同基因与异基因胚胎肝干细胞移植治疗小鼠肝硬化

背景：胚胎肝干细胞移植免疫相关研究较少,同基因与异基因胚胎肝干细胞移植对小鼠肝硬化的治疗作用,目前尚不清楚。 目的：观察同种同基因与同种异基因胚胎肝干细胞移植对小鼠肝硬化的治疗作用,以及治疗过程中免疫排斥反应发生情况。 方法：采用Ⅳ型胶原酶消化法分离纯化BALB/c与C57BL/6胚胎肝干细胞。104只健康BALB/c小鼠随机分为4 组：正常对照组不予任何处理;肝硬化组、同种同基因移植组、同种异基因移植组腹腔注射四氯化碳石蜡油溶液复制肝硬化模型,16周后分别经其尾静脉注射生理盐水,等量同种同基因胚胎肝干细胞和同种异基因胚胎肝干细胞。在移植4周后比较各组受体小鼠存活情况、肝功能恢复情况、肝纤维化程度、免疫细胞(CD4⁺T、CD8⁺T、NK、NKT)数目及比值、肝脏病理学变化。 结果与结论：同种同基因移植组和同种异基因移植组生存率均为100%,与肝硬化组小鼠存活率67%相比差异有显著性意义(P < 0.05);各组肝功能和肝纤维化指标差异无显著性意义(P > 0.05)。各组免疫学指标比较差异无显著性意义(P > 0.05)。肝脏组织病理学显示肝组织修复：同种异基因移植组>同种同基因移植组>肝硬化组。因此,经尾静脉移植胚胎肝干细胞能提高肝硬化小鼠的生存率、减轻肝细胞坏死程度;同种同基因与同种异基因胚胎肝干细胞移植未发现免疫排斥,对小鼠肝硬化有一定的治疗作用。     

Mesenchymal stromal cells for systemic sclerosis treatment

Systemic sclerosis (SSc) is a rare chronic autoimmune disease characterized by vasculopathy, dysregulation of innate and adaptive immune responses, and progressive fibrosis. SSc remains an orphan disease, with high morbity and mortality in SSc patients. The mesenchymal stromal cells (MSC) demonstrate in vitro and in vivo pro-angiogenic, immuno-suppressive, and anti-fibrotic properties and appear as a promising stem cell therapy type, that may target the key pathological features of SSc disease.This review aims to summarize acquired knowledge in the field of :1) MSC definition and in vitro and in vivo functional properties, which vary according to the donor type (allogeneic or autologous), the tissue sources (bone marrow, adipose tissue or umbilical cord) or inflammatory micro-environment in the recipient; 2) preclinical studies in various SSc animal models , which showed reduction in skin and lung fibrosis after MSC infusion; 3) first clinical trials in human, with safety and early efficacy results reported in SSc patients or currently tested in several ongoing clinical trials.     

Effect of allogeneic bone marrow–derived mesenchymal stem cells transplantation in a polyI:C-induced primary biliary cirrhosis mouse model

Primary biliary cirrhosis (PBC) is a slowly progressive autoimmune disease of unknown mechanism. We established a PBC animal model by injecting C57BL/6 mice with polyinosinic–polycytidylic acid sodium (polyI:C) to investigate the therapeutic effect of bone marrow–derived mesenchymal stem cells (BM-MSC) on this model. After 6 weeks of MSC infusion, serum aminotransferase and autoimmune antibodies declined, and histological examination by hematoxylin and eosin staining showed significant amelioration of monocytes infiltration around bile ducts of mice treated with BM-MSC. Interestingly, allogeneic BM-MSC transplantation markedly increased CD4⁺Foxp3⁺ regulatory T cells in peripheral blood as well as in lymph nodes when analyzed by flow cytometry. Further examination showed serum TGF-β1 increased but IFN-γ decreased significantly in PBC mice treated with MSC, while with no obvious change in IL-10 expression. Our results for the first time suggested that BM-MSC transplantation could regulate systemic immune response and enhance recovery in liver inflammation of PBC mice, raising the possibility for clinical application of allogeneic MSC in treatment of early-stage PBC patients.     

