Abnormal Development at Early Postimplantation Stage in Mouse Embryos After Preimplantation Genetic Diagnosis

Preimplantation genetic diagnosis (PGD) is an established procedure for the genetic analysis of embryos. To assess the effect of the procedure on early embryonic development, we generated a murine experimental system, including mice implanted with biopsied in vitro cultured embryos, control mice implanted with in vitro cultured embryos without biopsy, and mice with naturally conceived embryos. Embryos at the 7.5‐dpc stage were isolated from all three groups and the embryo implantation rate, the survival rate of implanted embryos, and the developmental stage of surviving embryos were carefully assessed and compared among all three groups. We found the implantation rate was similar between biopsied and control group embryos (67.92% vs. 66.67%). However, the survival rate of implanted embryos in the biopsied group (49.31%) was significantly lower than that of the control (60.91%) and normal groups (96.24%) at 7.5 dpc. In addition, the survival rate of control group embryos was significant lower than that of normal group embryos. Classification of the precise developmental stages of randomly selected live implanted embryos at 7.5 dpc revealed no differences among the three groups. Our results indicate that blastomere biopsy does not adversely affect embryo implantation. The PGD procedure, in particular blastomere biopsy, increases the rate of embryo death at 4.5–7.5 dpc, but does not affect the development of surviving 7.5 dpc embryos. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

李氏5号方对半乳糖老化小鼠海马神经元褪黑素受体和Nogo受体表达的影响

为了观察李氏5号方对半乳糖老化小鼠海马神经元褪黑素受体(MTR)和Nogo受体(NogoR)表达的影响。我们将昆明小鼠随机分为五组:正常对照组(C组)、D半乳糖模型组(D组)、李氏5号方大剂量药物治疗组(L组)、中剂量药物治疗组(M组)和小剂量药物治疗组(S组);D、L、M、S四组每日皮下注射半乳糖65mg/kg体重,持续三个月。应用免疫组织化学染色方法检测小鼠海马神经元MTR和NogoR的表达水平。NogoR免疫组化染色结果:D组海马阳性反应细胞着色浅,少有突起,细胞数目少,仅为正常小鼠的20%,三个剂量李氏5号方治疗组染色结果与C组相似。结果提示:半乳糖老化小鼠海马神经元MTR免疫反应阳性神经元数的上调可能是老化小鼠的一种代偿性反应;NogoR表达下调提示老化小鼠可能存在脑白质的损害,出于机体自我保护的需要,NogoR表达下调以避免神经元修复机制对大脑的进一步损害;不同剂量的李氏5号方水提取液对两种受体的调节作用极为明显,说明这两种受体的表达可受到外源性药物的干预。     

Nasal administration of interleukin‐33 induces airways angiogenesis and expression of multiple angiogenic factors in a murine asthma surrogate

The T‐helper cell type 2‐promoting cytokine interleukin‐33 (IL‐33) has been implicated in asthma pathogenesis. Angiogenesis is a feature of airways remodelling in asthma. We hypothesized that IL‐33 induces airways angiogenesis and expression of angiogenic factors in an established murine surrogate of asthma. In the present study, BALB/c mice were subjected to serial intranasal challenge with IL‐33 alone for up to 70 days. In parallel, ovalbumin (OVA) ‐sensitized mice were subjected to serial intranasal challenge with OVA or normal saline to serve as positive and negative controls, respectively. Immunohistochemical analysis of expression of von Willebrand factor and erythroblast transformation‐specific‐related gene, both blood vessel markers, and angiogenic factors angiogenin, insulin‐like growth factor‐1, endothelin‐1, epidermal growth factor and amphiregulin was performed in lung sections ex vivo. An established in‐house assay was used to test whether IL‐33 was able to induce microvessel formation by human vascular endothelial cells. Results showed that serial intranasal challenge of mice with IL‐33 or OVA resulted in proliferation of peribronchial von Willebrand factor‐positive blood vessels to a degree closely related to the total expression of the angiogenic factors amphiregulin, angiogenin, endothelin‐1, epidermal growth factor and insulin‐like growth factor‐1. IL‐33 also induced microvessel formation by human endothelial cells in a concentration‐dependent fashion in vitro. Our data are consistent with the hypothesis that IL‐33 has the capacity to induce angiogenesis at least partly by increasing local expression of multiple angiogenic factors in an allergen‐independent murine asthma surrogate, and consequently that IL‐33 or its receptor is a potential novel molecular target for asthma therapy.     

