前列腺素E_2对人单核THP-1细胞释放肿瘤坏死因子受体的调节作用

最近体外研究指出，前列腺素－F2（PGE2）可以抑制人单核细胞系THP－1产生肿瘤坏死因子（TNF）。PGF2不仅调控TNF的产生，而且也通过调节2种可溶性TNF受体（BP－55，BP－75）的释放对TNF活性进行调节，因后者可干扰TNF与细胞膜上的受体结合。该文用PGE2体外刺激TNF－1细胞，观察对TNF受体释放以及mRNA和表面受体表达的影响。将THP-1细胞接种于24孔组织培养板，用不同浓度PGE2活化一定时间后，收集上清，用ELISA检测可溶性TNF受体；用125I标记的rTNF－α测定细胞膜表面的TNF受体数量及其亲和力；用异硫酸氰胍／氯化…     

β受体配体~(125)碘氰心得静的标记及初步鉴定

1981年G.Engel创用~(125)碘氰心得靜(~(125)Iodocyano-Pindolol,简称~(125)ICYP),其优点为1,容易标记.2,较~(125)碘羟苄心得靜(~(125)Iodohydroxybenzylpindolol)特异性强对α及5-HT受体不起作用,对β受体结合力强3倍。3,比放射性高,只需用少量膜蛋白。4,较稳定,在20℃保存3个月,游离碘少于1％。1983年薛全福与Engel用此配体作大鼠肺β受体自显影发现3-7天即可得清晰图象,较以往用~3H标记配体大大缩短     

大鼠肝细胞泌乳素受体的研究

本文以~(125)I-oPRL为放射催标记配体,利用受体放射分析法,对哺乳期大鼠肝细胞泌乳素受体进行了研究。结果表明。大鼠肝细胞泌乳素受体与泌乳素的结合具有高度特异性和可饱和性,而且受反应时间和pH值大小的影响;这种结合并非简单的双分子反应,大鼠肝细胞泌乳素受体很可能存在两种亲和力高低不同的结合位点(Kd_1=3.20×10~(10)M,Kd_2=1.26×10~(-8)M)或负协同调节。本研究为将来深入开展泌乳素对肝脏机能调节机制的探讨打下了基础。     

LAIR-1/LAIR-2(CD305/CD306)与配体结合亲和力的比较

目的:LAIR-1/LAIR-2(CD305/306)与配体结合亲和力的比较。方法:应用不同浓度混合的LAIR-1和LAIR-2融合蛋白,同时或先后与配体表达细胞孵育后,用特异性LAIR-1或LAIR-2单克隆抗体(mAb)进行免疫荧光染色和流式细胞术(FCM)分析,比较LAIR-1和LAIR-2融合蛋白与细胞结合的百分率的变化及相互竞争情况。结果:LAIR-1和LAIR-2膜型配体的分布较广泛,人和小鼠LAIR受体与配体的结合具有相互交叉的特点。LAIR-2能够明显阻断LAIR-1与其膜型配体的结合,而LAIR-1对LAIR-2与其配体的结合无影响。结论:LAIR-1和LAIR-2可能结合相同的膜型配体,但LAIR-2结合配体的亲和力明显高于LAIR-1结合的亲和力。此结果为深入探讨LAIR家族免疫调节的分子机制提供重要的实验依据。     

人IL-2抗独特型单克隆抗体

本文报道吸附纯化兔抗人重组IL-2抗体(Id)的抗独特型单克隆抗体(抗-IdMcAb)6A12。McAb与Id结合可以抑制Id对IL—2的中和作用,表明其结合位点可能与Id上IL—2中和位点相同或邻近。6A12协同IL—2对PHA活化人T细胞的增殖作用,纯化的6A12 IgG也可与纯化的重组IL-2受体可溶性P~(55)亚单以结合;对IL-2依赖的鼠T细胞系(C TLL),纯化的6A 12IgG有抑制IL-2爱性作用,而当IL-2过量时可使抑制作用逆转。结果表明:6A12不可能是IL-2受体结合位点精确的“内都影像”,但它至少可与高亲和力IL-2受体复合物中配体结合位点P~(55)亚单位结合。特定配体的抗-Id抗体与配体的生理受体相互作用已有系统描述,对人重组白细胞干扰素A(rIFN-αA )抗体的抗-Id McAb研究已知:McAb 3B1似乎完全可以作为干扰素内部影像或Jerne称作的“补位诱导抗体(Ab2β)”。T淋巴细胞表面具有对IL-2亲和力高低不同的膜受体,IL-2与之结合调节T细胞生长。最近有关受体结构研究也表明。高亲和力IL-2受体复合物至少由2种多肽链构成,α链(Tac抗原或P~(55))及β链(70～75kD糖蛋白或P70),二者分别以低等(P~(55))和中等亲和力独立与IL-2结合。本文报道研制了人IL-2抗-Id MeAb 6A12,并对其生物学活性进行了研究。     