The immunomodulatory activity of human umbilical cord blood-derived mesenchymal stem cells in vitro

Bone marrow-derived mesenchymal stem cells (BM-MSC) are currently being investigated in preclinical and clinical settings because of their self-renewal and multipotent differentiative capacity or their immunosuppressive function. However, BM may be detrimental because of the highly invasive donation procedure and BM-MSC decline with age. Therefore, MSC derived from other sources have been considered as an alternative. However, there is only limited knowledge on their immunomodulatory properties. Human umbilical cord blood (UCB) cells are good substitutes for BM-MSC because of the immaturity of newborn cells. In this study, we successfully isolated MSC from UCB. The morphological phenotypes, cell cycle status, surface markers and differentiation potential of these clonally expanded cells are consistent with BM-MSC. Furthermore, UCB-MSC expanded in vitro retain low immunogenicity and an immunomodulatory effect. Flow cytometry analysis showed that UCB-MSC did not express CD40, CD40 ligand, CD80, CD86 and major histocompatibility complex class II molecules. We have demonstrated that UCB-MSC are incapable of inducing allogeneic peripheral blood mononuclear cell (PBMC) proliferation and have a dose-dependent inhibition of PBMC immune responses in mixed lymphocyte reactions (MLR) and phytohaemagglutinin activation assays, even after interferon-gamma treatment. Additionally, we have found that UCB-MSC can suppress the function of mature dendritic cells. Using transwell systems, we have demonstrated an inhibition mechanism that depends on both cell contact and soluble factors. Based on the findings we conclude that banked UCB could serve as a potential alternative source of MSC for allogeneic application in the future.     

Mouse bone marrow-derived mesenchymal stem cells acquire immunogenicity concurrent with differentiation to insulin-producing cells

BackgroundAlthough mesenchymal stem cells (MSCs) are regarded as immune-elusive and even immunosuppressive, recent evidence suggests that allogeneic immune response might is inevitable in the case of some lineages differentiated from MSCs. Regarding the importance of allogeneic IPCs and MSCs in pre-clinical and clinical studies, the present study aimed to investigate the possible changes in the immunogenicity of MSCs during the differentiation to IPCs in a murine model of allogeneic transplantation.Material and methodsTwo mouse strains, C57BL/6 (H2D^b) and BALB/c (H2D^d) were selected to establish an allogeneic cell transplantation model. Bone marrow MSCs were differentiated into IPCs and the expression of H2D, CD80, and Qa-2 molecules were evaluated via flowcytometry on MSCs and IPCs. The differentiated and undifferentiated MSCs were encountered to allogeneic splenocytes and the proliferation, CD44 activation marker, and cytokine release in the splenocytes were evaluated.ResultsIPCs exhibited increased expression of MHC-I and CD80 that elicited an allogenic response including the activation-induced proliferation of splenocytes, activation of CD4⁺ T cells, and IFNγ response.ConclusionsMSCs acquire immunogenicity after differentiation to functional IPCs, which might cause decreased efficacy in the case of allogeneic transplantation. Careful precautions might be critical for saving the IPCs against the detrimental allogeneic responses.     

Allogeneic injection of fetal membrane-derived mesenchymal stem cells induces therapeutic angiogenesis in a rat model of hind limb ischemia

Bone marrow-derived mesenchymal stem cells (BM-MSC) have been demonstrated to be an attractive therapeutic cell source for tissue regeneration and repair. However, it remains unknown whether or not allogeneic transplantation of mesenchymal stem cells (MSC) derived from fetal membranes (FM), which are generally discarded as medical waste after delivery, has therapeutic potential. FM-MSC were obtained from Lewis rats and had surface antigen expression and multipotent potential partly similar to those of BM-MSC. Compared with BM-MSC, FM-MSC secreted a comparable amount of hepatocyte growth factor despite a small amount of vascular endothelial growth factor. FM-MSC and BM-MSC both expressed major histocompatibility complex (MHC) class I but not MHC class II antigens and did not elicit allogeneic lymphocyte proliferation in mixed lymphocyte culture. FM-MSC or BM-MSC obtained from Lewis rats were injected into a MHC-mismatched August-Copenhagen-Irish rat model of hind limb ischemia. Three weeks after injection, blood perfusion and capillary density were significantly higher in the FM-MSC and BM-MSC groups than in the phosphate-buffered saline group, and allogeneic FM-MSC and BM-MSC were still observed. In nonischemic hind limb tissues, allogeneic FM-MSC and BM-MSC injection were associated with a comparatively small amount of T lymphocyte infiltration, compared with the injection of allogeneic splenic lymphocytes. In conclusion, allogeneic FM-MSC injection did not elicit a lymphocyte proliferative response and provided significant improvement in a rat model of hind limb ischemia, comparable to the response to BM-MSC. Thus, allogeneic injection of FM-MSC may be a new therapeutic strategy for the treatment of severe peripheral vascular disease. Disclosure of potential conflicts of interest is found at the end of this article.     