EZH2和Bmi-1基因在膀胱移行细胞癌组织中的表达及其意义

目的 观察EZH2和Bmi-1基因在膀胱正常组织和癌组织中的表达及在膀胱肿瘤发生和发展中的作用.方法 应用逆转录-聚合酶链反应(RT-PCR)和实时定量PCR的方法 ,检测EZH2和Bmi-1基因在膀胱正常黏膜(共15例)和膀胱癌中(共70例)的相对表达量,以膀胱癌T24细胞株作为阳性对照.结果 在膀胱正常黏膜中,EZH2和Bmi-1基因的mRNA不表达或低表达.而在膀胱癌组织中,两者的表达量明显高于对照组,差异有统计学意义(P＜0.01).EZH2基因在低度恶性倾向尿路上皮乳头状肿瘤(PUNLMP)组、低分级组和高分级组的表达量分别为:0.1082±0.1880、0.0740±0.0984、0.1885±0.2144,与对照组(0.0158±0.0208)比较,P值分别为＞0.05、＜0.01和＜0.01;在浸润性膀胱癌的表达(0.2069±0.2241)高于表浅性膀胱癌(0.0868±0.1233),其差异有统计学意义(P＜0.01).Bmi-1基因在PUNLMP组、低分级组和高分级组的表达量分别为:0.6654±0.7668、0.4955±0.4553、0.7986±0.9259与对照组(0.1745±0.0973)比较,P值分别为＞0.05、＞0.05和＜0.01;在浸润性膀胱癌组(0.8368±1.0041)的表达高于表浅性膀胱癌组(0.5505±0.4992),但差异无统计学意义(P＞0.05).结论 EZH2和Bmi-1基因在膀胱癌的发生中起重要作用,并且EZH2与膀胱癌的发生、进展相关.     

A Novel Rodent Orthotopic Forelimb Transplantation Model That Allows for Reliable Assessment of Functional Recovery Resulting From Nerve Regeneration

Improved nerve regeneration and functional outcomes would greatly enhance the utility of vascularized composite allotransplantation (VCA) such as hand and upper extremity transplantation. However, research aimed at achieving this goal has been limited by the lack of a functional VCA animal model. We have developed a novel rat midhumeral forelimb transplant model that allows for the characterization of upper extremity functional recovery following transplantation. At the final end point of 12 weeks, we found that animals with forelimb transplantation including median, ulnar and radial nerve coaptation demonstrated significantly improved grip strength and forelimb function as compared to forelimb transplantation without nerve approximation (grip strength: 1.71N ± 0.57 vs. no appreciable recovery; IBB scale: 2.6 ± 0.7? vs. 0.8 ± 0.40; p = 0.0005), and similar recovery to nerve transection‐and‐repair only (grip strength: 1.71N ± 0.57 vs. 2.03 ± 0.42.6; IBB scale: 2.6 ± 0.7 vs. 2.8 ± 0.8; p = ns). Moreover, all forelimb transplant animals with nerve coaptation displayed robust axonal regeneration with myelination and reduced flexor muscle atrophy when compared to forelimb transplant animals without nerve coaptation. In conclusion, this is the first VCA small‐animal model that allows for reliable and reproducible measurement of behavioral functional recovery in addition to histologic evaluation of nerve regeneration and graft reinnervation.     

Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance

Neutrophil extracellular traps (NETs) have been shown to worsen acute pulmonary injury including after lung transplantation. The breakdown of NETs by DNAse‐1 can help restore lung function, but whether there is an impact on allograft tolerance remains less clear. Using intravital 2‐photon microscopy, we analyzed the effects of DNAse‐1 on NETs in mouse orthotopic lung allografts damaged by ischemia‐reperfusion injury. Although DNAse‐1 treatment rapidly degrades intragraft NETs, the consequential release of NET fragments induces prolonged interactions between infiltrating CD4⁺ T cells and donor‐derived antigen presenting cells. DNAse‐1 generated NET fragments also promote human alveolar macrophage inflammatory cytokine production and prime dendritic cells for alloantigen‐specific CD4⁺ T cell proliferation through activating toll‐like receptor (TLR) — Myeloid Differentiation Primary Response 88 (MyD88) signaling pathways. Furthermore, and in contrast to allograft recipients with a deficiency in NET generation due to a neutrophil‐specific ablation of Protein Arginine Deiminase 4 (PAD4), DNAse‐1 administration to wild‐type recipients promotes the recognition of allo‐ and self‐antigens and prevents immunosuppression‐mediated lung allograft acceptance through a MyD88‐dependent pathway. Taken together, these data show that the rapid catalytic release of NET fragments promotes innate immune responses that prevent lung transplant tolerance.     

胆管癌靶向治疗研究进展

胆管癌(CCA)是源于胆管上皮细胞恶性增殖的较为少见的恶性肿瘤.因疾病早期无特异临床表现,诊断困难,一经诊断多为中晚期,无法手术.即使采取手术切除,术后生存率也较低.无手术指征的患者一线标准治疗(SOC)为吉西他滨联合顺铂,中位生存期小于1年.2020年4月,成纤维细胞生长因子受体(FGFR)抑制剂培米替尼获美国FDA...     

