scholarly journals Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species

2020 ◽  
Vol 48 (10) ◽  
pp. e57-e57
Author(s):  
Xiaozhen He ◽  
Wenfeng Chen ◽  
Zhen Liu ◽  
Guirong Yu ◽  
Youbang Chen ◽  
...  
Keyword(s):  
Genome Editing ◽  
Human Peripheral Blood ◽  
Point Mutations ◽  
Peripheral Blood Lymphocytes ◽  
Dna Double Strand Breaks ◽  
Site Specific ◽  
Low Efficiency ◽  
Multiple Species ◽  
Balancing Efficiency

Abstract Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and ‘cleaner’ knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

scholarly journals Characteristics of a Pathogenic Molecular Clone of an End-Stage Serum-Derived Variant of Simian Immunodeficiency Virus (SIVF359)

2001 ◽  
Vol 75 (19) ◽  
pp. 9328-9338 ◽  
Author(s):  
Lennart Holterman ◽  
Rob Dubbes ◽  
James Mullins ◽  
Gerald Learn ◽  
Henk Niphuis ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Peripheral Blood Lymphocytes ◽  
Peripheral Blood Mononuclear ◽  
Molecular Clone ◽  
Immunodeficiency Virus ◽  
End Stage

ABSTRACT End-stage simian immunodeficiency virus (SIV) isolates are suggested to be the most fit of the evolved virulent variants that precipitate the progression to AIDS. To determine if there were common characteristics of end-stage variants which emerge from accelerated cases of AIDS, a molecular clone was derived directly from serum following in vivo selection of a highly virulent SIV isolate obtained by serial end-stage passage in rhesus monkeys (Macaca mulatta). This dominant variant caused a marked cytopathic effect and replicated to very high levels in activated but not resting peripheral blood lymphocytes. Furthermore, although this clone infected but did not replicate to detectable levels in rhesus monocyte-derived macrophages, these cells were able to transmit infection to autologous T cells upon contact. Interestingly, although at low doses this end-stage variant did not use any of the known coreceptors except CCR5, it was able to infect and replicate in human peripheral blood mononuclear cells homozygous for the Δ32 deletion of CCR5, suggesting the use of a novel coreceptor. It represents the first pathogenic molecular clone of SIV derived from viral RNA in serum and provides evidence that not only the genetic but also the biological characteristics acquired by highly fit late-stage disease variants may be distinct in different hosts.

scholarly journals Human interferon-inducible protein-10 induces mononuclear cell infiltration in mice and promotes the migration of human T lymphocytes into the peripheral tissues and human peripheral blood lymphocytes-SCID mice

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1423-1431 ◽  
Author(s):  
DD Taub ◽  
DL Longo ◽  
WJ Murphy
Keyword(s):  
T Cell ◽  
Mononuclear Cell ◽  
Human Peripheral Blood ◽  
Peripheral Blood Lymphocytes ◽  
Cell Infiltration ◽  
Mononuclear Cell Infiltration ◽  
Human T Cell ◽  
Inducible Protein ◽  
Interferon Inducible Protein

The human cytokine, interferon-inducible protein-10 (IP-10), is a small glycoprotein secreted by activated monocytes, T cells, keratinocytes, astrocytes, and endothelial cells and is structurally related to the alpha subfamily of chemotactic cytokines called chemokines (Taub and Oppenheim, Cytokine 5:175, 1993). However, in contrast to other alpha chemokines that induce neutrophil migration, IP-10 has been shown to chemoattract monocytes and T lymphocytes in vitro, suggesting a role in T-cell-mediated immune responses. We therefore examined the effects of human IP-10 after in vivo administration. IP-10 induces significant mononuclear cell infiltration after subcutaneous injections in normal mice. In an effort to study the in vivo effects of IP-10 on human leukocyte migration, we then examined the ability of recombinant human IP-10 (rhIP-10) to induce human-T-cell infiltration using a human/severe combined immune deficiency (SCID) mouse model. SCID mice received an intraperitoneal injection of human peripheral blood lymphocytes (10(8) cells), followed by a subcutaneous injection of rhIP- 10 (1 micrograms/injection) in the hind flank for 4 hours or sequential injections for 3 days. The skin and underlying tissue from the rhIP-10 injection site were then biopsied and examined for the extent of mononuclear cell infiltration. rhIP-10 again induced significant mononuclear cell accumulation 72 hours after injection. Immunohistologic evaluation determined that a significant number of human CD3+ T cells were recruited in response to rhIP-10 injections. These results show that rhIP-10 is capable of inducing human T-cell migration in vivo and may play an important role in monocyte and lymphocyte recruitment into inflammatory sites.

scholarly journals Assessment of biosafety and toxicity of hydrophilic gel for implantation in experimental in vitro and in vivo models 