Mesenchymal Stem Cell-Mediated Immature Dendritic Cells Induce Regulatory T Cell-Based Immunosuppressive Effect

Immature dendritic cells (imDCs) are increasingly viewed as mediators of T-cell tolerance. We investigated factors enabling induction of regulatory T (Treg) cells through syngeneic imDC/mesenchymal stem cell (MSC) co-cultures in vitro and immunosuppressive effects of MSC-mediated imDCs (MSCs were excluded after 72 h co-culture) in vivo. In these experiments, we found that Foxp3⁺ Treg cell population remarkably increased after the T cell priming phase when splenocytes were co-cultured with both imDCs and MSCs, presumably inducing naïve T cells into Treg cells by MSCs and imDCs. In parallel, TGF-β secretion was markedly induced from the imDC+MSC+splenocyte culture supernatant to a significant level at 72-h co-culture, compared to the MSC or imDC+splenocyte co-culture. Based on these results, using a murine melanoma tumor model, we confirmed that the subcutaneous injection of B16 cells induced a perfect tumor incidence in allogeneic recipients when MSC-mediated DCs were coinjected. Consequently, these results suggested that immune tolerance with MSC-mediated DCs leads to immunosuppression induced by at least Foxp3-specific Treg cells. This tool may be useful in clinical trials due to the yet unknown side effects of stem cell therapy.     

An impairment of antibody production in adoptively restored, lethally irradiated thymectomized mice

Lethally irradiated, bone marrow protected, normal and thymectomized adult mice were given 140–250 × 10⁶ syngeneic spleen and lymph node cells from normal or sensitized donors. These mice were tested at intervals following irradiation for their ability to reject allogeneic and xenogeneic skin grafts and to produce haemagglutinins. The results show that adoptively restored, thymectomized mice are capable of responding normally to skin homografts of varying antigenic disparity and of producing haemagglutinins to xenogeneic antigens. However, the majority of the mice which received lymphoid cells from normal donors were unable to produce haemagglutinating antibody in response to allogeneic antigens. Both 19S and 7S antibody were produced by the thymectomized mice.     

骨髓间充质干细胞对致敏小鼠造血干/祖细胞移植植入的影响

目的: 前期的研究已经证实致敏小鼠造血干/祖细胞移植植入失败率高。本研究拟通过骨髓间充质干细胞(MSCs)进行干预,观察能否提高造血干、祖细胞移植的植入率。方法: 应用贴壁培养法体外培养正常小鼠骨髓MSCs,并分为6个实验组,包括实验组1:d11 MSCs干预的致敏组;实验组2: d0 MSCs干预的致敏组;实验组3:d11和d0 2次MSCs干预的致敏组;实验组4: 无MSCs干预的致敏小鼠对照组;实验组5:无MSCs干预的正常小鼠(非致敏小鼠)移植对照组;实验组6:无MSCs干预的正常小鼠不移植对照组。观察指标包括生存分析、移植效果分析(血象改变、骨髓细胞恢复及嵌合分析等)和移植物抗宿主病(GVHD)检测,最终评估MSCs干预对各实验组异基因造血干/祖细胞移植植入率的影响效果。结果: 与对照组(实验组4、5、6)比较,MSCs干预(实验组1、2、3)在2次异基因脾细胞注射法致敏的动物模型进行异基因造血干/祖细胞移植时,未能促进骨髓造血干/祖细胞移植的植入,也未能延长致敏动物移植后的生存时间。结论: 体内应用1×10⁶ MSCs干预,未能促进2次异基因1×10⁶ C57BL/6小鼠脾细胞输注法建立的重度致敏模型异基因造血干/祖细胞移植的植入。     

Human adipose‐tissue derived mesenchymal stem cells induce functional de‐novo regulatory T cells with methylated FOXP3 gene DNA

