A novel ganglioside, 9-O-acetyl GD1b, is recognized by serum antibodies in Guillain-Barré syndrome

A hitherto undescribed ganglioside was detected in a crude ganglioside fraction of bovine brain using an IgM M-protein binding to Ga1β 1,3Ga1NAc residue. We purified and identified it as 9-O-acetyl GD1b based on results of alkali treatment that yielded GD1b and results of fast atom bombardment-mass and gas chromatography-mass spectrometries. 9-O-acetyl GD1b was also found to be present in human peripheral nerve tissue. The reactivities of the serum antibodies from patients with Guillain-Barré syndrome to 9-O-acetyl GD1b, GD1b, and GM1 were determined by ELISA and TLC immunostaining. Nineteen of 85 serum samples from Guillain-Barré syndrome patients had antibodies that bound to 9-O-acetyl GD1b: 14 of the positive samples also reacted with GM1 and GD1b, three reacted with GM1 but not with GD1b, one with GD1b but not with GM1, and one with neither GM1 nor GD1b. These results show that a subset of patients with Guillain-Barré syndrome had antibodies that react with 9-O-acetyl GD1b; therefore, this ganglioside can serve as a target antigen against the antibodies present in Guillain-Barré syndrome.     

Disialoganglioside GD2 in human neuroectodermal tumor cell lines and gliomas

Summary Monoclonal antibodies (mAbs) recognizing the disialoganglioside II³(NeuAc)₂GgOse₃Cer (GD2) were produced by immunizing mice with the GD2-expressing neuroblastoma cell line LAN-1 and a prefusion boost with purified GD2 coupled to Salmonella minnesota. Two IgM mAbs were isolated which demonstrated high levels of reactivity (binding ratios in excess of 100) with GD2 by solid-phase radioimmunoassay and positivity in high-performance thin-layer chromatography (HPTLC) immunostain; only one (DMAb-20) was subsequently shown by analysis with a panel of defined ganglioside species to be specific for the minimum epitope of GD2, GalNAc1-4(NeuAc2-8NeuAc2-3)Gal-. DMAb-20 was used to evaluate the expression of GD2 by malignant glioma and medulloblastoma cell lines using cell surface radioimmunoassay, indirect membrane immunofluorescence, HPTLC immunostain, and densitometric analysis of extracted gangliosides from selected cell lines. Sixteen of 20 (80%) malignant glioma and 5 of 5 medulloblastoma cell lines reacted with DMAb-20; in agreement with previous studies, 5 of 5 neuroblastoma and 2 of 3 melanoma cell lines also reacted with DMAb-20. GD2 was proportionally increased in the glioma and medulloblastoma cell lines relative to levels in normal brain, as determined by densitometric analysis. In a phenotypic survey of malignant glioma biopsies, tumor cells in 24 of 30 (80%) cases stained positively with DMAb-20. Reactive astrocytes, both within and adjacent to tumors, were frequently intensely stained. Among the morphological variants of glioblastoma examined, the most intense staining with DMAb-20 was observed in neoplastic gemistocytes, with the weakest or absent staining in small cell glioblastomas. As GD2 is a commonly expressed surface antigen of gliomas and medulloblastomas, expression of which is retained in tissue culture, DMAb-20 will be useful in determining the functional role of GD2 in cell-cell interaction, adhesion, and invasion, and in defining altered growth control mechanisms of central nervous system neoplasms in in vitro models.Abbreviations xxGangliosides have been designated according to CBN recommendations [22] and to the coding system of Svennerholm [35] GD2 II³(NeuAc)₂GgOse₃Cer - GM3 II³NeuAc-LacCer - GD3 II³(NeuAc)₂-LacCer - GM2 II³NeuAcGgOse₃Cer - GM1 II³NeuAcGgOse₄Cer - GD1a II³NeuAcIV³NeuAcGgOse₄Cer - GD1b II³(NeuAc)₂GgOse₄Cer - GT1b IV³NeuAcII³(NeuAc)₂GgOse₄Cer - GQ1b IV³(NeuAc)₂II³(NeuAc)₂GgOse₄Cer - 3,8-LD1 IV³(NeuAc)₂nLeOse₄Cer Supported in part by NIH Grants R37 CA 11898, NS 20023, CA 32672, and T32-NS 07304, and by a grant from the Swedish Medical Research Council (Project 03X-627). Dr. Longee is an Association of Medical School Pediatric Department Chairmen, Inc., Pediatric Scientist Training Program Fellow supported by St. Jude Children's Research Hospital     

Graves病患者外周血细胞凋亡、NO、NOS水平的变化及其临床意义

探讨GD患者细胞凋亡、NO、NOS水平的变化及相互关系与甲状腺免疫紊乱的关系. GD组28例, 对照组21名. 分别测定细胞凋亡、NO、NOS的含量. GD组细胞凋亡、NO、NOS水平均明显高于对照组(P＜0.01).直线相关分析显示, 细胞凋亡与NO、NOS呈明显正相关(r=0.438, P＜0.01; r=0.496,P＜0.01).结论是: GD患者外周血细胞凋亡与NO、NOS的变化密切相关, 可介导免疫紊乱; 调控NO与NOS可能利于GD病情的转归.     

