C.B‐17 SCID mice develop epicardial calcinosis with unaltered cardiac function

Inbred mouse strains are the most widely used mammalian model organism in biomedical research owing to ease of genetic manipulation and short lifespan; however, each inbred strain possesses a unique repertoire of deleterious homozygous alleles that can make a specific strain more susceptible to a particular disease. In the current study, we report dystrophic cardiac calcinosis (DCC) in C.B‐17 SCID male mice at 10 weeks of age with no significant change in cardiac function. Acquisition of DCC was characterized by myocardial injury, fibrosis, calcification, and necrosis of the tissue. At 10 weeks of age, 38% of the C.B‐17 SCID mice from two different commercial colonies exhibited significant calcinosis on the ventricular epicardium, predominantly on the right ventricle. The frequency of calcinosis was more than 50% for mice obtained from Taconic's Cambridge City colony and 25% for mice obtained from Taconic's German Town colony. Interestingly, the DCC phenotype did not affect cardiac function at 10 weeks of age. No differences in echocardiography or electrocardiography were observed between the calcinotic and non‐calcinotic mice from either colony. Our findings suggest that C.B‐17 SCID mice exhibit DCC as early as 10 weeks of age with no significant impact on cardiac function. This strain of mice should be cautiously considered for the study of cardiac physiology.     

Multicentric Analytical and Inter-observer Comparability of Four Clinically Developed Programmed Death-ligand 1 Immunohistochemistry Assays in Advanced Clear-cell Renal Cell Carcinoma

BackgroundPrevious studies have suggested increased clinical benefit with inhibition of programmed death-ligand 1 (PD-L1)/programmed death-1 in patients with PD-L1–positive locally advanced/metastatic renal cell carcinoma (RCC). We examined the analytical and inter-observer comparability of PD-L1–positivity across 4 clinically developed immunohistochemistry assays in clear-cell RCC (CCRCC).Materials and MethodsRandomly selected archived, formalin-fixed, paraffin-embedded nephrectomy specimens from 201 patients with locally advanced CCRCC were screened using VENTANA SP142. From these, 30 cases were selected based on their tumor-infiltrating immune cell (IC) PD-L1 status (PD-L1-IC–positivity of < 1%, 1%-5%, or > 5%; 10 cases each). These cases were stained for PD-L1 using VENTANA SP142 and SP263, and DAKO 22C3 and 28-8, and scored for PD-L1 expression on IC and tumor cells (TC) by trained readers at 5 sites.ResultsAdjusted mean percentages of PD-L1-IC–positivity and PD-L1-TC–positivity varied from 4.0% to 4.9% and from 1.3% to 10.7%, respectively, between assays. Inter-assay differences in PD-L1-IC–positivity were small and non-significant (P = .1938 to .9963); for PD-L1-TC–positivity, significant differences were observed between VENTANA SP142 and the other assays (P ≤ .0001) and between VENTANA SP263 and DAKO 28-8 (P = .0248). Intra-class correlation values showed moderate-to-high inter-reader agreement for each assay for PD-L1-IC–positivity and for 3 assays for PD-L1-TC–positivity.ConclusionsIn this first multicenter analytical comparison study of PD-L1 assays in CCRCC, PD-L1–positivity could be assessed reproducibly using all 4 assays for IC and for 3 of the 4 assays for TC.     

A new mouse model for the neurodevelopmental ciliopathy Joubert syndrome

Recent recognition of the key role of primary cilia in orchestrating human development and of the dire consequences of their dysfunction on human health has placed this small organelle in the spotlight. While the causal link between mutations in ciliary genes and central nervous system malformations and dysfunction is well established, the mechanisms by which primary cilia dysfunction acts on development and function of the CNS remain partly unknown. The recent article by Bashford and Subramanian in The Journal of Pathology describes a new mouse model for the neurodevelopmental ciliopathy Joubert syndrome, supporting a role for ciliary-mediated Hedgehog signaling on proliferation, survival, and differentiation of cerebellar granule cell progenitors. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

2‐deoxy D‐glucose treatment does not elicit a hair growth response in alopecia areata

2‐deoxy D‐glucose (2DG) was tested for efficacy in treating alopecia areata using the C3H/HeJ skin graft model. 2DG has proven to be efficacious in treatment of various mouse models of autoimmunity with minimal serious side effects noted. This agent has been shown to normalize abnormally activated T‐cell populations while also preventing cell surface expression of NKG2D; key factors defining alopecia areata disease progression. Daily oral ingestion of 2DG via drinking water to mice with patchy or diffuse alopecia areata for 16 weeks failed to prevent expansion of alopecia or cause regrowth of hair in treated mice. Histologically, there were no differences between treated and control groups. These results indicate that, while 2DG is effective for some autoimmune diseases, it was not efficacious for the cell‐mediated autoimmune mouse disease, alopecia areata.     