白细胞介素6受体研究新进展

白细胞介素6(IL-6)的受体系统由配体结合?白细胞介素6受体(IL-6R)和信号传递链gp130组成,其中gp130虽无配体结合能力,但参与IL-6高亲和力结合位点的形成.这两条链均为跨膜的单链膜糖蛋白,属于细胞因子受体家族.IL-6受体的双链模式可能也适用于此家族中的其它成员.与其它细胞因子的可溶性受体不同的是,可溶性IL-6R反而能介导IL-6的生物学作用,在机体的免疫调节和与IL-6相关疾病的病理过程中发挥着重要作用.     

化学换能器

受体具有与配体(Ligands)相结合的能力,但酶也具有这种能力,其它物质如蛋白质、类脂质、神经节甙脂(Gangliosides)等也能在较弱程度上与配体相结合,所以,要证实一种受体的存在,这种结合就需满足以下一些标准: 标准: 1、这种结合一定要有高度亲和力。大多数受体反应的解离常数(Kd)都在100微微克分子(100pM)至10毫微克分子(10nM)之间。某些酶与配体的结合也有高度亲和力,这可用某些特异的酶抑制剂来作鉴别。这些酶抑制剂能减弱酶的活     

大鼠伏核、杏仁核、中脑导水管周围灰质内的CCK受体为CCK-B型受体:受体放射自显影分析

本工作用受体激动剂~(125)I-Bolton Hunter-CCK-8(~(125)I-BH-CCK)、CCK-A受体拮抗剂Devazepide、CCK-B受体拮抗剂L-365,260与脑组织切片共同孵育,以确定大鼠伏核、杏仁核、中脑导水管周围灰质等脑区内CCK受体的类型。结果显示:1μM未标记的CCK-8与~(125)I-BH-CCK及脑组织切片共同孵育,可完全竞争标记配体与受体的结合,说明这种结合是特异性的。30μM Devazepide可抑制脚间核处受体与标记配体的结合,但不能抑制伏核、杏仁核、中脑导水管周围灰质、大脑皮质及脊髓内受体与标记配体的结合;当剂量增大到90μM时,也只产生部分抑制。而用L-365,260只需90μM即可完全竞争伏核、杏仁核、中脑导水管周围灰质、大脑皮质及脊髓内受体与标记配体的结合。以上结果提示:脚间核部位的CCK受体为CCK-A受体;伏核、杏仁核、中脑导水管周围皮质、大脑皮质及脊髓内的CCK受体为CCK-B受体。     

鸟类及鼠类的胸腺、腔上囊及脾脏褪黑素受体的鉴定及意义

应用放射配体结合法检测大鼠、小鼠、仓鼠、鸡、鸭、鸽子及鹌鹑的胸腺、腔上囊及脾脏的[~(125)I]褪黑素(M)特异结合位点。结果显示鸟类及鼠类免疫器官均存在[~(125)I]M特异结合位点,并具低结合容量、高亲和力特点;动力学研究表明具可饱和性及可逆性,符合受体的结合及解离过程;特异性研究显示对M及激动剂有高度特异性,符合特异结合位点的全部条件,亚细胞分布的研究表明以细胞核含量最高,线粒体次之,且该结合位点具年龄依赖性降低。结果证实免疫器官存在根黑素受体(MR),免疫组织是M作用的靶器官,M对免疫系统的调节作用是通过免疫组织上MR直接作用的结果。     

