Genetic immune response and antibody repertoire of heterologous ChAdOx1-BNT162b2 vaccination in a Korean cohort

Heterologous ChAdOx1-BNT162b2 vaccination induces a stronger immune response than two doses of BNT162b2 or ChAdOx1. Yet, the molecular transcriptome, the germline allelic variants of immunoglobulin loci and anti-Omicron antibody levels induced by the heterologous vaccination have not been formally investigated. Moreover, there is a paucity of COVID vaccine studies including diverse genetic populations. Here, we show a robust molecular immune transcriptome and antibody repertoire in 51 office workers from the Republic of Korea after a heterologous ChAdOx1-BNT162b2 vaccination or a homologous ChAdOx1-ChAdOx1 vaccination. Anti-spike-specific IgG antibody levels in the heterologous group increased from 14,000 U/ml to 142,000 AU/ml within eight days after the BNT162b2 vaccination. In contrast, antibody levels in the homologous group increased two-fold after the second ChAdOx1 dose. Antibody titers against the Omicron spike protein as compared to the ancestral strain were reduced to a lesser extent in the heterologous group. RNA-seq conducted on immune cells demonstrated a stronger activation of interferon-induced genetic programs in the heterologous cohort. An increase of specific IGHV clonal transcripts encoding neutralizing antibodies was preferentially detected in the heterologous cohort. Enrichment of B cell and CD4+ T cell responses were observed following both heterologous and homologous vaccination using scRNA-seq, but clonally expanded memory B cells were relatively stronger in the ChAdOx1-BNT162b2 cohort. In summary, a heterologous vaccination with ChAdOx1 followed by BNT162b2 provides an innate and adaptive immune response exceeding that seen in homologous ChAdOx1 vaccinations but equivalent to that seen in homologous BNT162b2 vaccination.

Optimal Ratio of Wnt3a Expression in Human Mesenchymal Stem Cells Promotes Axonal Regeneration in Spinal Cord Injured Rat Model

Objective@#: Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI. @*Methods@#: Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9–10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×105 cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining. @*Results@#: Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups. @*Conclusion@#: Our results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.

Optimal Ratio of Wnt3a Expression in Human Mesenchymal Stem Cells Promotes Axonal Regeneration in Spinal Cord Injured Rat Model

Objective@#: Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI. @*Methods@#: Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9–10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×105 cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining. @*Results@#: Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups. @*Conclusion@#: Our results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.

hMSCs suppress neutrophil-dominant airway inflammation in a murine model of asthma

Although chronic eosinophilic inflammation is a common feature in patients with asthma, some patients have neutrophil-dominant inflammation, which is known to be associated with severe asthma.Human mesenchymal stem cells (hMSCs) have shown promise in treating various refractory immunological diseases. Thus, hMSCs may represent an alternative therapeutic option for asthma patients with neutrophil-dominant inflammation, in whom current treatments are ineffective. BALB/c mice exposed to ovalbumin and polyinosinic:polycytidylic acid (Poly I:C) to induce neutrophilic airway inflammation were systemically treated with hMSCs to examine whether the hMSCs can modulate neutrophilic airway inflammation. In addition, cytokine production was evaluated in co-cultures of hMSCs with either anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) obtained from asthmatic patients or cells of the human bronchial epithelial cell line BEAS-2B to assess the response to hMSC treatment. The total number of immune cells in bronchoalveolar lavage fluid (BALF) showed a dramatic decrease in hMSC-treated asthmatic mice, and, in particular, neutrophilic infiltration was significantly attenuated. This phenomenon was accompanied by reduced CXCL15 production in the BALF. BEAS-2B cells co-cultured with hMSCs showed reduced secretion of IL-8. Moreover, decreased secretion of IL-4, IL-13 and IFN-γ was observed when human PBMCs were cultured with hMSCs, whereas IL-10 production was greatly enhanced. Our data imply that hMSCs may have a role in reducing neutrophilic airway inflammation by downregulating neutrophil chemokine production and modulating T-cell responses.