Genetic toxicity assessment using liver cell models: past,present, and future

ABSTRACT

Genotoxic compounds may be detoxified to non-genotoxic metabolites while many pro-carcinogens require metabolic activation to exert their genotoxicity in vivo. Standard genotoxicity assays were developed and utilized for risk assessment for over 40 years. Most of these assays are conducted in metabolically incompetent rodent or human cell lines. Deficient in normal metabolism and relying on exogenous metabolic activation systems, the current in vitro genotoxicity assays often have yielded high false positive rates, which trigger unnecessary and costly in vivo studies. Metabolically active cells such as hepatocytes have been recognized as a promising cell model in predicting genotoxicity of carcinogens in vivo. In recent years, significant advances in tissue culture and biological technologies provided new opportunities for using hepatocytes in genetic toxicology. This review encompasses published studies (both in vitro and in vivo) using hepatocytes for genotoxicity assessment. Findings from both standard and newly developed genotoxicity assays are summarized. Various liver cell models used for genotoxicity assessment are described, including the potential application of advanced liver cell models such as 3D spheroids, organoids, and engineered hepatocytes. An integrated strategy, that includes the use of human-based cells with enhanced biological relevance and throughput, and applying the quantitative analysis of data, may provide an approach for future genotoxicity risk assessment.     

Inactivated influenza vaccine formulated with single-stranded RNA-based adjuvant confers mucosal immunity and cross-protection against influenza virus infection

Influenza vaccination is considered the most valuable means to prevent and control seasonal influenza infections, which causes various clinical symptoms, ranging from mild cough and fever to even death. Among various influenza vaccine types, the inactivated subunit type is known to provide improved safety with reduced reactogenicity. However, there are some drawbacks associated with inactivated subunit type vaccines, with the main ones being its low immunogenicity and the induction of Th2-biased immune responses. In this study, we investigated the role of a single-stranded RNA (ssRNA) derived from the intergenic region in the internal ribosome entry site of the Cricket paralysis virus as an adjuvant rather than the universal vaccine for a seasonal inactivated subunit influenza vaccine. The ssRNA adjuvant stimulated not only well-balanced cellular (indicated by IgG2a, IFN-γ, IL-2, and TNF-α) and humoral (indicated by IgG1 and IL-4) immune responses but also a mucosal immune response (indicated by IgA), a key protector against respiratory virus infections. It also increases the HI titer, the surrogate marker of influenza vaccine efficacy. Furthermore, ssRNA adjuvant confers cross-protective immune responses against heterologous influenza virus infection while promoting enhanced viral clearance. Moreover, ssRNA adjuvant increases the number of memory CD4⁺ and CD8⁺ T cells, which can be expected to induce long-term immune responses. Therefore, this ssRNA-adjuvanted seasonal inactivated subunit influenza vaccine might be the best influenza vaccine generating robust humoral and cellular immune responses and conferring cross-protective and long-term immunity.     