Melatonin promotes the in vitro development of pronuclear embryos and increases the efficiency of blastocyst implantation in murine

When a defect occurs in the in vitro development of a pronuclear embryo, the interruption of the subsequent implantation limits the success of assisted conception. This common problem remains to be solved. In this study, we observed that melatonin at its physiological concentration (10^?7 m ) significantly promoted the in vitro development of murine pronuclear embryos. This was indicated by the increased blastocyst rate, hatching blastocyst rate, and blastocyst cell number with melatonin treatment. In addition, when these blastocysts were implanted into female recipient mice, the pregnancy rates (95.0% versus control 67.8%), litter sizes (4.1 pups/litter versus control 2.7 pups/litter), and postnatal survival rates of offspring (96.84% versus control 81.24%) were significantly improved compared with their non‐melatonin‐treated counterparts. Mechanistic studies revealed that melatonin treatment upregulates gene expression of the antioxidant enzyme, superoxide dismutase (SOD), and the anti‐apoptotic factor bcl‐2 while downregulating the expression of pro‐apoptotic genes p53 and caspase‐3. Due to these changes, melatonin treatment reduces ROS production and cellular apoptosis during in vitro embryo development and improves the quality of blastocysts. The implantation of blastocysts with higher quality leads to more healthy offspring and increased pup survival.     

Candida tropicalis in human disease

Candida tropicalis is one of the more common Candida causing human disease in tropical countries; the frequency of invasive disease varies by geography causing 3–66% of candidaemia. C. tropicalis is taxonomically close to C. albicans and shares many pathogenic traits. C. tropicalis is particularly virulent in neutropenic hosts commonly with hematogenous seeding to peripheral organs. For candidaemia and invasive candidiasis amphotericin B or an echinocandin are recommended as first-line treatment, with extended-spectrum triazoles acceptable alternatives. Primary fluconazole resistance is uncommon but may be induced on exposure. Physicians in regions where C. tropicalis is common need to be mindful of this lesser-described pathogen.     

同基因混合G-CSF动员的异基因骨髓移植物诱导的抗白血病效应

Objective:To explore whether the graft-versus-leukemia (GVL) effects could be enhanced and acute graft-versus-host disease (aGVHD) could be relieved by syngeneic bone marrow mixed with G-CSF-primed H-2 haploidentical marrow grafting.Methods:Female L615 (H-2k) mice were recipient mice and male (615×BALB/c) F1 (6BF1) (H-2k×H-2d) mice were donors respectively.Donor mice were injected subcutaneously with G-CSF daily at 0.01 μg/g for 6 days,and splenocytes were harvested on day 7.A total of 615 mice were loaded with L615 tumor cells and received 8.5 Gy (60Co γ-ray) irradiation three days later,followed by a mixed bone marrow transplantation (MBMT).The allo-grafts consisted of a mixture of syngenetic plus G-CSF-mobilized (control diluents) H-2 haploidetical marrow cells.GVL effects were monitored by survival time and survival rate of recipient mice.GVHD was assessed by clinical signs of weight loss,ruffled fur,diarrhea and histological changes of skin,liver and small intestines.Allogeneic chimerism was detected using cytogenetic analysis.Enzyme-linked immunosorbent assay (ELISA) method was used to detect cytokines (IL-2,IL-4 and IFN-γ).Fluorescence-activated cell sorting (FACS) analysis was used to detect T-cell phenotype.Results:(1) The mice merely received L615 leukemia cells were all died of leukemia.The L615 mice of 3∶1 and 4∶1 MBMT groups survived longer than those post syngeneic BMT (P＜0.01).(2) The survival time of mice in the G-CSF-treated MBMT groups was longer than that of non-primed MBMT groups (P＜0.05).Administration of G-CSF-treated 6BF1 mice could markedly increase the survival rate of 3:1 and 4:1 MBMT mice (P＜0.01) with little or a little GVHD.(3) As the post-transplanted time prolonged,the rates of allogeneic chimerism were decreased.The chimerism rates decreased to zero when the mice died of leukemia relapse.(4) L3T4 cells and relative ratio in both subsets were significantly reduced in G-CSF-treated donor mice.After G-CSF-treated donors,splenocytes from recipients at day 14 post-MBMT showed an increased production of IL-4 and a decreased production of IL-2 and IFN-γ.Conclusion:Syngeneic bone marrow mixed with H-2 haploidentical marrow grafts had a potential way to increase GVL effects,and this GVL effects could be enhanced with little or a little GVHD by G-CSF preetreatment of donors.Improved survival in recipients of G-CSF-mobilized donors is associated with increased IL-4 production and decreased IL-2 and IFN-γ production.