For effective CP infusion in MERS, donor plasma with a neutralization activity of a PRNT titre ≥1:80 should be used. ELISA IgG could substitute for the neutralization test in resource-limited situations.

Purpose To develop new therapies for children with solid tumors, we tested the cytotoxicity of natural killer (NK) cells expanded by coculture with K562-mb15-41BBL cells. We sought to identify the most sensitive tumor subtypes, clarify the molecular interactions regulating cytotoxicity, and determine NK anti-tumor potential in vivo. Experimental Design We tested in vitro cytotoxicity of expanded NK cells against cell lines representative of Ewing sarcoma (EWS) (n=5), rhabdomyosarcoma (n=4), neuroblastoma (n=3) and osteosarcoma (n=3), and correlated the results with expression of inhibitory and activating NK receptor ligands. We also compared expanded and primary NK cells, determined the effects of activating-receptor ligation and of chemotherapeutic drugs, and assessed the therapeutic effect of NK cell infusions in xenografts. Results In 45 experiments, EWS and rhabdomyosarcoma cell lines were remarkably sensitive to expanded NK cells, with median cytotoxicities at 1:1 effector:target ratio of 87.2% and 79.1%, respectively. Cytotoxicity was not related to levels of expression of NK receptor ligands, nor was it affected by pretreatment of target cells with daunorubicin or vincristine, but was markedly inhibited by preincubation of NK cells with a combination of antibodies against the NK activating receptors NKGD2 and DNAM-1. Expanded NK cells were considerably more cytototoxic than unstimulated NK cells, and eradicated EWS cells engrafted in NOD/scid IL2RGnull mice. Conclusions Among pediatric solid tumors, EWS and rhabdomyosarcoma are exquisitely sensitive to expanded NK cells. The NK expansion method described here has been adapted to large-scale conditions, and supports a Phase I clinical study including patients with these malignancies.

No abstract

Sapovirus, a member of the Caliciviridae family, is an important cause of acute gastroenteritis in humans and pigs. Currently, the porcine sapovirus (PSaV) Cowden strain remains the only cultivable member of the Sapovirus genus. While some caliciviruses are known to utilize carbohydrate receptors for entry and infection, a functional receptor for sapovirus is unknown. To characterize the functional receptor of the Cowden strain of PSaV, we undertook a comprehensive series of protein-ligand biochemical assays in mock and PSaV-infected cell culture and/or piglet intestinal tissue sections. PSaV revealed neither hemagglutination activity with red blood cells from any species nor binding activity to synthetic histo-blood group antigens, indicating that PSaV does not use histo-blood group antigens as receptors. Attachment and infection of PSaV were markedly blocked by sialic acid and Vibrio cholerae neuraminidase (NA), suggesting a role for α2,3-linked, α2,6-linked or α2,8-linked sialic acid in virus attachment. However, viral attachment and infection were only partially inhibited by treatment of cells with sialidase S (SS) or Maackia amurensis lectin (MAL), both specific for α2,3-linked sialic acid, or Sambucus nigra lectin (SNL), specific for α2,6-linked sialic acid. These results indicated that PSaV recognizes both α2,3- and α2,6-linked sialic acids for viral attachment and infection. Treatment of cells with proteases or with benzyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside (benzylGalNAc), which inhibits O-linked glycosylation, also reduced virus binding and infection, whereas inhibition of glycolipd synthesis or N-linked glycosylation had no such effect on virus binding or infection. These data suggest PSaV binds to cellular receptors that consist of α2,3- and α2,6-linked sialic acids on glycoproteins attached via O-linked glycosylation.