The Decellularized Vascular Allograft as an Experimental Platform for Developing a Biocompatible Small-Diameter Graft Conduit in a Rat Surgical Model

PURPOSE: The present study was aimed to assess the feasibility of using decellularized aortic allograft in a rat small animal surgical model for conducting small diameter vascular tissue engineering research. MATERIALS AND METHODS: Decellularized aortic allografts were infra-renally implanted in 12 Sprague-Dawley (SD) adult rats. The conduits were harvested at 2 (n = 6) and 8 weeks (n = 6), and assessed by hematoxylin and eosin (H&E), van Gieson, Masson Trichrome staining, and immunohistochemistry for von Willebrand factor, CD 31+, and actin. RESULTS: Consistent, predictable, and reproducible results were produced by means of a standardized surgical procedure. All animals survived without major complications. Inflammatory immune reaction was minimal, and there was no evidence of aneurysmal degeneration or rupture of the decellularized vascular implants. However, the aortic wall appeared thinner and the elastic fibers in the medial layer showed decreased undulation compared to the normal aorta. There was also minimal cellular repopulation of the vascular media. The remodeling appeared progressive from 2 to 8 weeks with increased intimal thickening and accumulation of both collagen and cells staining for actin. Although the endothelial like cells appeared largely confluent at 8 weeks, they were not as concentrated in appearance as in the normal aorta. CONCLUSION: The results showed the present rat animal model using decellularized vascular allograft implants to be a potentially durable and effective experimental platform for conducting further research on small diameter vascular tissue engineering.

The Effect of IGF-I on Collagen Synthesis in Cultured Chondrocytes

Cartilage is one of the most commonly manipulated tissue in esthetic and reconstructive surgery. Cartilage has an important role in longitudinal bone growth. Anabolic hormones and locally produced peptide growth factors are known to influence this process Matrix composition changes through proliferation, maturation, and differentiation of chondrocytes, and endochondral ossification thereafter. Defined cartilage matrix is synthesized during the maturation of chondrocytes where the major change is the increment of type II collagen. Variable sulfated mucololysaccharides and hyaluronic acid are also synthesized during this maturation. IGF-I(insulin like growth factor-I), so called somatomedin C, is a prominent growth factor in serum. IGF-I is known to be involved in long growth. IGF-I is affected by pituitary growth hormone. There are few studies done on IGF-I effect in cartilage matrix formation and possible changes of collagen subtypes. This experiment was designed to see the IGF-I effect on the colagen synthesis of cultured chondrocytes. Optimal concentration of IGF-I for the experiment was determined using H3-thymidine incorporation into DNA. The IGF-I effect on collagen synthesis was studied using H3-proline. The IGF-I effect on the synthesis of subtypes of collagen was studied using SDS-PAGE and immunocytochemical staining. Chondrocytes were isolated from the ears of New Zealand white rabbit and cultured in 2 X 10(5) cells/300 microgram density. IGF-I increased DNA synthesis, and optimal concentration of IGF-I was determined by dose-relationship curve as 10ng/ml. Collagen synthesis was increased by IGF-I. Type II collagen was increased on SDS-PAGE with IGF-I and this gel electrophoresis showed type X collagen, also. The increase in type II collagen was confirmed with immunocytochemical staining, the reaction becoming stronger with the addition of IGF-I. Type I collagen was not changed with IGF-I on immunocytochemistry. We conclude that IGE-I is an important modulator influencing not only proliferation and maturation but also terminal different-iation of chondrocytes.

Fibroblast growth factor-induced Thymidylate Synthase activity and expression in the serum-starved UMR 106-01 osteoblast cells / 대한정형외과연구학회지

PURPOSE: In the present study, the effects of bFGF on the early responses of proliferation of UMR 106-01 osteoblast cells during cell cycle reentry from the latent(G0/G1) to the proliferative periods(S/M) were investigated. MATERIALS AND METHODS: The synchronized cell culture method using the serum starvation was utilized. After the addition of bFGF, the time courses of protein synthesis, DNA synthesis, thymidylate synthase(TS) activity, TS mRNA level and expression of c-fos were determined. RESULTS: 87% UMR 106-01 cells were synchronized to G0/G1 by serum starvation for seven days in the medium containing 0.1% serum. The protein level began to increase 3 hours after bFGF treatment and reached the maximum at 18 hours. TS activity began to increase 3 hours after the bFGF treatment and reached its peak at 6 hours while its mRNA level, determined by quantitative PCR, reached the maximum at 12 hours. The expression of c-fos protein, determined by western blot analysis and immunocytochemistry, increased 3 hours after bFGF treatment. On the contrary, these prominent changes and responses to bFGF were not observed in the case of using non-synchronized cells cultured in the medium containing 10% serum. CONCLUSION: Based on these data it can be concluded that bFGF-induced DNA synthesis in the early proliferative phase is due to increases in both TS activity and mRNA amount and that the increase in c-fos expression and TS activity occur before the increase in TS mRNA level.

