HIV-1 tracing method of systemic viremia in vivo using an artificially mutated virus pool

The appearance of human immunodeficiency virus type 1 (HIV-1) plasma viremia is associated with progression to symptomatic disease and CD4⁺ T cell depletion. To locate the source of systemic viremia, this study employed a novel method to trace HIV-1 infection in vivo. We created JRCSFξnef, a pool of infectious HIV-1 (strain JR-CSF) with highly mutated nef gene regions by random mutagenesis PCR and infected this mutated virus pool into both Jurkat-CCR5 cells and hematopoietic stem cell-transplanted humanized mice. Infection resulted in systemic plasma viremia in humanized mice and viral RNA sequencing helped us to identify multiple lymphoid organs such as spleen, lymph nodes, and bone marrow but not peripheral blood cells as the source of systemic viremia. Our data suggest that this method could be useful for the tracing of viral trafficking in vivo.     

Partial protection against P. vivax infection diminishes hypnozoite burden and blood-stage relapses

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A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of “randomly paired” scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.     

Hsp90 inhibitors suppress HCV replication in replicon cells and humanized liver mice

Persistent infection with hepatitis C virus (HCV) is a major cause of liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Here we report that inhibition of heat shock protein 90 (Hsp90) is highly effective in suppressing HCV genome replication. In HCV replicon cells, HCV replication was reduced by Hsp90 inhibitors and by knockdown of endogenous Hsp90 expression mediated by small-interfering RNA (siRNA). The suppression of HCV replication by an Hsp90 inhibitor was prevented by transfection with Hsp90 expression vector. We also tested the anti-HCV effect of Hsp90 inhibition in HCV-infected chimeric mice with humanized liver. Combined administration of an Hsp90 inhibitor and polyethylene glycol-conjugated interferon (PEG-IFN) was more effective in reducing HCV genome RNA levels in serum than was PEG-IFN monotherapy. These results suggest that inhibition of Hsp90 could provide a new therapeutic approach to HCV infection.     

Human-Specific Adaptations in Vpu Conferring Anti-tetherin Activity Are Critical for Efficient Early HIV-1 Replication In Vivo

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Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle

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Cancer‐associated POT1 mutations lead to telomere elongation without induction of a DNA damage response

Mutations in the shelterin protein POT1 are associated with chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, angiosarcoma, melanoma, and other cancers. These cancer‐associated POT1 (caPOT1) mutations are generally heterozygous, missense, or nonsense mutations occurring throughout the POT1 reading frame. Cancers with caPOT1 mutations have elongated telomeres and show increased genomic instability, but which of the two phenotypes promotes tumorigenesis is unclear. We tested the effects of CAS9‐engineered caPOT1 mutations in human embryonic and hematopoietic stem cells (hESCs and HSCs, respectively). HSCs with caPOT1 mutations did not show overt telomere damage. In vitro and in vivo competition experiments showed the caPOT1 mutations did not confer a selective disadvantage. Since DNA damage signaling is known to affect the fitness of HSCs, the data argue that caPOT1 mutations do not cause significant telomere damage. Furthermore, hESC lines with caPOT1 mutations showed no detectable telomere damage response while showing consistent telomere elongation. Thus, caPOT1 mutations are likely selected for during cancer progression because of their ability to elongate telomeres and extend the proliferative capacity of the incipient cancer cells.     

Establishment of peripheral blood mononuclear cell-derived humanized lung cancer mouse models for studying efficacy of PD-L1/PD-1 targeted immunotherapy

ABSTRACT

Animal models used to evaluate efficacies of immune checkpoint inhibitors are insufficient or inaccurate. We thus examined two xenograft models used for this purpose, with the aim of optimizing them. One method involves the use of peripheral blood mononuclear cells and cell line-derived xenografts (PBMCs-CDX model). For this model, we implanted human lung cancer cells into NOD-scid-IL2Rg?/? (NSI) mice, followed by injection of human PBMCs. The second method involves the use of hematopoietic stem and progenitor cells and CDX (HSPCs-CDX model). For this model, we first reconstituted the human immune system by transferring human CD34+ hematopoietic stem and progenitor cells (HSPCs-derived humanized model) and then transplanted human lung cancer cells. We found that the PBMCs-CDX model was more accurate in evaluating PD-L1/PD-1 targeted immunotherapies. In addition, it took only four weeks with the PBMCs-CDX model for efficacy evaluation, compared to 10–14 weeks with the HSPCs-CDX model. We then further established PBMCs-derived patient-derived xenografts (PDX) models, including an auto-PBMCs-PDX model using cancer and T cells from the same tumor, and applied them to assess the antitumor efficacies of anti-PD-L1 antibodies. We demonstrated that this PBMCs-derived PDX model was an invaluable tool to study the efficacies of PD-L1/PD-1 targeted cancer immunotherapies. Overall, we found our PBMCs-derived models to be excellent preclinical models for studying immune checkpoint inhibitors.     