Due to their immunomodulatory properties, mesenchymal stem cells (MSC) are interesting candidates for cellular therapy for autoimmune disorders, graft‐versus‐host disease and allograft rejection. MSC inhibit the proliferation of effector T cells and induce T cells with a regulatory phenotype. So far it is unknown whether human MSC‐induced CD4⁺CD25⁺CD127^–forkhead box P3 (FoxP3)⁺ T cells are functional and whether they originate from effector T cells or represent expanded natural regulatory T cells (nT_reg). Perirenal adipose‐tissue derived MSC (ASC) obtained from kidney donors induced a 2·1‐fold increase in the percentage of CD25⁺CD127^–FoxP3⁺ cells within the CD4⁺ T cell population from allostimulated CD25^–/dim cells. Interleukin (IL)‐2 receptor blocking prevented this induction. The ASC‐induced T cells (iT_reg) inhibited effector cell proliferation as effectively as nT_reg. The vast majority of cells within the iTreg fraction had a methylated FOXP3 gene Treg‐specific demethylated region (TSDR) indicating that they were not of nT_reg origin. In conclusion, ASC induce T_reg from effector T cells. These iT_reg have immunosuppressive capacities comparable to those of nT_reg. Their induction is IL‐2 pathway‐dependent. The dual effect of MSC of inhibiting immune cell proliferation while generating de‐novo immunosuppressive cells emphasizes their potential as cellular immunotherapeutic agent.     

Survival and tissue localization of xenogeneic human lymphocytes in hypothymic nude mice: observations in normal subjects and multiple sclerosis

The feasibility of using the hypothymic nude mouse as a transfer system for the study of cellular autoimmunity, by injection and tracing of 51chromium (Cr) or fluorescein labelled lymphocytes from humans, allogeneic or syngeneic mice was investigated. The localization of the injected cells was studied by radioisotopic counting or microscopy. For mouse cells there was better survival of syngeneic than allogeneic cells, and the pattern of distribution of injected mouse lymphocytes differed markedly from that of xenogeneic human lymphocytes. Thus, the lymph nodes and spleen were the predominant sites of localization of transferred syngeneic (BALB/c) lymphocytes, indicative of long recirculation of these cells, whereas the liver was the predominant site of localization of transferred human lymphocytes, and there was no evidence for recirculation of these cells. In studies on patients with multiple sclerosis, lymphocytes did not localize in neural tissue. The data indicate that the nude mouse is unlikely to be useful for the study of the tissue distribution of dispersed xenogeneic (human) lymphocytes.     

Autologous stem cell transplantation for systemic sclerosis

Systemic sclerosis (SSc) is a generalised autoimmune disease, of yet unknown origin, with two major clinical subsets: the limited (lcSSc) and the diffuse cutaneous (dcSSc) forms, which can be distinguished by the extent of skin involvement, the autoantibody profile and the pattern of organ involvement. With an incidence of 1/10⁵, SSc affects around 250,000 people in Europe and is responsible for significant morbidity with a 5-year mortality rate of at least 30% of all patients. In patients with rapidly progressive dcSSc, the 5-year mortality is estimated to be 40–50%. Hematopoietic stem cell transplantation (HSCT), mostly autologous but also allogeneic in some specific cases, has been employed worldwide since 1996 as a new therapeutic strategy in patients with a poor prognosis. In 2007, 150 HSCT procedures have been reported in the EBMT data base. We review herein both the short and the long-term reports from the various European and North American phase I–II studies, which have shown that autologous HSCT in selected patients with severe dcSSc results in sustained improvement of skin thickening and stabilisation of organ function up to seven years after transplantation. Based on these promising results, ongoing phase III trials have been designed in parrallel, both in Europe (ASTIS) and in North America (SCOTT) aiming to analyse the respective benefits from autologous HSCT respectively without or with high dose irradiation. This review reports the current data concerning the effects of HSCT on survival, skin, and major organ function in patients with severe dcSSc.     