Fibroblast growth factor-induced Thymidylate Synthase activity and expression in the serum-starved UMR 106-01 osteoblast cells / 대한정형외과연구학회지

PURPOSE: In the present study, the effects of bFGF on the early responses of proliferation of UMR 106-01 osteoblast cells during cell cycle reentry from the latent(G0/G1) to the proliferative periods(S/M) were investigated. MATERIALS AND METHODS: The synchronized cell culture method using the serum starvation was utilized. After the addition of bFGF, the time courses of protein synthesis, DNA synthesis, thymidylate synthase(TS) activity, TS mRNA level and expression of c-fos were determined. RESULTS: 87% UMR 106-01 cells were synchronized to G0/G1 by serum starvation for seven days in the medium containing 0.1% serum. The protein level began to increase 3 hours after bFGF treatment and reached the maximum at 18 hours. TS activity began to increase 3 hours after the bFGF treatment and reached its peak at 6 hours while its mRNA level, determined by quantitative PCR, reached the maximum at 12 hours. The expression of c-fos protein, determined by western blot analysis and immunocytochemistry, increased 3 hours after bFGF treatment. On the contrary, these prominent changes and responses to bFGF were not observed in the case of using non-synchronized cells cultured in the medium containing 10% serum. CONCLUSION: Based on these data it can be concluded that bFGF-induced DNA synthesis in the early proliferative phase is due to increases in both TS activity and mRNA amount and that the increase in c-fos expression and TS activity occur before the increase in TS mRNA level.

Effect of VCAM-1 expression in human endothelial cells by proinflammatory cytokines / 천식및알레르기

BACKGROUND: The expression of adhesion molecules contribute to development of systemic diseases. Vascular cell adhesion molecule-l(VCAM-1) is an endothelial cell membrane glycoprotein that has been implicated in leukocyte/endothelial cell interactions in inflammation. OBJECTIVE: The aim of this study was to characterize the surface expression and regulation of VCAM-1 on two different endothelial cells. METHOD: We examined the effects of the expression of VCAM-1 in two different endothelial cells, isolated from human umbilical cords and human glomerulus. Expression of VCAM-1 was measured by enzyme-linked immunosorbent assay(ELISA) and flow cytometry. RESULTS: In human umbilical cord endothelial cells(HUVECs), both interleukin-l B(IL-lB) and tumor necrosis factor-a (TNF-a) increased VCAM-1 expression. VCAM-1 expression increased by TNF-a was higher than that increased by IL-lB. In human glomerular endothelial cells(HGECs), IL-lB and TNF-a markedly increased VCAM-1 expression. Conclusion. The regulation of VCAM-1 appears to be somewhat different in HGECs compared with HUVECs. These differences between the responsiveness of the two cells may possibly indicate inherent differences in endothelial cell derived from different vascular beds.

Effects of Parathyroid Hormone and Insulin on Proliferation in Osteogenic Sarcoma UMR-106-01 Cells / 대한정형외과학회잡지

Parathyroid hormone(PTH), a major bone hormone, inhihits DNA and collagen syntheses in osteohlast-like cells in vitro, but increase the proliferation of osteoblast in vivo as secn in hyperparathyroidism. On the other hand, insulin is known to increase DNA and collagen syntheses and modify the effects of PTH in osteoblast-like cells. We have examined the effects of PTH and insulin in rat osteosarcoma UMR-l06-01 cells and whether PTH plays a role in the insulin-mediated bone formation. When 1 nM PTH and 10 nM insulin were administered to VMR-l06-01 ceils, the rates of DNA synthesis were 124% and 136% of the untreated control, respectively. When the two hormones were administered serially by exposing to 1 nM PTH for 7 days followed by 10 nM insulin lor 24h, the largest increase was observed. The protein synthesis was also increased remarkahly when the two hormones were aclministered serially: the[3H]-leucine incorporation rates, compared to the control group, were 75% and l62% with PTH ancl insulin administration, respectively, but the rate was 297% with the serial administration of the two. The collaeen synthesis, as measured by the (3H)-proline incorporation rates were 60% and l64% with PTH and insulin administration, respectively, but 351% with serial administration, again showing a dramatic effect. These results showed that 1 nM PTH decreased DNA and collagen syntheses in UMR-l06-01 cells after both a 24h and a more prolonged exposure. Similar exposures to insulin tended to increase the syntheses. The comhination of PTH and insulin tended to increase the syntheses. hut not beyond the effect of insulin alone. However, the sequential administration of PTH and insulin markedly increases ihose rales relative to the simultaneous adminstration of these two hormones. Thus, it is possihle that sequential stimulation of PTH and insulin in hone matrix exerts an synergistic effect on hone formation in vivo.