树鼩视网膜褪黑激素受体

视网膜具有合成,释放褪黑激素(MEL)的能力。MEL作为局部激素,可调节视网膜内多种神经递质的释放及视敏度。近年来应用~(125)IMEL作为放射性配体,证明鸟类视网膜存在MEL受体。本文报道了灵长类动物——树鼩视网膜MEL受体的结合特性。将成年树鼩断买处死后立即分离视网膜,制备匀浆、分离膜组份,进行放射配体受体结合     

CD40-mediated signaling in monocytic cells: up-regulation of tumor necrosis factor receptor-associated factor mRNAs and activation of mitogen-activated protein kinase signaling pathways

The biochemical pathways involved in CD40 signaling have been extensively studied in B cells and B cell lines, and appear to be primarily initiated by recruitment of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) signaling proteins to the CD40 cytoplasmic domain. Signaling pathways activated through CD40 in monocytes/macrophages have not been characterized as well as in B cells. Using human monocytes and the human monocytic cell line THP1, we examined signal transduction events induced by CD40 engagement with its ligand, CD154. In human monocytes, all TRAF mRNAs were expressed constitutively and CD40 ligation resulted in a strong up-regulation of TRAF1 mRNA. In THP1 cells, CD40 ligation induced expression of TRAF1 and TRAF5 mRNAs. Engagement of CD40 in both monocytes and THP1 cells led to the rapid and transient activation of the extracellular signal-regulated kinases (ERK) 1 and 2, and to low levels of JNK activation. No CD40-dependent activation of p38 mitogen-activated protein kinase (MAPK) was found. In CD154-stimulated monocytes and THP1 cells the upstream ERK1/2 activator, MAPK kinase (MEK) 1/2, and downstream substrate, c-Myc, were activated. By blocking activation of ERK1/2 with a MEK-specific inhibitor, PD98059, CD40-dependent secretion of the pro-inflammatory cytokines, TNF-alpha, IL-6 and IL-8, was demonstrated to be linked to the ERK1/2 pathway. The ERK1/2 pathway did not appear to be involved in up-regulating TRAF1 and TRAF5 mRNAs in THP1 cells. Collectively, these results suggest distinct differences between B cells and monocytic cells in CD40-dependent activation of MAPK pathways.     

MBL抑制白假丝酵母菌刺激THP1/CD14细胞产生TNF-α和IL-8

目的 探讨甘露聚糖结合凝集素(mannan-binding lectin,MBL)对白假丝酵母菌(Candida albicans)刺激的THP1/CD14细胞产生TNF-α和IL-8的影响.方法 以不同浓度人MBL预处理THP1/CD14细胞2 h后,再用热灭活的酵母相C.albicans和/或菌丝相C. albicans刺激细胞24 h,收集培养上清,以ELISA从蛋白水平分析其TNF-α和IL-8的产生.收集细胞提取总RNA,以RT-PCR从转录水平评估TNF-α和IL-8的表达,Western blot分析NF-KB的细胞核移位.结果 ELISA检测发现,两种相态的C. albicans均可刺激各组细胞分泌TNF-α和IL-8,酵母相C.albicans刺激细胞产生细胞因子水平稍高;高浓度MBL(10～20 mg/L)均可抑制两种相态的C. albicans诱导细胞分泌TNF-α和IL-8,低浓度MBL(1 mg/L)则几乎无影响;RT-PCR分析亦显示,与相应只用C. albicans刺激的实验组相比,高浓度MBL(20 mg/L)对两种相态的C. albicans诱导的TNF-α、IL-B的mRNA表达均有不同程度的抑制作用;Western blot分析显示,高浓度MBL(20 mg/L)对两种相态的C. albicans诱导的NF-KB细胞核移位有显著的抑制作用.结论 MBL可抑制C.albicans诱导的THP1/CD14细胞产生TNF-α和IL-8,提示MBL能够在抗C.albicans 免疫中起调控作用.

