Search

Objectives: Although haematopoietic stem cells (HSCs) migrate to injured gut, therapeutic success clinically remains poor. This has been partially attributed to limited local HSC recruitment following systemic injection. Identifying site specific adhesive mechanisms underpinning HSC-endothelial interactions may provide important information on how to enhance their recruitment and thus potentially improve therapeutic efficacy. This study determined (i) the integrins and inflammatory cyto/chemokines governing HSC adhesion to injured gut and muscle (ii) whether pre-treating HSCs with these cyto/chemokines enhanced their adhesion and (iii) whether the degree of HSC adhesion influenced their ability to modulate leukocyte recruitment. Methods: Adhesion of HPC-7, a murine HSC line, to ischaemia-reperfused (IR) injured mouse gut or cremaster muscle was monitored intravitally. Critical adhesion molecules were identified by pre-treating HPC-7 with blocking antibodies to CD18 and CD49d. To identify cyto/chemokines capable of recruiting HPC-7, adhesion was monitored following tissue exposure to TNF-α, IL-1β or CXCL12. The effects of pre-treating HPC-7 with these cyto/chemokines on surface integrin expression/clustering, adhesion to ICAM-1/VCAM-1 and recruitment in vivo was also investigated. Endogenous leukocyte adhesion following HPC-7 injection was again determined intravitally. Results: IR injury increased HPC-7 adhesion in vivo, with intestinal adhesion dependent upon CD18 and muscle adhesion predominantly relying on CD49d. Only CXCL12 pre-treatment enhanced HPC-7 adhesion within injured gut, likely by increasing CD18 binding to ICAM-1 and/or CD18 surface clustering on HPC-7. Leukocyte adhesion was reduced at 4 hours post-reperfusion, but only when local HPC-7 adhesion was enhanced using CXCL12. Conclusion: This data provides evidence that site-specific molecular mechanisms govern HPC-7 adhesion to injured tissue. Importantly, we show that HPC-7 adhesion is a modulatable event in IR injury and further demonstrate that adhesion instigated by injury alone is not sufficient for mediating anti-inflammatory effects. Enhancing local HSC presence may therefore be essential to realising their clinical potential. [ABSTRACT FROM AUTHOR]

Objectives: GPIIb integrin expression has been found on platelets and megakaryocytes, and more recently on immature hematopoietic progenitors. We set out to investigate expression of GPIIb in other hematopoietic cell lineages and, having detected it on mast cells, aimed to determine what possible role it might perform. Methods: We have made use of cultured human and murine bone marrow mast cells (BMMC) in order to characterize the expression of GPIIb. Further, BMMC cultures from wild type and GPIIb deficient (gpIIb-/-) mice were used for comparison of the adhesive properties mediated by this receptor. Finally, peritoneal mast cells were analyzed from both wild type and (gpIIb-/-) mice. Results: We demonstrate expression of GPIIb on cultured BMMC. Using cells derived from mice homozygous for a null allele of gbIIb we show that the absence of GPIIb has no effect on mast cells with respect to a number of measures of cell growth and differentiation. However, loss of GPIIb on BMMC results in an increase in surface expression of aV integrin, the alternative partner of GPIIIa. Conclusion: The results in this study demonstrate that GPIIb is expressed in human and murine mast cells. A function for GPIIb on mast cells is suggested by the altered adhesion of gbIIb-/- BMMC to fibronectin- and vitronectin-coated surfaces. Moreover, comparison of mast cells from the peritoneal cavity of wild type and gbIIb-/- mice indicates that GPIIb could influence the in vivo differentiation or homing of tissue mast cells. [Copyright &y& Elsevier]

Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activatorlike effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1,3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG 1/2 knockout pigs are promising tools for biomedical and translational research. [ABSTRACT FROM AUTHOR]

Objectives: Although haematopoietic stem cells (HSCs) migrate to injured gut, therapeutic success clinically remains poor. This has been partially attributed to limited local HSC recruitment following systemic injection. Identifying site specific adhesive mechanisms underpinning HSC-endothelial interactions may provide important information on how to enhance their recruitment and thus potentially improve therapeutic efficacy. This study determined (i) the integrins and inflammatory cyto/chemokines governing HSC adhesion to injured gut and muscle (ii) whether pre-treating HSCs with these cyto/chemokines enhanced their adhesion and (iii) whether the degree of HSC adhesion influenced their ability to modulate leukocyte recruitment. Methods: Adhesion of HPC-7, a murine HSC line, to ischaemia-reperfused (IR) injured mouse gut or cremaster muscle was monitored intravitally. Critical adhesion molecules were identified by pre-treating HPC-7 with blocking antibodies to CD18 and CD49d. To identify cyto/chemokines capable of recruiting HPC-7, adhesion was monitored following tissue exposure to TNF-α, IL-1β or CXCL12. The effects of pre-treating HPC-7 with these cyto/chemokines on surface integrin expression/clustering, adhesion to ICAM-1/VCAM-1 and recruitment in vivo was also investigated. Endogenous leukocyte adhesion following HPC-7 injection was again determined intravitally. Results: IR injury increased HPC-7 adhesion in vivo, with intestinal adhesion dependent upon CD18 and muscle adhesion predominantly relying on CD49d. Only CXCL12 pre-treatment enhanced HPC-7 adhesion within injured gut, likely by increasing CD18 binding to ICAM-1 and/or CD18 surface clustering on HPC-7. Leukocyte adhesion was reduced at 4 hours post-reperfusion, but only when local HPC-7 adhesion was enhanced using CXCL12. Conclusion: This data provides evidence that site-specific molecular mechanisms govern HPC-7 adhesion to injured tissue. Importantly, we show that HPC-7 adhesion is a modulatable event in IR injury and further demonstrate that adhesion instigated by injury alone is not sufficient for mediating anti-inflammatory effects. Enhancing local HSC presence may therefore be essential to realising their clinical potential. [ABSTRACT FROM AUTHOR]