The Effect of Erythropoietin on the Production of Endothelin in Human Glomerular and Umbilical Endothelial Cells / 대한신장학회잡지

The administration of erythropoietin for renal failure patients is frequently associated with a mild-to-marked rise in arterial blood pressure. The erythropoietin-induced hypertension has been variably attributed to the rise in erythrocyte concentration and/or a direct or indirect pressor action of erythropoietin on vascular smooth muscle. Especially erythropoietin-induced hypertension may be mediated by increased production of the potent vasoactive peptide endothelin. This study was designed to determine the effect of erythropoietin to the production of endothelin in human glomerular endothelial cells and human umbilical vein endothelial cells. ELISA method was used to measure the endothelin- 1, after 4 X 10(4) endothelial cells were grown to confluency on 24-well plates, in which erythropoietin 25u/ml was incubated for 24 hours. The results showed that the endothelin production in human glomerular endothelial cells was insignificant (116.0+/-14.0%, P>0.05, mean+/-S.E., n=5, each n means the mean of duplicate experiments), but the endothelin production in human umbilical endothelial cells was increased after 25u/ml erythropoietin treatment(126.5+/-15.2%, P<0.05, mean+/-S.E., n=4). In glomerular endothelial cells, TGF-beta(0.5ng/ml) increased the production of endothelin(218.8+/-57.2%, P<0.01, mean+/-S.E., n=5), also it looks like that TNF-alpha and thrombin might increase the production of endothelin according to the concentration. In conclusion, the responsiveness of endothelial cells to erythropoietin may be different according to the cell type, and glomerular endothelial cells could increase the production of endothelin, if appropriate stimuli were given.

The Effect of Cycolsporine A on the Expression of Von Willebrand Factor in Human Glomerular Endothelial Cells / 대한신장학회잡지

Cyclosporine(CsA) is a modulator of the immune system used therapeutically to prevent organ transplant rejection. However, it is nephrotoxic and causes thrombotic phenomena after renal and bone marrow transplantation. CsA nephrotoxicity in vivo is associated with elevated levels of von Willebrand factor(vWf), which is a multimeric plasm glycoprotein secreted by endothelial cells and platelets. CsA is not soluble in water and the intravenous form given to patients is dissolved in a vehicle called cremophor EL. The vehicle has been implicated in anaphylatic reactions and associated with the release of histamine in vivo. We hypothesized that CsA or cremophor might affect the release of vWf from human glomerular endothelial cells. vWf was measured in culture supernant using ELISA kit after speed vaccum for 3 hour. The expression of vWf in cultured human glomerular endothelial cells was relatively low compared to human plasm in vivo. CsA alone did not increase vWf release(100%, 99.9+/-0.7%, 99.1+/-2.9%, 106+/-21.5%, CsA 0, 0.1, 1, 10microM mean S.E., n=2), but cremophor EL increased vWf release (100%, 104.0+/-8.6%, 117.6+/-9.5%, 121.3+/-12.2%, mean+/-S.E., n=3). These results were same as the results of experiments under thrombin(1IU/ml) and histamine(10(-4)M). The increased expression of vWf in human glomerular endothelial cells in response to CsA seems to be related to cremophor, the solvent, rather than CsA itself in vitro culture experiments.

Culture of the Human Glomerular Endothelial Cells / 대한신장학회잡지

Whereas mesangial and epithelial cells from glomeruli are commonly grown in vitro, there has been major difficulties in developing homogenous cultures of human glomerular endothelial cells. This study defines the conditions for the reproducible isolation and growth of homogenous monolayers of human glomerular endothelial cells based on the method of Green DF et al published in 1992. Using the selective media and the sieving method, fibronectin was required as a surface matrix after adequate collagenase treatment, and endothelial cell growth factor and heparin was needed for the continuous growth of endothelial cells. The endothelial cell growth factor was isolated from the bovine hypothalamic extracts. Glomerular capillary endothelial cells exhibited a cobblestone morphology at confluence and stained homogenously with von Willebrand factor(factor VIII). The cytokeratin and the actin were not stained. This study might be helpful for in vitro study to know the biological characteristics of human glomerular endothelial cells under the predetermined condition.