Clinical and chest computed tomography features associated with severe Chlamydia psittaci pneumonia diagnosed by metagenomic next-generation sequencing: A multicenter, retrospective, observational study.

Chlamydia psittaci pneumonia is a rare disease with varying clinical presentations. Here, we aimed to investigate the clinical and chest computed tomography (CT) features of severe psittacosis pneumonia. Clinical data of 35 patients diagnosed with psittacosis pneumonia were retrospectively analyzed using metagenomic next-generation sequencing. The patients were classified into severe (n = 20) and non-severe (n = 15) groups. The median age of patients was 54 years, and 27 patients (77.1%) had a definite history of bird contact. Severe patients had more underlying comorbidities and were more prone to dyspnea and consciousness disorders than non-severe patients. The neutrophil count and D-dimer, lactate dehydrogenase, interleukin (IL)-2, IL-6, and IL-10 levels were higher, whereas the lymphocyte, CD3 + T cell, and CD4 + T cell counts, CD4+/CD8 + T cell ratio, and albumin level were substantially lower in severe patients than in non-severe patients. Chest CT findings of severe patients revealed large areas of pulmonary consolidation, and ground-glass opacities were observed in some patients, with a higher risk of involving multiple lobes of the lungs and pleural effusion. One patient died of multiple organ failure, whereas the condition of the other 34 patients improved, and they were discharged from the hospital. Patients with severe psittacosis pneumonia often have underlying comorbidities and are prone to developing dyspnea, consciousness disorder, and lesions in both lungs. Serum D-dimer, IL-2, IL-6, and IL-10 levels and lymphocyte, CD3 + T cell, and CD4 + T cell counts are associated with disease severity.

Chimeric Mouse Generation by ES Cell Blastocyst Microinjection and Uterine Transfer.

The ability to generate chimeric mice through microinjecting embryonic stem (ES) cells into blastocysts is a critical step for the conventional ES cell-mediated knockout technology. In recent years, designer nuclease-based methods, especially the CRISPR technology, have substantially decreased the needs for blastocyst microinjection. However, this method has still remained as a valuable technique for generating sophisticated genetic models as well as for stem cell research. In this chapter, we describe the detailed procedures used in our laboratory on how to use ES cells to produce chimeric mice, including derivation and inactivation of MEF feeder cells, culturing and handling of mouse ES cells, collection and microinjection of blastocysts, and finally implantation of injected blastocysts into the uteri of pseudopregnant surrogate mothers.

Using CRISPR/Cas9 for Gene Knockout in Immunodeficient NSG Mice.

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice are an immunodeficient strain that enables human cell xenografts. However, NSG mice possess a complex genetic background that would complicate cross-breeding with other inbred transgenic or knockout mouse strains to establish a congenic strain with a desired genetic modification in the NSG background. Newly developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology enables modification of the mouse genome at the zygote stage without the need for extensive cross-breeding or the use of embryonic stem cells. In this chapter, we use the knockout of the X-linked Cybb gene as an example to describe our procedures for genetically modifying NSG mice using the CRISPR/Cas9 method. Briefly, two sgRNAs were designed and made to target exon 1 and exon 3 of the Cybb gene, and either sgRNA was then microinjected together with Cas9 mRNA into fertilized eggs collected from NSG mice. The injected embryos are subsequently transferred into the oviducts of pseudopregnant surrogate mothers. Offspring born to the foster mothers were genotyped by PCR and DNA sequencing. In this chapter, we describe our experiment procedures in detail and report our genotyping results for demonstrating that NSG mice can be genetically modified using the CRISPR/Cas9 technology in a highly efficient manner.

Generation of Conditional Knockout Mice by Sequential Insertion of Two loxP Sites In Cis Using CRISPR/Cas9 and Single-Stranded DNA Oligonucleotides.

Conditional knockout (cKO) mice are extremely valuable for biomedical research because they enable detailed analyses of gene functions in a tissue- or temporally-specific fashion. The conventional method for generating cKO mice is time consuming and labor intensive, which involves making a large gene-targeting construct, transfecting and screening many embryonic stem (ES) cell clones, injecting positive ES clones into blastocysts to produce chimeric mice, and breeding the chimeras to transmit the targeted gene through the germline. Recently developed CRISPR technology has revolutionized the way genetically engineered organisms are created. Knockout and knockin mice can now be made by directly injecting zygotes with Cas9, sgRNA, and donor DNA. In theory, cKO mice can be generated by simultaneously inserting two loxP sites using two sgRNAs and two oligonucleotides as donors, but in practice the probability of obtaining cKO mice in one step is still very low, partly because the efficiency of oligo-mediated knockin is much lower than non-homologous end joining (NHEJ) and partly because co-cutting juxtaposed sites in one allele at the same time often leads to the deletion of the entire fragment between the two cutting sites. Therefore, many laboratories prefer to insert the two loxP sites sequentially, i.e., generating mice with one loxP first and then use embryos collected from these mice to insert the second loxP site. In this chapter, we describe our procedures and timeline using this sequential method to make a Six6 cKO mouse line as a demonstration of its feasibility.