用于病毒研究的人源化小鼠研究进展简

人源化小鼠从起初的入-鼠嵌合体到目前具有人体免疫活性的模型不断演进,已用于人免疫缺陷病毒、EB病毒、丙型肝炎病毒和登革病毒等病原体的感染、发病机制和防治的研究,取得了很大进展。我们简要介绍几种有代表性的人源化小鼠模型及其在病毒研究中的应用。     

Klebsiella oxytoca causes colonization resistance against multidrug-resistant K. pneumoniae in the gut via cooperative carbohydrate competition

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白细胞介素2免疫毒素研究进展

应用基因工程技术已研制出多种免疫毒素,IL-2免疫毒素是研究最为成功的例子之一,包括IL-2与绿脓杆菌外毒素的融合蛋白,IL-2与白喉毒素的融合蛋白等.这类毒素以IL-2为导向分子,IL-2受体阳性细胞为靶细胞,治疗移植排斥反应、自身免疫性疾病、T细胞淋巴瘤等疾病,在Ⅰ/Ⅱ期临床试验中已取得了较好的疗效.但是这类免疫毒素对人体均有较强的抗原性,使治疗时间和效果均受限制,为此研制了人源化的IL-2免疫毒素,能在体内外杀伤IL-2受体阳性细胞.     

Complete functional rescue of the ABCA1-/- mouse by human BAC transgenesis

Humanized mouse models are useful tools to explore the functional and regulatory differences between human and murine orthologous genes. We have combined a bioinformatics approach and an in vivo approach to assess the functional and regulatory differences between the human and mouse ABCA1 genes. Computational analysis identified significant differences in potential regulatory sites between the human and mouse genes. The effect of these differences was assessed in vivo, using a bacterial artificial chromosome transgenic humanized ABCA1 mouse model that expresses the human gene in the absence of mouse ABCA1. Humanized mice expressed human ABCA1 protein at levels similar to wild-type mice and fully compensated for cholesterol efflux activity and lipid levels seen in ABCA1-deficient mice. Liver X receptor agonist administration resulted in significant increases in HDL values associated with parallel increases in the hepatic ABCA1 protein and mRNA levels in the humanized ABCA1 mice, as seen in the wild-type animals. Our studies indicate that despite differences in potential regulatory regions, the human ABCA1 gene is able to functionally fully compensate for the mouse gene. Our humanized ABCA1 mice can serve as a useful model system for functional analysis of the human ABCA1 gene in vivo and can be used for the generation of potential new therapeutics that target HDL metabolism.     

治疗性单克隆抗体研究进展

杂交瘤技术使鼠源单克隆抗体（鼠单抗）被广泛用于人类疾病的诊断和研究,建立了治疗性抗体的第一个里程碑。但随后出现的人抗鼠抗体等副作用极大地限制了鼠单抗的临床应用。随着生物学技术的发展和抗体基因结构的阐明,应用DNA重组技术和抗体库技术对鼠单抗进行人源化改造,先后出现了嵌合抗体、改型抗体和全人抗体,同时也涌现了各种单抗衍生物,它们从不同角度克服了鼠单抗临床应用的不足,未人类疾病治疗带来新的曙光。我们就上述治疗性抗体人源化的研究进展做简要综述。     

Persistent hepatitis C virus infections and hepatopathological manifestations in immune-competent humanized mice

The majority of hepatitis C virus (HCV) infection develops chronic infection, which causes steatosis, cirrhosis and hepatocellular carcinoma. However, understanding HCV chronicity and pathogenesis is hampered by its narrow host range, mostly restricted to human and chimpanzee. Recent endeavour to infect a variety of humanized mice has not been able to achieve persistent HCV infection unless the essential innate immune responsive genes are knocked out. Nevertheless, such immune-compromised humanized mice still lacked HCV infection-induced hepatopathogenesis. Here we report that transgenic mice in ICR background harboring both human CD81 and occludin genes (C/O^Tg) are permissive to HCV infection at a chronicity rate comparable to humans. In this mouse model, HCV accomplishes its replication cycle, leading to sustained viremia and infectivity for more than 12 months post infection with expected fibrotic and cirrhotic progression. Host factors favorable for HCV replication, and inadequate innate immune-response may contribute to the persistence. Lastly, NS3/4 protease inhibitor telaprevir can effectively inhibit de novo RNA synthesis and acute HCV infection of C/O^Tg mice. Thus, chronic HCV infection with complete replication cycle and hepatopathologic manifestations is recapitulated, for the first time, in immune-competent mice. This model will open a new venue to study the mechanisms of chronic hepatitis C and develop better treatments.     

