Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

Loma Linda University

Pluripotent stem cells have the remarkable self-renewal ability and are capable of differentiating into multiple diverse cells. There is increasing evidence that the aging process can have adverse effects on stem cells. As stem cells age, their renewal ability deteriorates and their ability to differentiate into the various cell types is altered. Accordingly, it is suggested aging-induced deterioration of stem cell functions may play a key role in the pathophysiology of the various aging-associated disorders. Understanding the role of the aging process in deterioration of stem cell function is crucial, not only in understanding the pathophysiology of aging-associated disorders, but also in future development of novel effective stem cell-based therapies to treat aging-associated diseases. This review article first focuses on the basis of the various aging disease-related stem cell dysfunction. It then addresses the several concepts on the potential mechanism that causes aging-related stem cell dysfunction. It also briefly discusses the current potential therapies under development for aging-associated stem cell defects.

Effect of aging on stem cells.

Multiple sclerosis (MS) is caused by immune-mediated damage of myelin sheath. Current therapies aim to block such immune responses. However, this blocking is not sufficiently specific and hence compromises immunity, leading to severe side effects. In addition, blocking medications usually provide transient effects and require frequent administration, which further increases the chance to compromise immunity. In this regard, myelin-specific therapy may provide the desired specificity and a long-lasting therapeutic effect by inducing myelin-specific regulatory T (Treg) cells. Tolerogenic dendritic cells (TolDCs) are one such therapy. However, ex vivo generated TolDCs may be converted into immunogenic DCs in a proinflammatory environment. In this study, we identified a potential novel myelin-specific therapy that works with immunogenic DCs, hence without the in vivo conversion concern. We showed that immunization with DCs, engineered to overexpress 25-hydroxyvitamin D 1α-hydroxylase for de novo synthesis of a focally high 1,25-dihydroxyvitamin D concentration in the peripheral lymphoid tissues, induced Treg cells. In addition, such engineered DCs, when pulsed with a myelin antigen, led to myelin-specific suppression of ongoing experimental allergic encephalomyelitis (an MS animal model), and the disease suppression depended on forkhead-box-protein-P3(foxp3)+ Treg cells. Our data support a novel concept that immunogenic DCs can be engineered for myelin-specific therapy for MS.-Li, C.-H., Zhang, J., Baylink, D. J., Wang, X., Goparaju, N. B., Xu, Y., Wasnik, S., Cheng, Y., Berumen, E. C., Qin, X., Lau, K.-H. W., Tang, X. Dendritic cells, engineered to overexpress 25-hydroxyvitamin D 1α-hydroxylase and pulsed with a myelin antigen, provide myelin-specific suppression of ongoing experimental allergic encephalomyelitis.

Dendritic cells, engineered to overexpress 25-hydroxyvitamin D 1α-hydroxylase and pulsed with a myelin antigen, provide myelin-specific suppression of ongoing experimental allergic encephalomyelitis.

In the past there have been a multitude of studies that ardently support the role of arginase II (Arg II) in vascular and endothelial disorders; however, the regulation and function of Arg II in autoimmune diseases has thus far remained unclear. Here we report that a global Arg II null mutation in mice suppressed experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. During EAE, both Arg I and Arg II were induced in spinal cords, but only Arg II was induced in spleens and splenic dendritic cells (DCs). DC activation by lipopolysaccharide (LPS), CD40L or TLR8 agonist significantly enhanced Arg II expression without affecting Arg I expression. Conversely, DC differentiating cytokines [IL-4 and granulocyte macrophage-colony-stimulating factor (GM-CSF)] yielded opposite effects. In addition, Arg I and Arg II were regulated differentially during Th1 and Th17 cell polarization. Arg II deficiency in mice delayed EAE onset, ameliorated clinical symptoms and reduced myelin loss, accompanied by a remarkable reduction in the EAE-induced spinal cord expression of Th17 cell markers (IL-17 and RORγt). The abundance of Th17 cells and IL-23 cells in relevant draining lymph nodes was significantly reduced in Arg II knockout mice. In activated DCs, Arg II deficiency significantly suppressed the expression of Th17-differentiating cytokines IL-23 and IL-6. Interestingly, Arg II deficiency did not lead to any compensatory increase in Arg I expression in vivo and in vitro. In conclusion, Arg II was identified as a factor promoting EAE likely via an Arg I-independent mechanism. Arg II may promote EAE by enhancing DC production of Th17-differentiating cytokines. Specific inhibition of Arg II could be a potential therapy for multiple sclerosis.