Effect of human leukocyte antigen class II genes on Hashimoto’s thyroiditis requiring replacement therapy with levothyroxine in the Japanese population

Contribution of the human leukocyte antigen (HLA) subtype to Hashimoto’s thyroiditis (HT) that requires replacement therapy with levothyroxine remains unclear in the Japanese population. The frequencies of HLA DR–DQ haplotypes were compared between patients with HT requiring levothyroxine replacement therapy and the control individuals. We studied 82 patients with HT requiring levothyroxine replacement therapy. The frequencies of DRB1∗08:03–DQB1∗06:01 and DRB1∗09:01–DQB1∗03:03 haplotypes were significantly higher in HT patients, whereas those of DRB1∗13:02–DQB1∗06:04 and DRB1∗15:01–DQB1∗06:02 haplotypes were significantly lower in these patients than in the controls. Deduced from known linkage disequilibria, DRB1∗13:02–DQB1∗06:04 and DRB1∗15:01–DQB1∗06:02 haplotypes share the same DQA1∗01:02 allele. Since DQB1∗06:02 and DQB1∗06:04 molecules differ in the beta chain by 7 residues, these DQB1 genes are very similar. The DQA1∗01:02–DQB1∗06 (DQB1∗06:02 or DQB1∗06:04) haplotype might play a pivotal role in the resistance to HT.     

促甲状腺激素、甲状腺过氧化物酶抗体和促甲状腺激素受体抗体检测在甲状腺疾病中的应用价值

探讨促甲状腺激素（TSH）、甲状腺过氧化物酶抗体（TPOAb）、促甲状腺激素受体抗体（TRAb）在甲状腺疾病中的诊断价值。对44例Graves病（GD）、29例桥本甲状腺炎（HT）、13例亚甲炎患者和26例正常对照,采用电化学发光免疫分析法测定TSH、TPOAb、TRAb的含量,分析其在几组病例中的意义。三病例组的TSH、TPOAb、TRAb含量与正常对照组比均有显著差异（P〈0.01）;HT组TPOAb的测值和阳性率又显著高于GD组,而GD组TRAb的测值和阳性率都显著高于HT组,经统计学检验两者均有显著意义（P〈0.05）。结果说明,TSH是甲状腺功能的非常敏感的特异性参数,TPOAb、TRAb的检测对鉴别诊断GD和HT有着重要意义。     

Intratumoral immunosuppression profiles in 11q‐deleted neuroblastomas provide new potential therapeutic targets

High‐risk neuroblastoma (NB) patients with 11q deletion frequently undergo late but consecutive relapse cycles with fatal outcome. To date, no actionable targets to improve current multimodal treatment have been identified. We analyzed immune microenvironment and genetic profiles of high‐risk NB correlating with 11q immune status. We show in two independent cohorts that 11q‐deleted NB exhibits various immune inhibitory mechanisms, including increased CD4+ resting T cells and M2 macrophages, higher expression of programmed death‐ligand 1, interleukin‐10, transforming growth factor‐beta‐1, and indoleamine 2,3‐dioxygenase 1 (P < 0.05), and also higher chromosomal breakages (P ≤ 0.02) and hemizygosity of immunosuppressive miRNAs than MYCN‐amplified and other 11q‐nondeleted high‐risk NB. We also analyzed benefits of maintenance treatment in 83 high‐risk stage M NB patients focusing on 11q status, either with standard anti‐GD2 immunotherapy (n = 50) or previous retinoic acid‐based therapy alone (n = 33). Immunotherapy associated with higher EFS (50 vs. 30, P = 0.028) and OS (72 vs. 52, P = 0.047) at 3 years in the overall population. Despite benefits from standard anti‐GD2 immunotherapy in high‐risk NB patients, those with 11q deletion still face poor outcome. This NB subgroup displays intratumoral immune suppression profiles, revealing a potential therapeutic strategy with combination immunotherapy to circumvent this immune checkpoint blockade.