极速实时荧光聚合酶链反应与常规方法对新型布尼亚病毒检测的评价

目的探讨极速实时荧光聚合酶链反应(polymerase chain reaction,PCR)、实时荧光PCR、酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)和胶体金免疫层析法(gold immunochromatography assay,GICA)4种方法检测新型布尼亚病毒的特异度和灵敏度,为发热伴血小板减少综合征的早期诊断提供依据。方法采集2017年6月1日至9月30日山东大学附属济南市传染病医院86例临床诊断为发热伴血小板减少综合征患者的血清样本,分别应用极速实时荧光PCR、实时荧光PCR、ELISA和GICA 4种方法进行检测。统计学分析采用χ^2检验。结果86份患者血清标本中,极速实时荧光PCR、实时荧光PCR、IgM-ELISA、IgG-ELISA、IgM-GICA、IgG-GICA的新型布尼亚病毒阳性分别为82份(95.34%)、79份(91.86%)、41份(47.67%)、8份(9.3%)、19份(22.09%)和3份(3.49%)。极速实时荧光PCR特异度为100%,灵敏度达到1×103拷贝/mL,3次重复扩增试验显示其Ct值变异系数均<2%。在发热伴血小板减少综合征进展的1期、2期、3期病程中,极速实时荧光PCR的阳性检出率为41份(97.62%)、34份(94.44%)、7份(87.50%),实时荧光PCR的阳性检出率为39份(92.86%)、33份(91.67%)、7份(87.50%),在1期和2期两个病程,极速实时荧光PCR阳性检出率略高;IgM-ELISA阳性检出率从1期(28.57%)到3期(87.50%)显著增高,2期、3期与1期相比,差异均有统计学意义(χ^2=8.347、7.561,均P<0.01);IgM-GICA的阳性检出率从1期(14.29%)到2期(33.33%)也有增高,差异有统计学意义(χ^2=3.962,P<0.05),但与其他方法相比,其检出率偏低。1期,实时荧光PCR阳性检出率显著高于ELISA(IgM和IgG)和GICA(IgM和IgG),差异均有统计学意义(χ^2=33.740、55.080、49.010、64.340,均P<0.01)。2期,实时荧光PCR的阳性检出率高于ELISA(IgM和IgG)和GICA(IgM和IgG),差异均有统计学意义(χ^2=7.700、46.720、23.700、50.630,均P<0.01)。3期,极速实时荧光PCR、实时荧光PCR和IgM-ELISA表现出同样高的阳性检出率,远高于IgG-ELISA和GICA(IgM和IgG)。实时荧光PCR阳性检出率和IgG-ELISA、IgM-GICA、IgG-GICA之间差异均有统计学意义(均χ^2=6.250,P<0.05)。结论极速实时荧光PCR在新型布尼亚病毒的早期检测中有更高的灵敏度和特异度,且重复性好、稳定度高,与传统实时荧光PCR相比大大缩短了扩增时间,对发热伴血小板减少综合征的早期快速诊断具有重要价值。     

ELAM-1在鼻咽癌裸鼠移植瘤中的表达及其与转移的关系

目的 建立裸鼠鼻咽癌转移模型并探讨 E-选择素（ELAM-1）与鼻咽癌转移的相关性。方法 将鼻咽癌5-8F细胞悬液注射于裸鼠左后肢爪垫，观察裸鼠状态、成瘤情况并测量裸鼠体重及移植瘤长短径；采用连续病理切片苏木精-伊红染色观察移植瘤及转移情况，将16只人鼻咽癌荷瘤裸鼠分为转移组和非转移组；采用免疫组织化学法检测两组移植瘤组织中ELAM-1的表达。 结果 16只裸鼠均成瘤，成瘤率为100.0%，其中10只裸鼠出现转移瘤，转移率为62.5%。建模前，两组裸鼠体重差异无统计学意义［（13.83±0.56）g vs （14.62±0.30） g，t=1.026，P=0.071］。建模后4~7周，裸鼠瘤体体积呈指数增长，且转移组移植瘤增长速度较非转移组快，非转移组裸鼠瘤体体积小于转移组［（198.91 ± 163.29） mm³ vs （268.76 ±174.31） mm³，t=4.376，P=0.005］。ELAM-1在鼻咽癌裸鼠移植瘤、淋巴结转移灶及远处转移灶中的表达均为阳性，主要表达于细胞膜。转移组移植瘤光密度值高于非转移组（0.4497±0.0705 vs 0.0435±0.0082，t=4.388，P＝0.001）。结论 本研究成功构建稳定性好、转移率高的鼻咽癌裸鼠移植瘤转移模型，且ELAM-1在裸鼠移植瘤中高表达，可促进鼻咽癌裸鼠移植瘤生长和转移。     

△LuxS的表型分析及其在GBS毒力调控机制研究中的应用

目的采用LuxS缺失突变株模型对B型链球菌中数量感应相关分子LuxS的功能及与其相关的自诱导因子-2数量感应通路进行研究，以探索其毒力调控机制。方法采用RT-PCR法、菌落印迹分析、生长曲线测定、cAMP因子测定等方法对该缺失突变菌株的表型特性进行了研究。最后应用哈氏弧菌作为报告菌株，通过生物发光测定法分析了突变株对B型链球菌诱导报告菌株中的生物发光活性的影响。结果发现此缺失突变可导致B型链球菌中scpB基因表达的上调；与野生株相比，突变株的生物发光诱导活性比野生株降低了大约两倍。结论本研究结果证实了LuxS在GBS中AI-2数量感应通路中的重要性，并为GBS中毒力调控机制的进一步研究提供了新的线索。