Deficient arginase II expression without alteration in arginase I expression attenuated experimental autoimmune encephalomyelitis in mice.

An indispensable role of macrophages in bone repair has been well recognized. Previous data have demonstrated the copresence of M1 macrophages and mesenchymal stem cells (MSCs) during the proinflammatory stage of bone repair. However, the exact role of M1 macrophages in MSC function and bone repair is unknown. This study aimed to define the role of M1 macrophages at bone injury sites via the function of 1,25-Dihydroxyvitamin D (1,25[OH]2D) in suppressing M1 but promoting M2 differentiation. We showed that 1,25(OH)2D suppressed M1 macrophage-mediated enhancement of MSC migration. Additionally, 1,25(OH)2D inhibited M1 macrophage secretion of osteogenic proteins (i.e., Oncostatin M, TNF-α, and IL-6). Importantly, the 1,25(OH)2D-mediated suppression of osteogenic function in M1 macrophages at the proinflammatory stage was associated with 1,25(OH)2D-mediated reduction of MSC abundance, compromised osteogenic potential of MSCs, and impairment of fracture repair. Furthermore, outside the proinflammatory stage, 1,25(OH)2D treatment did not suppress fracture repair. Accordingly, our data support 2 conclusions: (a) M1 macrophages are important for the recruitment and osteogenic priming of MSCs and, hence, are necessary for fracture repair, and (b) under vitamin D-sufficient conditions, 1,25(OH)2D treatment is unnecessary and can be detrimental if provided during the proinflammatory stage of fracture healing.

1,25-Dihydroxyvitamin D suppresses M1 macrophages and promotes M2 differentiation at bone injury sites.

Current therapies for gut inflammation have not reached the desired specificity and are attended by unintended immune suppression. This study aimed to provide evidence for supporting a hypothesis that direct in vivo augmentation of the induction of gut-homing regulatory T (Treg) cells is a strategy of expected specificity for the treatment of chronic intestinal inflammation (e.g., inflammatory bowel disease). We showed that dendritic cells (DCs), engineered to de novo produce high concentrations of both 1,25-dihydroxyvitamin D, the active vitamin D metabolite, and retinoic acid, an active vitamin A metabolite, augmented the induction of T cells that express both the regulatory molecule Foxp3 and the gut-homing receptor CCR9 in vitro and in vivo. In vivo, the newly generated Ag-specific Foxp3 T cells homed to intestines. Additionally, transfer of such engineered DCs robustly suppressed ongoing experimental colitis. Moreover, CD4 T cells from spleens of the mice transferred with the engineered DCs suppressed experimental colitis in syngeneic hosts. The data suggest that the engineered DCs enhance regulatory function in CD4 T cell population in peripheral lymphoid tissues. Finally, we showed that colitis suppression following in vivo transfer of the engineered DCs was significantly reduced when Foxp3 Treg cells were depleted. The data indicate that maximal colitis suppression mediated by the engineered DCs requires Treg cells. Collectively, our data support that DCs de novo overproducing both 1,25-dihydroxyvitamin D and retinoic acid are a promising novel therapy for chronic intestinal inflammation.

In Vivo Generation of Gut-Homing Regulatory T Cells for the Suppression of Colitis.

Cyclooxygenase 2 (COX-2) is essential for normal tissue repair. Although COX-2 is known to enhance the differentiation of mesenchymal stem cells (MSCs), how COX-2 regulates MSC differentiation into different tissue-specific progenitors to promote tissue repair remains unknown. Because it has been shown that COX-2 is critical for normal bone repair and local COX-2 overexpression in fracture sites accelerates fracture repair, this study aimed to determine the MSC subsets that are targeted by COX-2. We showed that CD90 mouse skeletal stem cells (mSSCs; i.e., CD45Tie2AlphaV MSCs) were selectively recruited by macrophage/monocyte chemoattractant protein 1 into fracture sites following local COX-2 overexpression. In addition, local COX-2 overexpression augmented osteoblast differentiation and suppressed chondrocyte differentiation in CD90 mSSCs, which depended on canonical WNT signaling. CD90 depletion data demonstrated that local COX-2 overexpression targeted CD90 mSSCs to accelerate fracture repair. In conclusion, CD90 mSSCs are promising targets for the acceleration of bone repair.

Cyclooxygenase 2 augments osteoblastic but suppresses chondrocytic differentiation of CD90 skeletal stem cells in fracture sites.