Abstract：

Objective To investigate the effects of mannan-binding lectin (MBL) on IL-8 and TNF-α production induced by Candida albicans ( C. albicans) in human THP1/CD14 monocytes. Methods The THP1/CD14 cells were stimulated for 24 h with heat-inactivated yeast form or hyphal form cells of C. albicans strain at the indicated ratios after pretreated with human natural MBL at concentrations ranging from 1 to 20 mg/L for 2 h. The content of IL-8 and TNF-α in culture supernatants were detected by ELISA,and the levels of IL-8 and TNF-α mRNA expressions in these cells were determined by RT-PCR. Western blot was used to detect C. albicans-induced NF-κB translocation in THP1/CDI4 cells. Results ELISA showed that secretion of IL-8 and TNF-α from THP1/CD14 cells could be induced by both yeast cells and hyphal cells. Hyphal cells proved to be much less efficient than yeast cells in stimulating production of IL-8and TNF-α by THP1/CD14 cells. The productions of IL-8 and TNF-α by THP1/CD14 cells induced with C.albicans were profoundly inhibited by MBL at higher concentrations ( 10-20 mg/L) but not MBL at lower concentrations ( 1 mg/L). RT-PCR analysis also indicated that the mRNA expressions of IL-8 and TNF-αt in THP1/CD14 cells were decreased to various extents by MBL at higher concentration, compared to the corresponding THP1/CD14 cells stimulated with C. albicans only. Similarly, MBL at higher concentration ( 20mg/L) decreased the NF-κB translocation in THP1/CD14 cells. Conclusion MBL may inhibit IL-8 and TNF-α production induced by dimorphism C. albicans in THP1/CD14 cells, suggesting that MBL can play some roles on the regulation of C. albicans immune response.     

Identification of the scavenger receptors SREC-I, Cla-1 (SR-BI), and SR-AI as cellular receptors for Tamm-Horsfall protein

Tamm-Horsfall protein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in urine. An important role for THP in antibacterial host defense but also in inflammatory disorders of the urogenital tract has been suggested. In line with this, THP has been shown recently to potently activate macrophages and dendritic cells (DC) via the toll-like receptor 4 (TLR4) pathway. We show here that THP interacts specifically with surface structures on DC and provides evidence that they are distinct from TLR4. Using retroviral expression cloning, we have identified one such receptor as the scavenger receptor (SR) expressed by endothelial cells I (SREC-I). In addition, we found that two other receptors for acetylated low-density lipoprotein (AcLDL), namely scavenger receptors AI (SR-AI) and Cla-1 (SR-BI), also serve as receptors for THP. SREC-I/THP interaction is of high affinity (16.8+/-6.8 nM), whereas Cla-1 and SR-AI have lower affinities for THP (396 nM+/-114 nM and 802 nM+/-157 nM, respectively). The interaction of THP with these molecules is fully blocked by AcLDL. However, AcLDL only partially blocks binding of THP to DC, and a series of experiments did not support a role in DC activation for SR interacting with THP and AcLDL. Thus, our data point to the existence of additional receptors for THP, which mediate TLR4-dependent DC activation. Interaction and up-take of THP by SR might play an important role in local host defense and could contribute to inflammatory kidney diseases associated with THP-specific antibody responses.     

Pharmacological inhibition of Mannheimia haemolytica lipopolysaccharide and leukotoxin-induced cytokine expression in bovine alveolar macrophages

The inflammatory cytokines tumor necrosis factor-alpha (TNFalpha), interleukin-1 beta (IL-1beta), and interleukin-8 (IL-8) are believed to contribute to the pathogenesis of lung injury in bovine pneumonic mannheimiosis (BPM) caused by Mannheimia (Pasteurella) haemolytica. Inflammatory cytokines may, therefore, represent therapeutic targets to be modulated for the purpose of treating or preventing this important disease of cattle. The purpose of this study was to evaluate the ability of six pharmacological agents to suppress the expression of TNFalpha, IL-1beta, and IL-8 genes and proteins in bovine alveolar macrophages (AM) exposed to M. haemolytica lipopolysaccharide (LPS) and leukotoxin (LktA) in vitro. The compounds tested included dexamethasone (DEX), tetrahydropapaveroline (THP), pentoxifylline (PTX), rolipram (ROL), SB203580 (SB), and thalidomide (THL). Cytokine expression was induced by the addition of purified M. haemolytica LPS and LktA to AM cell cultures following pretreatment with inhibitor compounds. Secretion of TNFalpha, IL-1beta, and IL-8 proteins into the cell culture supernatant was measured using enzyme-linked immunosorbent assays, and steady-state accumulation of cytokine-specific mRNA was measured by northern blot analysis. Dose-dependent inhibition of cytokine secretion occurred in response to pretreatment of AM with DEX (TNFalpha, IL-1beta, IL-8), THP (TNFalpha, IL-1beta, IL-8), PTX (TNFalpha, IL-1beta, IL-8), ROL (TNFalpha, IL-1beta), and SB (TNFalpha, IL-8). Significant dose-dependent inhibition of cytokine mRNA expression occurred in response to pretreatment with DEX (TNFalpha, IL-1beta, IL-8), THP (TNFalpha, IL-1beta, IL-8), and PTX (TNFalpha). DEX was the most effective inhibitor by far; pretreatment with this compound yielded greater than 95% inhibition of cytokine gene and protein expression over a broad range of concentrations. These findings demonstrate that DEX, THP, PTX, ROL, and SB are capable of suppressing inflammatory cytokine secretion by bovine AM in vitro. If pulmonary cytokine secretion may be similarly inhibited in vivo, anti-cytokine therapy may represent a novel strategy for the management of BPM.     

