不同浓度内生致热原的发热效应,脑脊液中cAMP的反应及“热限”的研究

为研究cAMP在发热机制及“EP热限”中的作用,用家兔血细胞加微量内毒素体外培育制备内生致热原(EP),在72只家兔的实验中观察不同剂量EP静脉注射后4小时发热效应;在65只家兔的实验中观察不同剂量EP静脉注射后1小时发热效应及血浆和脑脊液中cAMP含量变化。实验结果表明:(1) 在一定范围内,EP的发热效应随注射剂量的递增而增强,但当发热达到一定强度后,便不再随EP剂量的继续增加而加强,在剂量—效应曲线上出现“平坡”,作者称之为“EP热限”。(2)注射EP后1小时,血浆中cAMP无明显变化,而脑脊液中cAMP的浓度却明显增加,且与发热效应呈非常显著的正相关关系。(3) 当注射大剂量EP出现“热限”时,脑脊液中cAMP的增长也受限。作者推论:脑脊液中cAMP的变化不是来自血浆,cAMP很可能是EP发热的重要中枢介质,cAMP的增长受限可能是“EP热限”的重要成因。     

柴胡注射液对家兔内生致热原性发热及其脑脊液cAMP含量变化的效应

本实验共分二部分进行。第一部分,观察柴胡注射液解热效应,第二部分,观察柴胡注射液解热时对脑脊液cAMP含量变化的影响。用家兔内生致热原复制发热模型,剂量(1×10~6 cells/ml)0.5ml/kg。目前认为:cAMP是一种重要的中枢性发热介质,脑脊液中cAMP含量与发热效应呈正相关。本研究结果表明,柴胡注射液(250mg/kg)对内生致热原性发热有明显抑制作用;同时,脑脊液中cAMP含量也明显低于发热对照组。作者推论:柴胡注射液可能通过某些环节减少体温调节中枢神经元cAMP合成和释放,从而抑制发热效应。     

内生致热原制备的实验研究

本研究应用Ficoll-Hypaque分离技术,从离体兔血液获得单核细胞,用生理盐水配成含单核细胞0.65～1.0×10~6cell/ml的悬液。以精制大肠杆菌内毒素激活,37—38℃振荡孵育10小时。离心收集上清液进行超滤。收集液中含单核细胞内生致热原(monocytic endogenous pyrogen)。实验证明:通过Ficoll-Hypaque分离技术可获得高浓度的单核细胞内生致热原,该内生致热原给动物静脉注射引起明显的发热。     

cAMP热限及其与致热原热限的关系的研究

本实验室最新的研究资料表明,中枢内cAMP的生成受限可能是致热原性发热时热限的重要成因之一。本研究结果显示:(1)在一定范围内,家兔侧脑室注射Db-cAMP(Dibutyryl cyclic AMP,双丁酰环腺苷—磷酸)可以引起依赖于剂量的发热效应,而大剂量时可出现cAMP热限;(2)非热限剂量Db-cAMP加非热限剂量EP(endogenous pyrogen,内生致热原)负荷,表现出两者致热效应的叠加;(3)Db-cAMP热限加热剂量EP负荷,发热高度没有突破EP热限或Db-cAMP热限水平;Db-cAMF热限加热限剂量ET(endotxin,内毒素)负荷,发热高度接近ET热限,但明显高于Db-cAMP热限水平。作者推论,cAMP热限的产生可能与中枢内cAMP的作用点达到饱和有关。在EP热限或ET热限的成因中,既有中枢内cAMP生成受限,又可能同时有中枢内cAMP作用点达到饱和、或cAMP后继介质生成受限。ET热限的构成,除cAMP以外,同时可能还有其它因素参与。     

内生致热原的纯化实验研究

本实验采用家兔单核细胞致热原(monocytic endogenous pyrogen)粗制品,通过超滤、Sephadex G75、QAE-Sephadex A50和等电聚焦电泳方法进一步纯化内生致热原。实验结果表明:凝胶层析纯化内生致热原,有两个致热原性蛋白吸收峰,其致热活性0.3μg蛋白和5.0μg蛋白均引起0.6℃的发热效应。等电聚焦电泳获得的致热原活性蛋白质的等电点,分别为5.1和3.6。     

热限及热限加致热原负荷时脑脊液及血浆中cAMP含量变化的研究

为探讨内毒素(ET)和内生致热原(EP)热限的机制,在家兔静脉注射非热限与热限剂量的EF或EP后及热限加不同致热原负荷时,用竟争性蛋白结合法检测脑脊液和血浆样品中cAMP的含量。结果发现:(1).脑脊液中cAMP含量随EF性发热效应的增强而升高,到达EP热限后则不再上升,而血浆cAMP含量无类似变化。(2).脑脊液和血浆中cAMP含量均随ET性发热效应的增强而上升,到达ET热限后均不再升高。(3).虽然ET热限的发热水平明显高于EP热限,且EP热限加ET负荷使发热水平超过EP热限;但各组之间脑脊液中cAMP含量却无明显差异。上述结果表明,中枢cAMP生成受限可能既是构成EP热限的重要因素,又是ET热限的一个重要成因。但是除cAMP外,ET热限的构成还与其它因素有关。     