In central lymphoid tissues, mature lymphocytes are generated and pathogenic autoreactive lymphocytes are deleted. However, it is currently known that a significant number of potentially pathogenic autoreactive lymphocytes escape the deletion and populate peripheral lymphoid tissues. Therefore, peripheral mechanisms are present to prevent these potentially pathogenic autoreactive lymphocytes from harming one's own tissues. One such mechanism is dictated by regulatory T (Treg) cells. So far, the most extensively studied Treg cells are CD4Foxp3 Treg cells. However, recent clinical trials for the treatment of immune-mediated diseases using CD4 Foxp3 Treg cells met with limited success. Accordingly, it is necessary to explore the potential importance of other Treg cells such as CD8 Treg cells. In this regard, one extensively studied CD8 Treg cell subset is Qa-1(HLA-E in human)-restricted CD8 Treg cells, in which Qa-1(HLA-E) molecules belong to a group of non-classical major histocompatibility complex Ib molecules. This review will first summarize the evidence for the presence of Qa-1-restricted CD8 Treg cells and their regulatory mechanisms. Major discussions will then focus on the potential clinical translation of Qa-1-restricted CD8 Treg cells. At the end, we will briefly discuss the current status of human studies on HLA-E-restricted CD8 Treg cells as well as potential future directions.

Towards Clinical Translation of CD8 Regulatory T Cells Restricted by Non-Classical Major Histocompatibility Complex Ib Molecules.

Vitamin D deficiency, determined by blood levels of 25-hydroxyvitamin D [25(OH) D, i.e. the major vitamin D form in blood], has been shown to associate with all-cause mortalities. We recently demonstrated that blood levels of 1,25-dihydroxyvitamin D [1,25(OH)D, i.e. the active vitamin D] were significantly lower in non-survivors compared to survivors among sepsis patients. Unexpectedly, despite the well documented roles of 1,25(OH)D in multiple biological functions such as regulation of immune responses, stimulation of antimicrobials, and maintenance of barrier function, 1,25(OH)D supplementation failed to improve disease outcomes. These previous findings suggest that, in addition to 1,25(OH)D deficiency, disorders leading to the 1,25(OH)D deficiency also contribute to mortality among sepsis patients. Therefore, this study investigated the mechanisms leading to sepsis-associated 1,25(OH)D deficiency.

Mechanistic study of the cause of decreased blood 1,25-Dihydroxyvitamin D in sepsis.

In the present study we sought to improve the efficacy and safety of our Sca1 PDGFB stem cell gene therapy for osteoporosis in ovariectomized (OVX) mouse model. This therapy is administered by marrow transplantation. We established the promise of this approach by previously showing that this therapy in normal mice increase bone density, increased endosteal cortical and trabecular bone formation, caused de novo trabecular bone formation, increased cortical thickness and improve bone strength. In the current study we produced a fusion gene, PDGFB-DSS6. We reasoned that the DSS6, calcium binding protein would trap the PDGFB at the bone surface and thereby limit the amount of PDGFB required to produce an optimal bone formation response, i.e. efficacy with a lower engraftment. The result shows that indeed with a very low level of engraftment we achieved a large increase in bone formation in the OVX model of bone loss. Serum analysis for biochemical marker of new bone formation showed an approximate 75% increase in alkaline phosphatase levels in Sca1PDGFB-DSS6 group as compared to other groups. Quantitative analysis of bone by microCT showed a massive increase in trabecular bone density and trabecular connectivity of the femur in the metaphysis in Sca1 PDGFB-DSS6 group. The increased cortical porosity produced by OVX was replaced by the Sca1 PDGFB-DSS6 therapy but not by the positive control Sca1 PDGFB. Additionally, an increase in the femur bone strength was also observed specifically in Sca1 PDGFB-DSS6 as compared to other treatment groups, emphasizing the functional significance of the observed anabolic action is on bone formation. In future work we will focus on nontoxic preconditioning of our marrow transplantation procedure and also on transcriptional control of therapeutic gene expression to avoid excess bone formation.

Samiksha Wasnik

Department of Medicine,
Division of Regenerative Medicine,
Department of Medicine, Division of Regenerative Medicine,
Division of Regenerative Medicine, Department of Medicine

Loma Linda University

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Samiksha Wasnik

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Medicine,Division of Regenerative Medicine,Department of Medicine, Division of Regenerative Medicine,Division of Regenerative Medicine, Department of Medicine

Loma Linda University

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Department of Medicine,
Division of Regenerative Medicine,
Department of Medicine, Division of Regenerative Medicine,
Division of Regenerative Medicine, Department of Medicine