Polarized expression of Tamm-Horsfall protein by renal tubular epithelial cells activates human granulocytes

In renal bacterial infections granulocytes are of major importance in the primary immune defense against invading pathogens. However, the mechanisms of granulocytic activation in renal interstitial invasion have not been clarified. Renal tubular epithelial cell mechanisms inducing granulocytic activation and bacterial killing may include tubular cell expression of Tamm-Horsfall protein (THP), a urinary protein that is known to enhance cytokine expression in monocytes. We studied the role of THP in granulocytic activation. A strong binding of THP to human granulocytes was demonstrated by fluorescence-activated cell sorter analysis. Urinary THP and supernatants of THP-expressing cultured tubular epithelial cells (MDCK) enhanced interleukin-8 (IL-8) expression by human granulocytes. Renal tubular cells growing polarized on polycarbonate membranes were used to study apical versus basal THP expression. By electron microscopy THP immunoreactivity was exclusively found on the apical surfaces of tubular cells and was absent on the basolateral cell membrane. In the apical cell culture compartment we found significantly more stimulatory activity for granulocytic IL-8 expression. CD62L, a selectin less expressed in activated granulocytes, was decreased in granulocytes incubated with urinary THP and in supernatants of THP-producing renal tubular cells but not in supernatants from THP-negative cells. Again, the effect on CD62L expression was found only in apical culture media and was absent in the basal compartment. In summary our data give evidence that renal tubular cell THP expression may be relevant in kidney diseases since THP is a potent activator of human granulocytes. The regulation of apical versus basal THP expression and release in vivo may be crucial in the induction of the inflammatory response, e.g., in bacterial renal diseases.     

Regulation of FcεRI expression on human monocytic cells by ligand and IL-4

BACKGROUND: The Fc epsilonRI subunit composition and kinetic of expression differ between antigen-presenting cells and mast cells. Up to now, there has been no human in vitro model available that mimics the characteristics on monocytes. OBJECTIVE: The characterization of a natural human monocytic cell line (THP1), which expresses Fc epsilonRI, and the comparison to primary human monocytes and other monocytic cell lines, which only express Fc epsilonRI after transfection with the human Fc epsilonRI alpha-chain gene. METHODS: Surface receptor expression was characterized by flow cytometry, the human Fc epsilonRI alpha-chain gene was introduced by electroporation, and induction of Fc epsilonRI alpha-chain message was detected by semiquantitative RT PCR. RESULTS: Here we show that the parental human cell line THP1, but none of the other cell lines tested, displays surface Fc epsilonRI in response to IL-4 or incubation with receptor ligand (IgE, antibody). Transfection of Fc epsilonRI alpha-chain resulted in receptor expression on all cell lines, all of which increased surface Fc epsilonRI in the presence of IgE. Only the THP1-alpha transfectant, however, further increased receptor levels in response to IL-4, resulting from mRNA induction for the Fc epsilonRI-alpha, but not the beta- or gamma-subunit. CONCLUSION: Based on THP1, U937 and HL60 and their alpha-chain transfectants we present a model system for the study of Fc epsilonRI regulation and signalling on human cells. THP1 in particular, due to its responsiveness to both ligand and IL-4, even without prior manipulation, is ideally suited to address questions on Fc epsilonRI modulation in an 'allergic environment'.     

The interaction of Tamm-Horsfall protein with the extracellular matrix.