2021 ◽  
Author(s):  
Natalia Bezdieniezhnykh ◽  
Alexandra Lykhova ◽  
Tamara Kozak ◽  
Taras Zadvornyi ◽  
Olena Voronina ◽  
...  
Keyword(s):  
Human Peripheral Blood ◽  
Clinical Manifestations ◽  
Peripheral Blood Lymphocytes ◽  
Model Systems ◽  
In Vivo Studies ◽  
In Vivo Models ◽  
Pharmacologically Active ◽  
Hydrophilic Gel

Abstract Background: The assessment of biosafety of pharmacologically active substances is crucial for determining the feasibility of their medical use. There are controversial issues regarding the use of substances of different origins as implants. Methods: We have conducted the comprehensive studies to determine the in vivo toxicity and in vitro genotoxicity of new generation of hydrophilic gel for implantation (production name of the substance "Activegel") to detail its characteristics and assess its biosafety. Results: In vivo studies have shown the absence of clinical manifestations of intoxication in animals and no abnormalities in their physiological condition, general and biochemical blood tests. Evaluation of the site of the gel application showed no inflammatory reaction and evidenced on normal state of tissues of animal skin. The results of the genotoxicity test indicated that the gel did not affect the parameters of DNA comets and, accordingly, had no genotoxic effect on human peripheral blood lymphocytes. When studying the effect of the gel on malignantly transformed cells in vitro, it was found that the gel for implantation did not change the proliferative activity and viability of human breast cancer cells. Conclusions: Comprehensive in vitro and in vivo study using various experimental model systems showed that the hydrophilic gel for implantation "Activegel" is non-toxic.

scholarly journals Activation of Fc receptor-bearing lymphocytes by immune complexes. II. Killer lymphocytes mediate Fc ligand-induced lymphokine production.

1981 ◽  
Vol 154 (6) ◽  
pp. 1868-1880 ◽  
Author(s):  
M E Neville ◽  
H W Lischner
Keyword(s):  
Nk Cells ◽  
Immune Complex ◽  
Immune Complexes ◽  
Human Peripheral Blood ◽  
Peripheral Blood Lymphocytes ◽  
Fc Receptor ◽  
Lymphokine Production ◽  
Cell Antibody ◽  
Activated T Lymphocytes

Cells that participate in immune complex-induced production of leukocyte migration inhibitory factor (LIF) activity can be concentrated in a population making up 2-4% of human peripheral blood lymphocytes in which greater than 90% of the cells are active in a single cell antibody-dependent cellular cytotoxicity assay. When so concentrated, such killer (K) cell preparations are as efficient in producing LIF activity as mitogen activated T lymphocytes. Other Fc receptor (FcR)-bearing lymphocytes, including natural killer (NK) cells, do not produce measurable LIF activity when incubated with immune complexes (additional evidence that the K and NK cells among ligands to the FcR of the appropriate lymphocytes, possibly without need for exogenous receptor bridging, is the only requirement for their activation to immune complex-induced lymphokine production (ICLP). It is probable that ICLP by K cells palays a role in antibody-mediated effector functions in vivo.

scholarly journals Nonclinical Toxicology and Toxicokinetic Profile of an Oral Lanthionine Synthetase C-Like 2 (LANCL2) Agonist, BT-11

2019 ◽  
Vol 38 (2) ◽  
pp. 96-109 ◽  
Author(s):  
Andrew Leber ◽  
Raquel Hontecillas ◽  
Victoria Zoccoli-Rodriguez ◽  
Marion Ehrich ◽  
Jennifer Davis ◽  
...  
Keyword(s):  
Human Peripheral Blood ◽  
Clinical Signs ◽  
Systemic Exposure ◽  
Micronucleus Assay ◽  
Peripheral Blood Lymphocytes ◽  
In Vivo Studies ◽  
Repeat Dose ◽  
Toxicity Studies ◽  
Oral Doses