精氨酸加压素对家兔血细胞生成内生致热原的影响

本文观察了精氨酸加压素对体外培育的兔全血细胞产生内生致热原的影响。结果表明，ＡＶＲ对内毒素诱生ＥＰ过程有明显抑制作用；而ＡＶＰ单独与血细胞培养无降温物质产生；同时，所用剂量的ＡＶＰ对ＥＰ性发热无直接抑制作用。因此作者认为，ＡＶＰ１能够抑制ＥＴ诱生家兔血细胞生成ＥＰ，这可能是其降低或阻断ＥＴ性发热的主要机制之一。     

针刺对家兔内生致热原性发热的效应

用126只大耳白家兔分成三部分进行实验,每部分动物均分成实验组和对照组。第一部分和第二部分实验,均用内生致热原复制发热模型。前者实验动物,针刺“百会穴”,后者实验动物,针“刺非穴位”,均观察对发热的影响。第三部分实验动物,均不注射内生致热原,但针刺实验组动物“百会穴”,观察对正常体温的影响实验结果表明:(一) 针刺内生致热原发热家兔“百会穴”,具有明显的退热作用。在体温上升期,发热高度降低,抑制体温上升、加大退热幅度和加快退热速度;(二) 针刺内生致热原发热家兔“非穴位”。没有显著的退热作用;(三) 针刺正常家兔“百会穴”,体温不因针刺而发生显著波动。     

热限及热限加致热原负荷时发热效应的研究

为研究内毒素(ET)和内生致热原(EP)热限的形成机制,观察家兔静脉注射不同剂量ET和EP的发热效应。确定ET及EP的热限剂量后对ET与EP热限的发热水平以及热限加不同致热原负荷时发热水平的变化进行观察比较。实验表明:1.ET热限水平明显高于EP热限水平,提示ET性发热及热限的形成机制与EP性发热及热限形成机制有所不同。2.EP热限加ET负荷使发热高度突破了EP热限,而ET热限加EP负荷则未能使发热高度超过ET热限的水平。表明ET热限包含了EP热限,即EP热限是构成ET热限的重要成因,但不是全部。ET性发热及热限的机制中还有EP以外的因素参与。     

低温保存内生致热原对其致热活性的影响

本实验用家兔全血加精制大肠杆菌内毒素，体外培养提取粗制家兔内生致热原。给大鼠静脉注射复制发热模型，观察了不同温度保存和不同时间保存的ＥＰ对其致热活性的影响。结果表明：４℃保存３天，－４０℃保存３天，７天，３０天和１８０天的ＥＰ与４℃保存１天的ＥＰ比较，其发热第一时相发热峰值和１小时体温反应指数均无显著性差异（Ｐ＜０．０５）。发热第二时相△Ｔ和第二时相１小时ＴＲＩ，在４℃保存３天和－４０℃保存３天，     

Further comparisons of endogenous pyrogens and leukocytic endogenous mediators.

It was recently shown (Murphy et al., Infect. Immun. 34:177-183), that rabbit macrophages produce two biochemically and immunologically distinct endogenous pyrogens. One of these has or copurifies with substances having a molecular weight of 13,000 and a pI of 7.3. This protein was produced by blood monocytes or inflammatory cells elicited in 16-h rabbit peritoneal exudates. These acute peritoneal exudates were produced by the intraperitoneal injection of large volumes of saline containing shellfish glycogen. When the leukocytes in these exudates were washed and incubated at 37 degrees C in saline, they released an endogenous pyrogen. The injection of this pyrogen into rabbits, rats, or mice caused the biological manifestations which have been attributed to leukocytic endogenous mediator. These effects were increases in blood neutrophils, the lowering of plasma iron and zinc levels, and the increased synthesis of the acute-phase proteins. The other rabbit endogenous pyrogen seems to be a family of proteins with isoelectric points between 4.5 and 5.0. These proteins are produced by macrophages in the lung, liver, or in chronic peritoneal exudates. In these experiments, the lower-isoelectric-point endogenous pyrogens were produced by macrophages from the peritoneal cavity of rabbits that had been injected 4 days earlier with 50 ml of light mineral oil. These rabbit pyrogens were found to have leukocytic endogenous mediator activity in mice but to be completely inactive in rats. When injected into rabbits, these proteins produced fever, lowered plasma iron, increased blood neutrophils, but failed to elevate plasma fibrinogen.     

