Adjuvant intravesical bacillus Calmette‐Guérin (BCG) therapy is a well‐established and successful adjuvant immunotherapy in the treatment of superficial bladder cancer. Although the function of natural killer (NK) cells in other immunotherapeutic regimens (e.g., lymphokine‐activated killer [LAK] cell or interleukin‐2 [IL‐2] therapy) has been established, the contribution of NK cells to effective BCG immunotherapy is not clear. We used a human in vitro system to analyze the role of NK cells in BCG‐induced cellular cytotoxicity. After stimulation of mononuclear cells with BCG for 7 days, these BCG‐activated killer (BAK) cells displayed substantial cytotoxicity against bladder tumor cells. Magnetic depletion experiments and fluorescence activated cell sorting revealed that NK cells were the major effector cell population. To address NK cell function in vivo, we studied a syngeneic orthotopic murine bladder cancer model and compared BCG immunotherapy in C57BL/6 wild‐type mice, NK‐deficient beige mice and mice treated with anti‐NK1.1 monoclonal antibody. Four weekly instillations of viable BCG significantly prolonged survival in wild‐type mice compared with control mice treated with solvent alone. In contrast, BCG therapy was completely ineffective in NK‐deficient beige mice and in mice treated with anti‐NK1.1 monoclonal antibody. These findings suggest a key role for NK cells during BCG immunotherapy. © 2001 Wiley‐Liss, Inc.

BACKGROUND: Fusarium asiaticum is one of predominant pathogens of Fusarium head blight (FHB) in China. Pydiflumetofen (Pyd) is a novel succinate dehydrogenase inhibitor (SDHI) which has been commercialized in China for the controlling of wheat FHB since 2019. In the current study, a risk assessment of the pydiflumetofen-resistance selected in Fusarium asiaticum was investigated. RESULTS: One Pyd MR mutant [resistance factor (RF) < 80] and four Pyd HR mutants (RF > 3000) were generated by fungicidetaming from 1000 mycelial discs of the wild-type strain 2021. Nucleotide sequences alignment results of FaSdh from the wild-type strain and resistant mutants showed that all the mutations were categorized into three genotypes, i.e. FaSdhB H248Y from Pyd MR mutant, both FaSdhC 1 A64V and FaSdhC 1 R67K from Pyd HR mutants. All the resistant mutants possessed no fitness penalty based on the data of mycelial linear growth, conidiation and virulence. In addition, the FaSdhC 1 A64V mutants showed positive cross-resistance between pydiflumetofen and boscalid or thifluzamide, but no cross-resistance between pydiflumetofen and Y13149 or Y12196, while the FaSdhC 1 R67K mutants exhibited positive cross-resistance between pydiflumetofen and boscalid, thifluzamide or Y12196, and no cross-resistance between pydiflumetofen and Y13149. Furthermore, positive cross-resistance between the five tested SDHIs was detected in the FaSdhB H248Y mutants. CONCLUSION: The results suggest a moderate to high resistance risk of F. asiaticum to pydiflumetofen, and provide essential data for monitoring the emergence of resistance and resistance management strategies for pydiflumetofen, which will be useful for scientific application of this fungicide in China.