Data on clinical isolates of Kodamaea (Pichia) ohmeri, an emerging fungal pathogen, are scarce. Over the past 5 years, we identified yeast isolates from nine patients with fungemia as K. ohmeri by using the API 20C system. Here, we reanalyzed these isolates first by sequencing the internal transcribed spacer 2 (ITS2) regions and then by growing the isolates on CHROMagar Candida medium and subjecting them to pulsed-field gel electrophoresis (PFGE). Based on their ITS2 sequences, six of the nine isolates were confirmed as K. ohmeri, while the others were identified as Candida haemulonii (n ‫؍‬ 2) and Candida parapsilosis (n ‫؍‬ 1). PFGE karyotyping of the K. ohmeri isolates revealed similar major bands, and their colonies showed a characteristic color change from pink to blue when grown on CHROMagar Candida medium for more than 48 h. For K. ohmeri, the ranges of MICs of fluconazole, voriconazole, caspofungin, and micafungin were 2 to 32 g/ml, 0.03 to 0.5 g/ml, 0.125 to 0.25 g/ml, and 0.03 to 0.06 g/ml, respectively. Restriction endonuclease analysis of genomic NotI-digested DNA (REAG-N) from isolates from different patients produced unique patterns, suggesting that the fungemia had occurred sporadically. This study determined that ITS2 sequence data, PFGE karyotypes, and CHROMagar Candida chromogenic culture medium are reliable diagnostic tools for identifying K. ohmeri while REAG-N is useful for genotyping the clinical isolates of K. ohmeri.

Korean RHD allele frequencies are comparable to Chinese and Japanese frequencies. It is concluded that the RHDel allele may actually not exist but is identical to RHD(K409K). A practical RHD genotyping strategy applicable to D- donors in all East Asian populations was devised. The strategy requires four PCR-SSP procedures only for RHD intron 4 and exon 7 as well as RHD(K409K) and non-RHD(K409K).

We examined the changes in genotypes and azole susceptibilities among sequential bloodstream isolates of Candida glabrata during the course of fungemia and the relationship of these changes to antifungal therapy. Forty-one isolates were obtained from 15 patients (9 patients who received antifungal therapy and 6 patients who did not) over periods of up to 36 days. The isolates were analyzed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and tested for antifungal susceptibility to fluconazole, itraconazole, and voriconazole. PFGE typing consisted of electrophoretic karyotyping and restriction endonuclease analysis of genomic DNA by use of NotI (REAG-N). The 41 isolates yielded 23 different karyotypes and 11 different REAG-N patterns but only 3 MLST types. The sequential strains from each patient had identical or similar REAG-N patterns. However, they had two or three different karyotypes in 6 (40%) of 15 patients. The isolates from these six patients exhibited the same or similar azole susceptibilities, and five patients did not receive antifungal therapy. Development of acquired azole resistance in sequential isolates was detected for only one patient. For this patient, an isolate of the same genotype obtained after azole therapy showed threeor fourfold increases in the MICs of all three azole antifungals and exhibited increased expression of the CgCDR1 efflux pump. This study shows that karyotypic changes can develop rapidly among sequential bloodstream strains of C. glabrata from the same patient without antifungal therapy. In addition, we confirmed that C. glabrata could acquire azole resistance during the course of fungemia in association with azole therapy.

This study aimed to assess the efficacy and safety of treatment with avelumab, an anti-programmed death ligand 1 (PD-L1) antibody, in patients with relapsed or refractory extranodal natural killer (NK)/T-cell lymphoma (ENKTL). In this phase II trial, 21 patients with relapsed or refractory ENKTL were treated with 10 mg/kg of avelumab on days 1 and 15 of a 28-day cycle (ClinicalTrials.gov Identifier: NCT03439501). The primary end point was the complete response (CR) rate based on the best response. Targeted sequencing and immunohistochemistry were performed using pretreatment tumor tissue, and blood samples were drawn pre- and post-treatment for measurement of cytokines and soluble programmed cell death protein 1 (PD1), PD-L1, and PD-L2. The CR rate was 24% (5/21) and the overall response rate was 38% (8/21). Although nonresponders showed early progression, five responders currently continue to receive treatment and have maintained their response. Most treatment-related adverse events were grade 1 or 2; no grade 4 adverse events were observed. Treatment responses did not correlate with mutation profiles, tumor mutation burden, serum levels of cytokines, or soluble PD1/PD-L1 and PD-L2. However, the response to avelumab was significantly associated with the expression of PD-L1 by tumor tissue (P = 0.001). Therefore, all patients achieving CR showed high PD-L1 expression, and their tumor subtyping based on PD-L1 expression correlated with treatment response. In conclusion, avelumab showed single-agent activity in a subset of patients with relapsed or refractory ENKTL. The assessment of PD-L1 expression on tumor cells might be helpful for identifying responders to avelumab.