor prognosis. It is characterized by insidious onset, rapid progression, a high degree of malignancy and early metastasis. At present, radical surgery is considered the only curative option for treatment, however, the majority of patients with pancreatic cancer are diagnosed too late to undergo surgery. The sensitivity of pancreatic cancer to chemotherapy or radiotherapy is also poor. As a result, there is no standard treatment for patients with advanced pancreatic cancer. Cryoablation is generally considered to be an effective palliative treatment for pancreatic cancer. It has the advantages of minimal invasion and improved targeting, and is potentially safe with less pain to the patients. It is especially suitable in patients with unresectable pancreatic cancer. However, our initial findings suggest that cryotherapy combined with 125-iodine seed implantation, immunotherapy or various other treatments for advanced pancreatic cancer can improve survival in patients with unresectable or metastatic pancreatic cancer. Although these findings require further in-depth study, the initial results are encouraging. This paper reviews the safety and efficacy of cryoablation, including combined approaches, in the treatment of pancreatic cancer.

Intraperitoneal perfusion of cytokine-induced killer cells with local hyperthermia for advanced hepatocellular carcinoma.

AIM: To study the effect and tolerance of intraperitoneal perfusion of cytokine-induced killer (CIK) cells in combination with local radio frequency (RF) hyperthermia in patients with advanced primary hepatocellular carcinoma (HCC). METHODS: Patients with advanced primary HCC were included in this study. CIK cells were perfused intraperitoneal twice a week, using 3.2 × 109 to 3.6 × 109 cells each session. Local RF hyperthermia was performed 2 h after intraperitoneal perfusion. Following an interval of one month, the next course of treatment was administered. Patients received treatment until disease progression. Tumor size, immune indices (CD3⁺, CD4⁺, CD3⁺CD8⁺, CD3⁺CD56⁺), alpha-fetoprotein (AFP) level, abdominal circumference and adverse events were recorded. Time to progression and overall survival (OS) were calculated. RESULTS: From June 2010 to July 2011, 31 patients diagnosed with advanced primary HCC received intraperitoneal perfusion of CIK cells in combination with local RF hyperthermia in our study. Patients received an average of 4.2 ± 0.6 treatment courses (range, 1-8 courses). Patients were followed up for 8.3 ± 0.7 mo (range, 2-12 mo). Following combination treatment, CD4⁺, CD3⁺CD8⁺ and CD3⁺CD56⁺ cells increased from 35.78% ± 3.51%, 24.61% ± 4.19% and 5.94% ± 0.87% to 45.83% ± 2.48% (P = 0.016), 39.67% ± 3.38% (P = 0.008) and 10.72% ± 0.67% (P = 0.001), respectively. AFP decreased from 167.67 ± 22.44 to 99.89 ± 22.05 ng/mL (P = 0.001) and abdominal circumference decreased from 97.50 ± 3.45 cm to 87.17 ± 4.40 cm (P = 0.002). The disease control rate was 67.7%. The most common adverse events were low fever and slight abdominal erubescence, which resolved without treatment. The median time to progression was 6.1 mo. The 3-, 6- and 9-mo and 1-year survival rates were 93.5%, 77.4%, 41.9% and 17.4%, respectively. The median OS was 8.5 mo. CONCLUSION: Intraperitoneal perfusion of CIK cells in combination with local RF hyperthermia is safe, can efficiently improve immunological status, and may prolong survival in HCC patients.

Cryosurgery in combination with brachytherapy of iodine-125 seeds for pancreatic cancer.

A major limit of cryoablation is incomplete destruction of cells in the border zone of the cryogenic lesion in which the tissue temperature is warmer than (-)20 °C. The use of iodine-125 seed implantation is likely to be complementary to cryosurgery for treatment of pancreatic cancer. The procedure of cryosurgery and iodine-125 seed implantation is performed with percutaneous approaches under guidance of ultrasound and/or CT. The number of iodine-125 seeds implanted for every patient was 34 in median. Forty-nine patients with locally advanced pancreatic cancer received cryosurgery with combination of iodine-125 seed implantation. During a median follow-up of 18 months, the median of over all survival was 16.2 months. The 6-, 12-, 24- and 36-month overall survival rates were 94.9%, 63.1%, 22.8% and 9.5%, respectively. Compared with patients with cryosurgery alone, combination treatment shows higher the 6- and 12-month survival rates and longer the median survival.

A pilot study on combination of cryosurgery and (125)iodine seed implantation for treatment of locally advanced pancreatic cancer.