SUMMARYIntravesical BCG therapy is effective in the treatment of superficial bladder cancer. Both clinical and experimental results suggest a role for cytokines and delayed-type hypersensitivity (DTH) in BCGinduced antitumour immunity. We characterized the modulatory effects of BCG on bladder cytokine expression and determined the relationship between DTH and BCG antitumour activity. The bladders of mice were instilled with BCG through a catheter. Bladder tissue RNA and urine were collected for evaluation of cytokine expression using reverse transcriptase-polymerase chain reaction (RT-PCR) and/or ELISA. IFN-g and TNF-a , the two major cytokines associated with DTH, were efficiently induced by BCG. IL10, an important down-regulator of DTH, was also induced by BCG. Constitutive levels of IL4 and IL5 were observed, but neither IL4 nor IL5 were modulated by BCG. Similar results were observed in the kinetic analysis of urinary cytokines in patients after intravesical BCG therapy. Production of Th1 (T helper type 1) cytokines (IFN-g , IL2 and IL12) preceded that of the Th2 (T helper type 2) cytokine IL10. A tendency toward higher ratios of IFN-g versus IL10 for BCG responders also was observed. In animal studies the absence of IL10 abrogated either by antibody inhibition or the use of genetically modified, IL10 deficient (IL10 -/-) mice resulted in enhanced DTH responses. Under conditions of enhanced DTH, a significant enhancement in antitumour activity was observed. These data demonstrate that DTH and its associated mononuclear infiltration and cytokine production are important to the antitumour activity of intravesical BCG therapy, and suggest that effects to diminish IL10 production may have therapeutic value.

The attachment of mycobacteria to fibronectin is well documented (1, 2). All tested species including Mycobacterium bovis strain BCG, Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium avium, Mycobacterium leprae, and Mycobacterium smegmatis were observed to attach to fibronectin (3-5). In studies on M. bovis BCG-mediated immunotherapy, which is the treatment of choice for superficial bladder cancer, fibronectin attachment was shown to be necessary for the expression of antitumor activity (2). In addition, the attachment and internalization of M. bovis BCG, M. avium, and M. leprae by epithelial cells and Schwann cells also were shown to be dependent on bacterial attachment to fibronectin (4, 6, 7). Thus, an understanding of the interaction between mycobacterial proteins mediating attachment to fibronectin is needed.Two distinct mycobacterial proteins or protein complexes, fibronectin attachment protein (FAP) 1 and proteins of the antigen 85 complex, have been linked to mycobacterial attachment to fibronectin (8, 9). The interaction of proteins from the antigen 85 complex with fibronectin has been characterized using recombinant 85A, 85B, and 85C proteins (10 -12). The best characterized of the complex is the 85B protein. In the present study, the amino acids required for fibronectin binding of the FAP-A-(269 -292) peptide were determined us-

Chili anthracnose caused by Colletotrichum spp. is an annual production concern for growers in China. Sterol C14-demethylation inhibitors (DMIs, such as tebuconazole) have been widely used to control this disease for more than three decades. In the current study, of 48 isolates collected from commercial chili farms in Jiangsu Province of China during 2018 and 2019, 8 single-spore isolates were identified as Colletotrichum gloeosporioides and the rest were identified as C. acutatum. To determine whether the DMI resistance of isolates develops in the field, mycelial growth of the 48 isolates was measured in culture medium with and without tebuconazole. In all, 6 of the 8 C. gloeosporioides isolates were resistant to tebuconazole, but all 40 of the C. acutatum isolates were sensitive to tebuconazole. The fitness cost of resistance was low based on a comparison of fitness parameters between the sensitive and resistant isolates of C. gloeosporioides. Positive cross-resistance was observed between tebuconazole and difenconazole or propiconazole, but not prochloraz. Alignment results of the CgCYP51 amino acid sequences from the sensitive and resistant isolates indicated that mutations can be divided into three genotypes. Genotype I possessed four substitutions (V18F, L58V, S175P, and P341A) at the CgCYP51A gene but no substitutions at CgCYP51B, while genotype II had five substitutions (L58V, S175P, A340S, T379A, and N476T) at CgCYP51A, concomitant with three substitutions (D121N, T132A, and F391Y) at CgCYP51B. In addition, genotype III contained two substitutions (L58V and S175P) at CgCYP51A, concomitant with one substitution (T262A) at CgCYP51B. Molecular docking models illustrated that the affinity of tebuconazole to the binding site of the CgCYP51 protein from the resistant isolates was decreased when compared with binding site affinity of the sensitive isolates. Our findings provide not only novel insights into understanding the resistance mechanism to DMIs, but also some important references for resistance management of C. gloeosporioides on chili.

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