Cutting Edge: ß-Catenin-Interacting Tcf1 Isoforms Are Essential for Thymocyte Survival but Dispensable for Thymic Maturation Transitions.

T cell factor 1 (Tcf1) is essential for T cell development; however, it remains controversial whether ß-catenin, a known coactivator of Tcf1, has a role. Tcf1 is expressed in multiple isoforms in T lineage cells, with the long isoforms interacting with ß-catenin through an N-terminal domain. In this study, we specifically ablated Tcf1 long isoforms in mice (p45-/-mice) to abrogate ß-catenin interaction. Although thymic cellularity was diminished in p45-/- mice, transition of thymocytes through the maturation stages was unaffected, with no overt signs of developmental blocks. p45-/- thymocytes showed increased apoptosis and alterations in transcriptome, but these changes were substantially more modest than in thymocytes lacking all Tcf1 isoforms. These data indicate that Tcf1-ß-catenin interaction is necessary for promoting thymocyte survival to maintain thymic output. Rather than being dominant-negative regulators, Tcf1 short isoforms are adequate in supporting developing thymocytes to traverse through maturation steps and in regulating the expression of most Tcf1 target genes.

Heparin Promotes Cardiac Differentiation of Human Pluripotent Stem Cells in Chemically Defined Albumin-Free Medium, Enabling Consistent Manufacture of Cardiomyocytes.

Cardiomyocytes can be differentiated from human pluripotent stem cells (hPSCs) in defined conditions, but efficient and consistent cardiomyocyte differentiation often requires expensive reagents such as B27 supplement or recombinant albumin. Using a chemically defined albumin-free (E8 basal) medium, we identified heparin as a novel factor that significantly promotes cardiomyocyte differentiation efficiency, and developed an efficient method to differentiate hPSCs into cardiomyocytes. The treatment with heparin helped cardiomyocyte differentiation consistently reach at least 80% purity (up to 95%) from more than 10 different hPSC lines in chemically defined Dulbecco's modified Eagle's medium/F-12-based medium on either Matrigel or defined matrices like vitronectin and Synthemax. One of heparin's main functions was to act as a Wnt modulator that helped promote robust and consistent cardiomyocyte production. Our study provides an efficient, reliable, and cost-effective method for cardiomyocyte derivation from hPSCs that can be used for potential large-scale drug screening, disease modeling, and future cellular therapies. Stem Cells Translational Medicine 2017;6:527-538.

Induced pluripotent stem cell transplantation in the treatment of porcine chronic myocardial ischemia.

BACKGROUND: This study was designed to test the effects of induced pluripotent stem cell (iPSC) in the treatment of chronic myocardial ischemia. METHODS: The reprogramming of passage 3 myocardial fibroblasts was performed by using the lentiviral vector containing 4 human factors: OCT4, SOX2, KLF4, and c-MYC. The iPSC colonies at P12-17 were allogeneically transplanted into ischemic myocardium of 10 swine by direct injection. Cohorts of 2 animals were sacrificed at 2, 4, 6, 8, and 12 weeks after injection. RESULTS: No signs of graft versus host disease were evident at any time points. At 2 weeks, clusters of SSEA-4-positive iPSCs were detected in the injected area. At 4 to 8 weeks, these cells started to proliferate into small spheres surrounded by thin capsules. At 12 weeks the cell clusters still existed, but decreased in size and numbers. The cells inside these masses were homogeneous with no sign of differentiation into any specific lineage. Increased smooth muscle actin or vWF positive cells were found inside and around the iPSC clusters, compared with non-injected areas. By real-time polymerase chain reaction, the levels of VEGF, basic FGF, and ANRT expression were significantly higher in the iPSC-treated myocardium compared with untreated areas. These results suggest that iPSCs contributed to angiogenesis. CONCLUSIONS: Allogeneically transplanted pig iPSCs proliferated despite an ischemic environment in the first 2 months and survived for at least 3 months in immunocompetent hosts. Transplanted iPSCs were also proangiogenic and thus might have beneficial effects on the ischemic heart diseases.

Purification of plasmid and BAC transgenic DNA constructs.