AIM: To study the therapeutic value of combination of cryosurgery and (125)iodine seed implantation for locally advanced pancreatic cancer. METHODS: Forty-nine patients with locally advanced pancreatic cancer (males 36, females 13), with a median age of 59 years, were enrolled in the study. Twelve patients had liver metastases. In all cases the tumors were considered unresectable after a comprehensive evaluation. Patients were treated with cryosurgery, which was performed intraoperatively or percutaneously under guidance of ultrasound and/or computed tomography (CT), and (125)iodine seed implantation, which was performed during cryosurgery or post-cryosurgery under guidance of ultrasound and/or CT. A few patients received regional celiac artery chemotherapy. RESULTS: Thirteen patients received intraoperative cryosurgery and 36 received percutaneous cryosurgery. Some patients underwent repeat cryosurgery. (125)Iodine seed implantation was performed during freezing procedure in 35 patients and 3-9 d after cryosurgery in 14 cases. Twenty patients, 10 of whom had hepatic metastases received regional chemotherapy. At 3 mo after therapy, CT was repeated to estimate tumor response to therapy. Most patients showed varying degrees of tumor necrosis. Complete response (CR) of tumor was seen in 20.4% patients, partial response (PR), in 38.8%, stable disease (SD), in 30.6%, and progressive disease (PD), in 10.2%. Adverse effects associated with cryosurgery included upper abdomen pain and increased serum amylase. Acute pancreatitis was seen in 6 patients one of whom developed severe pancreatitis. All adverse effects were controlled by medical management with no poor outcome. There was no therapy-related mortality. During a median follow-up of 18 mo (range of 5-40), the median survival was 16.2 mo, with 26 patients (53.1%) surviving for 12 mo or more. Overall, the 6-, 12-, 24- and 36-mo survival rates were 94.9%, 63.1%, 22.8% and 9.5%, respectively. Eight patients had survival of 24 mo or more. The patient with the longest survival (40 mo) is still living without evidence of tumor recurrence. CONCLUSION: Cryosurgery, which is far less invasive than conventional pancreatic resection, and is associated with a low rate of adverse effects, should be the treatment of choice for patients with locally advanced pancreatic cancer. (125)Iodine seed implantation can destroy the residual surviving cancer cells after cryosurgery. Hence, a combination of both modalities has a complementary effect.

[Clinical and pathological study on effects of Qianggan Capsule combined lamivudine on hepatic fibrosis in patients with chronic hepatitis B].

OBJECTIVE: To study the therapeutic effects of Qianggan Capsule (QC) combined Lamivudine on hepatic fibrosis in patients with chronic hepatitis B. METHODS: Eighty-five patients were randomly divided into two groups, group A (40 cases) were treated with QC and Lamivudine and group B (45 cases) were treated with QC alone both for 6 months. Hepatic fibrosis related indexes and pathologic examination of liver biopsy were performed within 3 months before treatment and in 1 month after treatment. RESULTS: Serum levels of hyaluronic acid, collagen N and laminin decreased markedly after treatment in both groups (P < 0.05). Hepatic histopathological examination showed that the total effective rate of impovement in activity of inflammation-necrosis and fibrosis was 80.0% and 70.0% in group A, 57.8% and 75.6% in group B, respectively, the combined treatment showed a better effect in improving the activity of inflammation-necrosis than QC alone (P < 0.05), but with no significant difference to the latter in improving fibrosis. CONCLUSION: QC combined Lamivudine could markedly reduce the activity of hepatic inflammation-necrosis, QC alone could also improve hepatic fibrosis.

[Expression of bcl-2 gene in spleen deficiency syndrome in colorectal carcinoma and the regulatory effect of Jianpikangfu decoction].

OBJECTIVE: To investigate the correlation between spleen deficiency syndrome in colorectal carcinoma and bcl-2 gene expression, and observe the regulatory effect of Jianpikangfu decoction. METHODS: Forty-five advanced colorectal carcinoma patients with spleen deficiency were randomized into Jianpikangfu decoction treatment group with also symptomatic treatment with western medicine and control group in which the patients were given expectant treatment with western medicine. The activity of salivary amylase and bcl-2 expression in the tumor tissues were detected before and after the treatment. RESULTS: Jianpikangfu decoction in combination with western medicine treatment produced more obvious inhibition of reduction in salivary amylase activity than exclusive western medicine treatment (t=7.822, P<0.01), and significantly lowered the positivity rate of bcl-2 expression (chi2=4.286, P<0.05) in the tumor tissues, which, however, displayed no obvious changes in response to exclusive western medicine treatment. CONCLUSION: Jianpikangfu decoction can inhibit the decrease in salivary amylase activity and regulate bcl-2 gene expression in colorectal carcinoma patients with spleen deficiency syndrome.