Tamm-Horsfall protein (THP) is the major glycoprotein component of urine, yet its biological role remains obscure. Recent reports have suggested that a concanavalin A (Con A)-binding fraction of THP from pregnancy urine can bind the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1). In order to investigate this claim in relation to THP from normal adult urine we raised monoclonal antibodies to THP and sought THP/TNF-alpha interactions in three separate assay systems. We found no evidence that THP binds to TNF-alpha under physiological conditions, but we observed that it exerts a weak, probably not physiologically relevant, but reproducible inhibitory effect on the toxicity of TNF-alpha for monolayers of L929 cells, even when the cells are pretreated with the THP, and washed before addition of the cytokine. Since our preparations of THP do not interact directly with TNF-alpha we postulated an interaction with the cells themselves, or with their extracellular matrix. The THP was found by ELISA, immunoblotting and immunohistology, to bind to as yet unidentified components of the extracellular matrix in a manner dependent on cations, pH and carbohydrates. These data, considered in the light of the published amino acid sequence and biochemical properties, suggest that THP is a member of a structural glycoprotein family known to modulate cell adhesion.     

单核因子在Tamm-Horsfall蛋白所致肾小管间质肾炎纤维化形成中的作用

将Wistar大鼠的腹腔巨噬细胞(Mφ)分别与Tamm-Horsfall蛋白(THP,A组)、去糖基THP(B组)或RPMI1640(C组)在体外共育4小时,而后更换培养液继续孵育24小时。用生物学方法测定发现唯A组Mφ上清液含肿瘤坏死因子和IL-1样活性。将三组Mφ上清液分别注入各自大鼠右肾,6周后A组右肾全部发生了炎症和纤维化,伴有肾小管功能改变,而B、C组肾间质无明显异常。由此表明,THP对单核、Mφ的活化作用及由此而产生的生物活性介质可能在某些肾小管间质疾病纤维化的形成中具有重要意义。     

Identification and characterization of a novel bacterial virulence factor that shares homology with mammalian Toll/interleukin-1 receptor family proteins

Many important bacterial virulence factors act as mimics of mammalian proteins to subvert normal host cell processes. To identify bacterial protein mimics of components of the innate immune signaling pathway, we searched the bacterial genome database for proteins with homology to the Toll/interleukin-1 receptor (TIR) domain of the mammalian Toll-like receptors (TLRs) and their adaptor proteins. A previously uncharacterized gene, which we have named tlpA (for TIR-like protein A), was identified in the Salmonella enterica serovar Enteritidis genome that is predicted to encode a protein resembling mammalian TIR domains, We show that overexpression of TlpA in mammalian cells suppresses the ability of mammalian TIR-containing proteins TLR4, IL-1 receptor, and MyD88 to induce the transactivation and DNA-binding activities of NF-kappaB, a downstream target of the TIR signaling pathway. In addition, TlpA mimics the previously characterized Salmonella virulence factor SipB in its ability to induce activation of caspase-1 in a mammalian cell transfection model. Disruption of the chromosomal tlpA gene rendered a virulent serovar Enteritidis strain defective in intracellular survival and IL-1beta secretion in a cell culture infection model using human THP1 macrophages. Bacteria with disrupted tlpA also displayed reduced lethality in mice, further confirming an important role for this factor in pathogenesis. Taken together, our findings demonstrate that the bacterial TIR-like protein TlpA is a novel prokaryotic modulator of NF-kappaB activity and IL-1beta secretion that contributes to serovar Enteritidis virulence.     

The 5' coding sequence of IL-2 receptor alpha chain mRNA mediates mRNA stabilization by HTLV-1 Rex.

The interleukin-2 receptor alpha chain (IL-2R alpha) is a T-cell growth factor receptor and is known to be induced in helper T cells by infection with human T-cell leukaemia virus type-1 (HTLV-1). The Rex protein of HTLV-1 has been shown to stabilize IL-2R alpha mRNA. Although the 3' untranslated region of many RNA has been regarded as a key element for stabilization, we found that the first 300 bases of the IL-2R alpha protein coding sequence were necessary for stabilization of the mRNA. As the first 201 bases were not sufficient for this effect, we conclude that the bases at position 201-300 downstream of the AUG start are important for stabilization.