Natural endogenous adjuvants

It has long been known that immunization with a protein by itself is often not sufficient to stimulate immunity, and may instead induce tolerance. To elicit productive immune responses exogenous adjuvants need to be co-injected with an antigen. One important class of adjuvants are the unique (non-mammalian) components of microbes. It is now believed that an adjuvant is required for immunity because the immune system evolved to respond to dangerous situations such as infections, and the presence of an adjuvant is the mechanism used to identify these situations. However, there are some circumstances where immune responses are generated in the apparent absence of any microbial or other exogenous adjuvant. Such situations include immune responses to transplants, tumors, autoimmunity and possibly certain viral infections. It has been postulated that in these situations the danger signals come from endogenous adjuvants that are released from dying cells. There is abundant evidence that dead cells are immunogenic, and recently it has been shown that cells contain endogenous adjuvant activities that are released after death. Some actual and putative endogenous adjuvants, such as monosodium urate and heat shock proteins, have been identified and there are others whose identities are not yet known. The potential biological roles of this class of adjuvants are discussed.     

Natural endogenous adjuvants

It has long been known that immunization with a protein by itself is often not sufficient to stimulate immunity, and may instead induce tolerance. To elicit productive immune responses exogenous adjuvants need to be co-injected with an antigen. One important class of adjuvants are the unique (non-mammalian) components of microbes. It is now believed that an adjuvant is required for immunity because the immune system evolved to respond to dangerous situations such as infections, and the presence of an adjuvant is the mechanism used to identify these situations. However, there are some circumstances where immune responses are generated in the apparent absence of any microbial or other exogenous adjuvant. Such situations include immune responses to transplants, tumors, autoimmunity and possibly certain viral infections. It has been postulated that in these situations the danger signals come from endogenous adjuvants that are released from dying cells. There is abundant evidence that dead cells are immunogenic, and recently it has been shown that cells contain endogenous adjuvant activities that are released after death. Some actual and putative endogenous adjuvants, such as monosodium urate and heat shock proteins, have been identified and there are others whose identities are not yet known. The potential biological roles of this class of adjuvants are discussed.     

Pharmacology of endogenous neuroactive steroids

Neuroactive steroids are potent endogenous neuromodulators with rapid actions in the central nervous system. Neuroactive steroids have been claimed to have specific physiological roles in normal or pathological brain function. This article reviews the emerging evidence that progesterone-, deoxycorticosterone-, and testosterone-derived endogenous neuroactive steroids play an important role in the modulation of neural excitability and brain function. Neuroactive steroids such as allopregnanolone and allotetrahydrodeoxycorticosterone (THDOC) are extremely potent positive allosteric modulators of GABAA receptors with sedative, anxiolytic, and anticonvulsant properties.The sulfated neuroactive steroids pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS), which are negative GABAA receptor modulators, induce anxiogenic and proconvulsant effects. Thus, natural fluctuations in neuroactive steroid levels during the menstrual cycle and stress could affect several nervous system functions. There is strong evidence that allopregnanolone and THDOC are involved in the pathophysiology of premenstrual syndrome, catamenial epilepsy, major depression, and stress-sensitive brain disorders. Neuroactive steroids PS and DHEAS have been shown to modulate memory functions. However, the significance of the testosterone-derived neuroactive steroid 3alpha-androstanediol is not well understood. Like naturally occurring neuroactive steroids, synthetic derivatives such as ganaxolone have been proven in preclinical and clinical studies to be effective anticonvulsants with great potential for human use. Future research on inhibition or stimulation of specific neuroactive steroid synthesizing enzymes could provide an improved understanding and novel approaches for the treatment of anxiety, epilepsy, and depression.     

Deconstructing endogenous pain modulations

A pathway from the midbrain periaqueductal gray (PAG) through the ventromedial medulla (VMM) to the dorsal horn constitutes a putative endogenous nociceptive modulatory system. Yet activation of neurons in both PAG and VMM changes the responses of dorsal horn cells to non-noxious stimuli and elicits motor and autonomic reactions that are not directly related to nociception. Activation of mu-opioid receptors in VMM and PAG also modifies processes in addition to nociceptive transmission. The descending projections of VMM neurons are not specific to nociception as VMM projects to the spinal superficial dorsal horn where thermoreceptors as well as nociceptors terminate. In addition, experiments with pseudorabies virus demonstrate multi-synaptic pathways from VMM to sympathetic and parasympathetic target organs. VMM neurons respond to both noxious and unexpected innocuous stimuli of multiple modalities, and change their discharge during behaviors unrelated to pain such as micturition/continence and sleep/wake. In conclusion, all available evidence argues against the idea that PAG and VMM target nociception alone. Instead these brain stem sites may effect homeostatic adjustments made necessary by salient situations including but not limited to injury.