Pronuclear microinjection is the most used method for generating transgenic mice. The quality of DNA to be microinjected is a key determinant of the success rate of this method. DNA purity is a critical factor because trace amounts of many substances, when microinjected into the pronucleus of the fertilized egg, can kill or prevent the further development of the embryo. Avoiding all contaminants is not a trivial issue, because most transgenic fragments need to be purified from agarose gels. Small particles and viscous materials in the DNA solution can also dramatically reduce the efficiency of microinjection because they tend to clog the injection needles. DNA shearing or breakage during purification and microinjection is also a potential problem, particularly when linearized bacterial artificial chromosomes (BAC) DNAs are used. The overall quantity and the final DNA concentration are also important considerations, because egg -pronuclei are very sensitive to the amount of foreign DNA. In this chapter, we first discuss the general guidelines and cautions for preparing microinjection-quality DNA, and then describe in detail two -protocols, one for gel purification of transgenic fragments from plasmid vectors and the other for isolating high-quality BAC DNA from bacteria.

Pronuclear microinjection and oviduct transfer procedures for transgenic mouse production.

Transgenic mouse technology is a powerful method for studying gene function and creating animal models of human diseases. Currently, the most widely used method for generating transgenic mice is the pronuclear microinjection method. In this method, a transgenic DNA construct is physically microinjected into the pronucleus of a fertilized egg. The injected embryos are subsequently transferred into the oviducts of pseudopregnant surrogate mothers. A portion of the mice born to these surrogate mothers will harbor the injected foreign gene in their genomes. These procedures are technically challenging for most biomedical researchers. Inappropriate experimental procedures or suboptimal equipment setup can substantially reduce the efficiency of transgenic mouse production. In this chapter, we describe in detail our microinjection setup as well as our standard microinjection and oviduct transfer procedures.

[Changes of the elastic fibers and collagen fibers during the development and progression of experimentally induced tongue carcinoma in hamsters].

OBJECTIVE: To investigate the relationship between the extracellular matrix (ECM) and neoplastic progression in hamster with tongue cancer. METHODS: Forty-eight specimens of hamster tongue cancer were divided into control group (n=6) and experimental group (n=42). The pathological grade of the specimens was assessed (including 3 stages, namely atypical hyperplasia, carcinoma in situ and early invasive carcinoma). The sections of the tongue were stained with Masson and aldehyde-fuchsin (AF) staining for microscopic observation of the elastic fiber and collagen fiber changes. RESULTS: Within the connective tissue cores (CTC) of the papillae in the control group was a framework of numerous and fine Gomrori's aldehyde fuchsin-positive elastic fibers. But in the stages of dysplasia and carcinoma in situ, these elastic fibers decreased and further diminished in the CTC in early invasive carcinoma. In dysplasia and carcinoma in situ stages, most of the elastic fibers collapsed with scattered elastic fibers, and the elastic fibers decreased significantly in early invasive carcinoma. The control group showed a significantly greater number of elastic fibers in the experimental group. The collagen fiber was obviously increased and irregularly arranged in dysplasia and carcinoma in situ stage; in early invasive carcinoma, the collagen fibers became thicker with deposition in the lamina propria. CONCLUSION: An excessive deposition of collagen fiber and reduction of the elastic fibers is an important factor contributing to the development of tongue carcinoma in hamsters.

Strategies and considerations for distributing and recovering mouse lines.

As more and more genetically modified mouse lines are being generated, it becomes increasingly common to share animal models among different research institutions. Live mice are routinely transferred between animal facilities. Due to various issues concerning animal welfare, intellectual property rights, colony health status and biohazard, significant paperwork and coordination are required before any animal travel can take place. Shipping fresh or frozen preimplantation embryos, gametes, or reproductive organs can bypass some of the issues associated with live animal transfer, but it requires the receiving facilities to be able to perform delicate and sometimes intricate procedures such as embryo transfer, in vitro fertilization (IVF), or ovary transplantation. Here, we summarize the general requirements for live animal transport and review some of the assisted reproductive technologies (ART) that can be applied to shipping and reviving mouse lines. Intended users of these methods should consult their institution's responsible official to find out whether each specific method is legal or appropriate in their own animal facilities.

GA binding protein regulates interleukin 7 receptor alpha-chain gene expression in T cells.

The interleukin 7 receptor alpha-chain (IL-7Ralpha) is essential for T cell development in both humans and mice and for B cell development in mice. Whereas the transcription factor PU.1 regulates IL-7Ralpha expression in mouse pro-B cells via a GGAA motif, we demonstrate here that GA binding protein (GABP) bound to this site and was essential in the regulation of IL-7Ralpha expression in T cells, where PU.1 is not expressed. Moreover, IL-7Ralpha expression was diminished substantially in thymocytes but was normal on B220(+) fetal liver cells from mouse embryos with diminished expression of GABPalpha. Thus, GABP is essential for the regulation of IL-7Ralpha expression in T cells, and the differential regulation of IL-7Ralpha in distinct lymphoid lineages is achieved at least in part by differential recruitment of factors to the same GGAA motif.