Abbreviations

11q‐del

11q‐deleted

ADCC

antibody‐dependent cellular cytotoxicity

CDC

complement‐dependent cytotoxicity

COJEC

chemotherapeutic agents cisplatin, vincristine, carboplatin, etoposide, and cyclophosphamide

CTLA‐4

cytotoxic T lymphocyte antigen 4

EFS

event‐free survival

FISH

fluorescence in situ hybridization

HR

hazard ratio

ICI

immune checkpoint inhibitor

IDO1

indoleamine 2,3‐dioxygenase 1

IFN‐γ

interferon‐γ

IL‐10

interleukin 10

INRG

International Neuroblastoma Risk Group

miR

microRNA

MLPA

multiplex ligation‐dependent probe amplification

MMR

mismatch repair

MNA

MYCN amplification

MS

metastatic special stage

MSI

microsatellite instability

NB

neuroblastoma

NCA

numerical chromosome aberrations

NOS

nitric oxide synthase

OS

overall survival

PD‐1

programmed cell death protein 1

PD‐L1

programmed death‐ligand 1

SCA

segmental chromosome aberrations

TAM

tumor‐associated macrophages

Tfh

follicular helper T cells

TGF‐β

tumor growth factor‐β

TMB

tumor mutational burden

TME

tumor microenvironment

TNF‐α

tumor necrosis factor‐α

Treg

regulatory T cells

     

ASC amino acid transporter 2, defined by enzyme‐mediated activation of radical sources,enhances malignancy of GD2‐positive small‐cell lung cancer

Ganglioside GD2 is specifically expressed in small‐cell lung cancer (SCLC) cells, leading to enhancement of malignant phenotypes, such as cell proliferation and migration. However, how GD2 promotes malignant phenotypes in SCLC cells is not well known. In this study, to reveal the mechanisms by which GD2 increases malignant phenotypes in SCLC cells, we used enzyme‐mediated activation of radical sources combined with mass spectrometry in GD2⁺ SCLC cells. Consequently, we identified ASC amino acid transporter 2 (ASCT2), a major glutamine transporter, which coordinately works with GD2. We showed that ASCT2 was highly expressed in glycolipid‐enriched microdomain/rafts in GD2⁺ SCLC cells, and colocalized with GD2 in both proximity ligation assay and immunocytostaining, and bound with GD2 in immunoprecipitation/TLC immunostaining. Malignant phenotypes of GD2⁺ SCLC cells were enhanced by glutamine uptake, and were suppressed by L‐γ‐glutamyl‐p‐nitroanilide, a specific inhibitor of ASCT2, through reduced phosphorylation of p70 S6K1 and S6. These results suggested that ASCT2 enhances glutamine uptake in glycolipid‐enriched microdomain/rafts in GD2⁺ SCLC cells, leading to the enhancement of cell proliferation and migration through increased phosphorylation of the mTOR complex 1 signaling axis.     

Paternal treatment with cisplatin impairs reproduction of adult male offspring in rats

This study evaluated the acute and persistent effects of paternal cisplatin-treatment on progeny. Wistar male rats (45 days old) were assigned into 2 groups. Control and cisplatin (CP: 1 mg/kg-d, 5 days/week, for 3 weeks, ip.). Male rats at 66 (end of treatment, acute effects evaluation) and 140 days old (after recovery period, persistent effects evaluation) were mated with females. Fetal and post-natal developments of the offspring sired by treated-male mated in both ages were evaluated, including fertility of adult male offspring. No adverse effects in fetal development or puberty onset were seen in CP offspring. However, testicular descent was delayed and postnatal growth was impaired in these animals (acute effect). Moreover, seminal vesicle weight and epididymal sperm count from adult progeny were affected (acute effects) by paternal CP-exposure. The only persistent effect found was alterations in the spermatogenesis. We conclude that paternal CP-administration during peri-puberty affects reproductive endpoints of the progeny.     

A transplantable TH‐MYCN transgenic tumor model in C57Bl/6 mice for preclinical immunological studies in neuroblastoma

Current multimodal treatments for patients with neuroblastoma (NBL), including anti‐disialoganglioside (GD2) monoclonal antibody (mAb) based immunotherapy, result in a favorable outcome in around only half of the patients with advanced disease. To improve this, novel immunocombinational strategies need to be developed and tested in autologous preclinical NBL models. A genetically well‐explored autologous mouse model for NBL is the TH‐MYCN model. However, the immunobiology of the TH‐MYCN model remains largely unexplored. We developed a mouse model using a transplantable TH‐MYCN cell line in syngeneic C57Bl/6 mice and characterized the immunobiology of this model. In this report, we show the relevance and opportunities of this model to study immunotherapy for human NBL. Similar to human NBL cells, syngeneic TH‐MYCN‐derived 9464D cells endogenously express the tumor antigen GD2 and low levels of MHC Class I. The presence of the adaptive immune system had little or no influence on tumor growth, showing the low immunogenicity of the NBL cells. In contrast, depletion of NK1.1+ cells resulted in enhanced tumor outgrowth in both wild‐type and Rag1^?/? mice, showing an important role for NK cells in the natural anti‐NBL immune response. Analysis of the tumor infiltrating leukocytes ex vivo revealed the presence of both tumor associated myeloid cells and T regulatory cells, thus mimicking human NBL tumors. Finally, anti‐GD2 mAb mediated NBL therapy resulted in ADCC in vitro and delayed tumor outgrowth in vivo. We conclude that the transplantable TH‐MYCN model represents a relevant model for the development of novel immunocombinatorial approaches for NBL patients.