Co-infusion of donor bone marrow with host mesenchymal stem cells treats GVHD and promotes vascularized skin allograft survival in rats

We investigated the effect of autologous mesenchymal stem cells (MSC) on multiple unmodified donor bone marrow (BM) infusions and vascularized skin graft outcome. BM-derived rat MSC were examined for phenotype and function. MSC/MSC-conditioned-medium suppressed IFN-γ production by T cells and modified DC function. Infusions of MSC with one-time BM improved vascularized skin graft survival, while with one–two-times BM reversed graft versus host disease (GVHD). Mixed chimerism was enhanced in recipients given two–four-times BM with MSC infusions. Interestingly, four-times BM infusions with MSC delayed GVHD onset, reduced host tissue damage and enhanced vascularized skin allograft survival compared to four-times BM alone. These data demonstrate that, the co-infusion of MSC with unmodified BM limit the toxicity of allogeneic BM transplantation, enhance mixed chimerism and improve vascularized skin graft survival. These findings provide insights for the development of autologous MSC-based BM transplantation and prevention of graft rejection or treatment of autoimmunity.     

Termination of Immunologic Tolerance with Tolerated Antigen Complexed with Specific Antibody

Female NTH albino and BALB/c mice were tolerized with chicken egg lysozyme or capsular polysaccharide of Diplococcus pneumoniae type III (SIII). Isolated spleen cells from tolerant mice were incubated in vitro with antigen-antibody complexes formed with the tolerated antigen and specific xenogeneic, allogeneic and syngeneic immune serum. The spleen cells were washed and returned to the individual donor mice and their immune response to the tolerated antigen was assayed. In all cases one or more concentrations of specific antibody when complexed with the tolerated antigen was able to terminate the tolerant state. The successful use of specific antibodies raised in a syngeneic strain of mice (BALB/c) to terminate immunologic tolerance in other BALB/c mice with demonstrated T and B cell tolerance, precludes T cell recognition of a foreign antibody as a mechanism to explain these results.     

Human mesenchymal stem cells for the management of systemic sclerosis. Systematic review

IntroductionSistemic Sclerosis (SSc) is a heterogeneous autoimmune disease with a high rate of progression and therapeutic failure, and treatment is a challenge, new therapeutic proposals being needed, being mesenchymal stem cells (MSCs) considered as alternative therapy for SSc for its immunomodulatory capacity. We evaluated the efficacy and safety of human MSC (hMSC) in patients with SSc through a systematic literature review (SLR).MethodsSLR (PRISMA guideline) on MEDLINE/OVID, LILACS, EMBASE, and Cochrane/OVID bases (until July 2020, without limits). All types of clinical studies were considered: patients ≥18 years old with SSc and treatment with hMSC. Exclusion criteria: animal models, autologous/allogenic hematopoietic stem cell transplants, narrative reviews, letters to the editor. MeSH and “Key word” terms were used. The level of evidence and the quality rating were rated [Joanna Briggs Institute (JBI) lists]. Registration in PROSPERO repository (ID CRD42020185245) The Synthesis Without Meta-analysis (SWiM) guideline was followed.ResultsWe initially identified 508 articles, of which 11 were finally included (8 case series and 3 case reports). The 11 articles included 101 patients (85 female, age range 18–75 years). The level of evidence was mostly 4 (JBI); the quality of evidence was met (≥50% of JBI items). SWiM showed that vascular skin involvement (digital ulcers, necrosis, and gangrene) and associated pain were the predominant outcomes, while improvements were found in almost all cases. One patient died in the first month, and the frequency of complications was low. Expanded hMSCs were used in 24 patients and other cell sources in the remaining patients.ConclusionThere is too little reported data to reach definite conclusions about the use of hMSC in SSc. Further studies with better epidemiological designs are needed to evaluate the benefit of hMSCs in SSc patients.     

Allogeneic mesenchymal stem cells do not protect NZB �� NZW F1 mice from developing lupus disease