A metabolomic view of how the human gut microbiota impacts the host metabolome using humanized and gnotobiotic mice

Defining the functional status of host-associated microbial ecosystems has proven challenging owing to the vast number of predicted genes within the microbiome and relatively poor understanding of community dynamics and community–host interaction. Metabolomic approaches, in which a large number of small molecule metabolites can be defined in a biological sample, offer a promising avenue to ‘fingerprint'' microbiota functional status. Here, we examined the effects of the human gut microbiota on the fecal and urinary metabolome of a humanized (HUM) mouse using an optimized ultra performance liquid chromatography–mass spectrometry-based method. Differences between HUM and conventional mouse urine and fecal metabolomic profiles support host-specific aspects of the microbiota''s metabolomic contribution, consistent with distinct microbial compositions. Comparison of microbiota composition and metabolome of mice humanized with different human donors revealed that the vast majority of metabolomic features observed in donor samples are produced in the corresponding HUM mice, and individual-specific features suggest ‘personalized'' aspects of functionality can be reconstituted in mice. Feeding the mice a defined, custom diet resulted in modification of the metabolite signatures, illustrating that host diet provides an avenue for altering gut microbiota functionality, which in turn can be monitored via metabolomics. Using a defined model microbiota consisting of one or two species, we show that simplified communities can drive major changes in the host metabolomic profile. Our results demonstrate that metabolomics constitutes a powerful avenue for functional characterization of the intestinal microbiota and its interaction with the host.     

Paracrine Interactions within the Pancreatic Islet Determine the Glycemic Set Point

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Development of a serum-free medium for the production of humanized antibody from chinese hamster ovary cells using a statistical design

Summary To develop serum-free (SF) media for the production of humanized antibody from recombinant Chinese hamster ovary (rCHO) cells, a statistical optimization approach based on a Plackett-Burman design was adopted. A basal medium was prepared by supplementing α-minimal essential medium (α-MEM) with Fe(NO₃)₃·9H₂O, CuCl₂, ZnSO₄·7H₂O, and Na₂SeO₃ which are generally contained in SF medium formulations. Insulin, transferrin, and ethanolamine were also supplemented to the basal medium to determine their optimal concentrations. From this statistical analysis, serine, phenylalanine, and tyrosine were identified as important determinants for cell growth. Also, putrescine, linoleic acid, and hydrocortisone were shown to be important for both cell growth and antibody production. The SF medium was formulated by supplementing the basal medium with components showing positive effects on cell growth and/or antibody production. Cell growth and antibody production in this SF medium were comparable to those in α-MEM supplemented with 5% dialyzed fetal bovine serum. Taken together, the results obtained here show that a Plackett-Burman design facilitates the development of SF media for rCHO cells aimed at producing a humanized antibody.     

论医院绩效管理的人性化管理及其柔性化操作

医院绩效管理中存在倾向于刚性管理并热衷于惩罚方式的现象,其结果是无意中起了引导员工为逃避惩罚而隐瞒相关信息的作用,从而使医院员工不能共同分享与讨论各种经验教训,不利于医院进行持续质量改进。而有效预防与解决这一问题的主要策略是在绩效管理中进行人性化管理,并以柔性化操作处理那些不易进行刚性考核或易在刚性考核中产生消极后果的问题。     

Pharmacokinetics and biodistribution of genetically engineered antibodies

The engineering of monoclonal antibodies has created a new generation of pharmaceuticals with the desired pharmacokinetics and biodistribution properties. For radioimmunotherapy and radioscintigraphy, optimum tumor targeting can be achieved using engineered constructs that provide high antigen affinity and specificity, effective tumor penetration, circulation properties that allow high tumor uptake with acceptable doses to the normal tissues, and fast clearance allowing low background. Recent advances have made possible the development of antibodies with these properties.     

Aging impairs human bone marrow function and cardiac repair following myocardial infarction in a humanized chimeric mouse

Ventricular remodeling following myocardial infarction (MI) is a major cause of heart failure, a condition prevalent in older individuals. Following MI, immune cells are mobilized to the myocardium from peripheral lymphoid organs and play an active role in orchestrating repair. While the effect of aging on mouse bone marrow (BM) has been studied, less is known about how aging affects human BM cells and their ability to regulate repair processes. In this study, we investigate the effect aging has on human BM cell responses post‐MI using a humanized chimeric mouse model. BM samples were collected from middle aged (mean age 56.4 ± 0.97) and old (mean age 72.7 ± 0.59) patients undergoing cardiac surgery, CD34^+/− cells were isolated, and NOD‐scid‐IL2rγ^null (NSG) mice were reconstituted. Three months following reconstitution, the animals were examined at baseline or subjected to coronary artery ligation (MI). Younger patient cells exhibited greater repopulation capacity in the BM, blood, and spleen as well as greater lymphoid cell production. Following MI, CD34⁺ cell age impacted donor and host cellular responses. Mice reconstituted with younger CD34⁺ cells exhibited greater human CD45⁺ recruitment to the heart compared to mice reconstituted with old cells. Increased cellular responses were primarily driven by T‐cell recruitment, and these changes corresponded with greater human IFNy levels and reduced mouse IL‐1β in the heart. Age‐dependent changes in BM function led to significantly lower survival, increased infarct expansion, impaired host cell responses, and reduced function by 4w post‐MI. In contrast, younger CD34⁺ cells helped to limit remodeling and preserve function post‐MI.