BT-11 is an orally active, gut-restricted investigational therapeutic targeting the lanthionine synthetase C-like 2 pathway with lead indications in ulcerative colitis (UC) and Crohn disease (CD), 2 manifestations of inflammatory bowel disease (IBD). In 5 mouse models of IBD, BT-11 is effective at oral doses of 8 mg/kg. BT-11 was also efficacious at nanomolar concentrations in primary human samples from patients with UC and CD. BT-11 was tested under Good Laboratory Practice conditions in 90-day repeat-dose general toxicity studies in rats and dogs, toxicokinetics, respiratory, cardiovascular and central nervous system safety pharmacology, and genotoxicity studies. Oral BT-11 did not cause any clinical signs of toxicity, biochemical or hematological changes, or macroscopic or microscopic changes to organs in 90-day repeat-dose toxicity studies in rats and dogs at doses up to 1,000 mg/kg/d. Oral BT-11 resulted in low systemic exposure in both rats (area under the curve exposure from t = 0 to t = 8 hours [AUC0-8] of 216 h × ng/mL) and dogs (650 h × ng/mL) and rapid clearance with an average half-life of 3 hours. BT-11 did not induce changes in respiratory function, electrocardiogram parameters, or behavior with single oral doses of 1,000 mg/kg/d. There was no evidence of mutagenic or genotoxic potential for BT-11 up to tested limit doses using an Ames test, chromosomal aberration assay in human peripheral blood lymphocytes, or micronucleus assay in rats. Therefore, nonclinical studies show BT-11 to be a safe and well-tolerated oral therapeutic with potential as a potent immunometabolic therapy for UC and CD with no-observed adverse effect level >1,000 mg/kg in in vivo studies.

scholarly journals Nivalenol induced apoptosis in human peripheral blood lymphocytes in vitro and mouse peripheral blood lymphocytes in vivo

1997 ◽  
Vol 1997 (45) ◽  
pp. 33-37 ◽  
Author(s):  
Naoto YOSHINO ◽  
Mari TAKIZAWA ◽  
Hiroki OKUMURA ◽  
Tomomi IHARA ◽  
Masao SUGAMATA ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Peripheral Blood Lymphocytes ◽  
Blood Lymphocytes ◽  
Human Peripheral Blood Lymphocytes ◽  
Induced Apoptosis

scholarly journals Complement bridges between cells analysis of a possible cell-cell interaction mechanism.

1977 ◽  
Vol 146 (6) ◽  
pp. 1484-1499 ◽  
Author(s):  
M P Dierich ◽  
B Landen
Keyword(s):  
Human Peripheral Blood ◽  
Interaction Mechanism ◽  
Cell Interaction ◽  
Peripheral Blood Lymphocytes ◽  
Lymphoid Cells ◽  
Temperature Dependent ◽  
Complement Components ◽  
Human Peripheral Blood Lymphocytes ◽  
C3 Receptors

Different leukocytes (Raji, Daudi, Rael lymphoid cells; human peripheral blood lymphocytes, and guinea pig granulocytes), which had been coated with C3 by incubation of 37 degrees C for 20 min in a C3 solution, were demonstrated to form rosettes with erythrocytes coated with complement components (EAC142). The percentage of rosettes was dependent of the amount of C3 present on the cells. Loading of the lymphoid cells with C3 was a time- and temperature-dependent process. C3b was unable to serve the same purposes, although C3 and C3b occupied the C3 receptors on the lymphoid cells to a comparable degree. C3 functions in a similar manner. The C42 enzyme can be replaced by trypsin, so that bridging units may consist of C3 + C42, C5 + C42 OR C3 + trypsin, and C5 + trypsin. Bridging units can be constructed also from C4 + C1. It is suggested that enzymes on one cell liberate labile binding groups of complement components on adjacent cells, thus inducing coupling of the two cells. The possibility is raised that this type of cell interlinkage may play a role in vivo, since there is accumulating evidence that complement components are expressed in the plasma membrane of different cells.

scholarly journals Precision genome editing in the CRISPR era

2017 ◽  
Vol 95 (2) ◽  
pp. 187-201 ◽  
Author(s):  
Jayme Salsman ◽  
Graham Dellaire
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Point Mutations ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Genetic Changes ◽  
New Era ◽  
Homology Directed Repair ◽  
Repair Template ◽  
Non Homologous End Joining

With the introduction of precision genome editing using CRISPR–Cas9 technology, we have entered a new era of genetic engineering and gene therapy. With RNA-guided endonucleases, such as Cas9, it is possible to engineer DNA double strand breaks (DSB) at specific genomic loci. DSB repair by the error-prone non-homologous end-joining (NHEJ) pathway can disrupt a target gene by generating insertions and deletions. Alternatively, Cas9-mediated DSBs can be repaired by homology-directed repair (HDR) using an homologous DNA repair template, thus allowing precise gene editing by incorporating genetic changes into the repair template. HDR can introduce gene sequences for protein epitope tags, delete genes, make point mutations, or alter enhancer and promoter activities. In anticipation of adapting this technology for gene therapy in human somatic cells, much focus has been placed on increasing the fidelity of CRISPR–Cas9 and increasing HDR efficiency to improve precision genome editing. In this review, we will discuss applications of CRISPR technology for gene inactivation and genome editing with a focus on approaches to enhancing CRISPR–Cas9-mediated HDR for the generation of cell and animal models, and conclude with a discussion of recent advances and challenges towards the application of this technology for gene therapy in humans.

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