Mesenchymal stem cell (MSC) therapy has shown promise clinically in graft‐versus‐host disease and in preclinical animal models of T helper type 1 (Th1)‐driven autoimmune diseases, but whether MSCs can be used to treat autoimmune disease in general is unclear. Here, the therapeutic potential of MSCs was tested in the New Zealand black (NZB) × New Zealand white (NZW) F₁ (NZB/W) lupus mouse model. The pathogenesis of systemic lupus erythematosus involves abnormal B and T cell activation leading to autoantibody formation. To test whether the immunomodulatory activity of MSCs would inhibit the development of autoimmune responses and provide a therapeutic benefit, NZB/W mice were treated with Balb/c‐derived allogeneic MSCs starting before or after disease onset. Systemic MSC administration worsened disease and enhanced anti‐double‐stranded DNA (dsDNA) autoantibody production. The increase in autoantibody titres was accompanied by an increase in plasma cells in the bone marrow, an increase in glomerular immune complex deposition, more severe kidney pathology, and greater proteinuria. Co‐culturing MSCs with plasma cells purified from NZB/W mice led to an increase in immunoglobulin G antibody production, suggesting that MSCs might be augmenting plasma cell survival and function in MSC‐treated animals. Our results suggest that MSC therapy may not be beneficial in Th2‐type T cell‐ and B cell‐driven diseases such as lupus and highlight the need to understand further the appropriate application of MSC therapy.     

Mesenchymal Stem Cells versus Mesenchymal Stem Cells Combined with Cord Blood for Engraftment Failure after Autologous Hematopoietic Stem Cell Transplantation: A Pilot Prospective,Open-Label,Randomized Trial

Engraftment failure (EF) after autologous hematopoietic stem cell transplantation is a serious complication. We prospectively evaluated the effects and safeties of mesenchymal stem cells (MSCs) alone and MSCs combined with cord blood (CB) for EF. Twenty-two patients were randomized to receive MSCs (MSC group; n = 11) or MSCs plus CB (CB group; n = 11). Patients with no response (NR) to MSCs received the therapeutic schedule in the CB group, and those patients with partial response (PR) in the MSC group and patients without complete remission (CR) in the CB group received another cycle of MSC treatment. Patients who did not achieve CR after 2 cycles of treatments received other treatments, including allogeneic HSCT. After the first treatment cycle, response was seen in 7 of 11 patients in the MSC group and in 9 of 11 in the CB group (P = .635), with a significant difference in neutrophil reconstruction between the 2 groups (P = .030). After 2 treatment cycles, 16 patients achieved CR, 3 achieved PR, and 3 had NR. No patient experienced graft-versus-host disease (GVHD). With a median follow-up of 345 d (range, 129 to 784 d) post-transplantation, 18 patients remained alive and 4 had died (3 from primary disease relapse and 1 from cytomegalovirus pneumonia). The 2-year overall survival, disease-free survival, and cumulative incidence of tumor relapse post-transplantation were 75.2% ± 12.0%, 79.5% ± 9.4%, and 20.5% ± 9.4%, respectively. Our data indicate that the 2 strategies are effective for EF and do not result in GVHD or increase the risk of tumor relapse, but the MSC plus CB regimen has a superior effect on neutrophil reconstruction.     

MAPK signaling has stage-dependent osteogenic effects on human adipose-derived stem cells in vitro

Overview: The use of pro-osteogenic growth factors, such as BMP2, in human adipose-derived stem cell (ASC) osteogenesis is well described. Because these growth factors work via signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) cascade, a study of the relationship between MAPK signaling and ASC osteogenesis was conducted. Materials and Methods: ERK, JNK, and p38MAPK activation were measured in ASCs osteo-induced using either dexamethasone or vitamin D3 and correlated with mineralization. Activation and mineralization were also measured without dexamethasone or using the glucocorticoid, cortisone. The expression of the MAPK phosphatase, MKP1, and its relationship to mineralization was also assessed. The effect of decreasing MAPK activation on mineralization through the use of exogenous inhibitors was examined along with siRNA-knockdown and adenoviral overexpression of ERK1/2. Finally, the effect of ERK1/2 overexpression on ASCs induced on PLGA scaffolds was assessed. Results: ASC mineralization in dexamethasone or vitamin D3-induced ASCs correlated with both increased ERK1/2 and JNK1/2 activation. ASCs induced without dexamethasone also mineralized, with JNK1/2 signaling possibly mediating this event. No link between cortisone induction and MAPK signaling could be ascertained. ASCs treated with ERK, JNK, or p38MAPK inhibitors showed decreased osteogenic gene expression and diminished mineralization. Mineralization levels were also affected by viruses designed to inhibit or augment ERK1/2 expression and activity. Finally, ASC mineralization appeared to be a balance between the MAPK kinase activity and MKP1. Conclusions: It is likely that MAPK signaling plays a significant role in ASC osteogenesis, affecting differentiation in kinase- and stage-specific manners.