主动免疫AT1受体对自发性高血压大鼠血管重构的影响

目的观察血管紧张素Ⅱ的Ⅰ型(AT1)受体细胞外不同肽段主动免疫自发性高血压大鼠(SHR)对血压及血管重构的影响.方法2月龄SHR在血压升高后,以合成的AT1受体细胞外的多肽片段ATR12185、ATR10014和ATR12181作为抗原,分别主动免疫,用药组给予氯沙坦(10 mg/(kg·d))灌胃.同龄Wistar大鼠免疫干预组同SHR,对照组只用免疫佐剂.ELISA法检测抗体滴度,大鼠尾动脉血压计测量血压.试验结束前,测定肠系膜三级动脉中层及内径,观察肠系膜动脉病理改变,以及胸主动脉超微病理结构改变.结果SHR于2月龄血压开始升高;所有免疫组动物均产生了针对特定短肽的抗体.ATR12181免疫组SHR收缩压于免疫后1月降低,并持续至实验结束[(145.4±8.5)mm Hg,n=7],与SHR对照组[(197.0±7.7)mm Hg,n=7,P＜0.05]相比,收缩压降低,与氯沙坦治疗组[(139.3±17.2)mm Hg,n=7,P＞0.05]相比,无显著性差异,而ATR12185和ATR10014免疫组SHR收缩压无明显变化.Wistar大鼠免疫前后收缩压无明显变化.ATR12181免疫组SHR肠系膜三级动脉中膜厚度/管腔半径(1.10±0.18)及中膜面积/管腔面积(3.21±0.98),分别较SHR对照组(1.39±0.21)及(4.62±1.21)降低.结论用ATR12181主动免疫SHR,可降低SHR血压,降低肠系膜三级动脉中膜厚度/管腔半径及中膜面积/管腔面积,逆转主动脉超微结构的损害.     

血管紧张素AT1受体短肽对自发性高血压大鼠血压、心脏及血管的影响

目的观察血管紧张素Ⅱ的Ⅰ型(AT1)受体细胞外的不同肽段对主动免疫自发性高血压大鼠(SHR)血压及靶器官的影响.方法4周龄SHR随机分成5组,每组7只.免疫组有3组,在SHR血压升高后,以合成的AT1受体细胞外的多肽片段ATR12185、ATR10014和ATR12181作为抗原,分别主动免疫,SHR用药组给予氯沙坦(10mg·kg-1·d-1)灌胃治疗,对照组饮水灌胃.同龄Wistar大鼠随机分成4组,每组7只,免疫干预组同SHR,对照组只用免疫佐剂.ELISA法检测免疫组动物的抗体滴度,用大鼠尾动脉血压计观察大鼠血压的变化.试验结束前,测定心脏及左心室重量、肠系膜三级动脉中层及内径,观察心肌及肠系膜三级动脉病理改变,以及心肌超微病理结构改变.结果SHR于6周龄血压开始升高;所有免疫组动物均产生了针对特定短肽的抗体.ATR12181免疫组SHR收缩压于免疫后一个月降低,并持续至实验结束[(145.42±8.46)mmHg,n=7],与SHR对照组[(197.0±7.7)mmHg,n=7,P＜0.05]相比,收缩压明显降低,与氯沙坦治疗组[(139.3±17.2)mmHg,n=7]相比,P＞0.05],而ATR12185免疫组[(183.42±19.26)mm Hg,n=7]和ATR10014免疫组(191.6±15.0)mm Hg,n=7]SHR收缩压无明显变化.Wistar大鼠免疫前后收缩压无明显变化,试验结束时,ATR12181收缩压[(116.6±5.5)mm Hg,n=7],ATR12185收缩压[(116.0±9.5)mm Hg,n=7],ATR10014收缩压[(121.57±4.79)mm Hg,n=7],与对照组[(113.14±9.52)mm Hg,n=7,P＞0.05]无差别.ATR12181免疫组SHR心脏/体重(4.66±0.50)mg/g以及左心室/体重(3.6±0.5)mg/g较SHR对照组心脏/体重(5.16±0.11)mg/g、左心室/体重(4.2±0.1)mg/g降低,P＜0.05,与氯沙坦心脏/体重(4.4±0.6)mg/g及左心室/体重(3.5±0.3)mg/g相比,无显著性差异,P＞0.05.同时肠系膜三级动脉中膜厚度/管腔半径及中膜面积/管腔面积降低.结论用ATR12181主动免疫SHR,可降低SHR血压,降低心脏重量,逆转心脏超微结构的损害,同时降低肠系膜三级动脉中膜厚度/管腔半径及中膜面积/管腔面积降低.     

依那普利对自发性高血压大鼠心脏局部血管紧张素Ⅱ浓度和左心室结构的影响

目的观察自发性高血压大鼠(SHR)心脏局部血管紧张素Ⅱ(AngⅡ)浓度、左心室结构以及依那普利对其影响.方法12周龄SHR 40只,随机分为依那普利[30 mg/(kg·d)]治疗组(SHR-d组)和对照组(SHR组)两组,另设同周龄的正常血压大鼠20只(WKY组)作为对照,共观察12周.第1周和第12周测血压和体重,处死后分别测量左心室重量、左心室室壁厚度,放免法检定心肌AngⅡ浓度,应用病理学图像分析法对各组大鼠左室心肌细胞的长、短径、截面积和心肌胶原体积比例(CVF)、心肌血管周围胶原面积和管腔面积比例(PVCA)进行测量.结果1)SHR-d组血压、AngⅡ浓度显著低于SHR组而接近WKY组(P＜0.05).2)SHR-d组CVF(1.98%±1.57%vs 5.11%±2.25%)、PVCA(0.68%±0.19%vs 1.20%±0.19%)、LV/BW比值(3.14±0.31 vs4.09±0.21)mg/g,左心室室壁厚度(0.32±0.05 vs 0.43±0.03)cm均明显低于SHR组(P＜0.05).3)SHR-d组的心肌细胞长、短径和截面积数值均较SHR组下降,但未降到正常.结论应用依那普利能有效抑制SHR心脏局部AngⅡ生成,能部分逆转SHR的左室肥厚.     

自发性高血压大鼠肾小球胶原含量的早期变化

采用胶原特异性的苦味酸天狼猩红（PSR）染色法和计算机图像分析技术，检测不伴肾小球硬化的27周龄雄性自发性高血压大鼠（SHR）和同样年龄、性别的Wistar—Kyoto大鼠（WKY）肾小球胶原含量。结果显示：SHR肾小球胶原面积和胶原密度均显著高于WKY（SHR：1763±403um2，024±0．06；WKY：1224±443um2，0．18±0．05，P＜0．05）。提示（1）SHR肾小球胶原沉积增加，（2）累积的肾小球胶原可能是肾小球硬化的早期病变。     

复合离子盐对高血压大鼠肾脏功能及结构的影响

目的研究复合离子盐对自发性高血压大鼠(SHR)肾脏功能及结构的影响,并探讨其可能的机制.方法 38只8周龄雄性SHR随机分为4组:8%食盐摄入组(HS group);1%复合离子盐摄入组(CIS group);1%复合离子盐 2.25% L-Arg摄入组(CIS L-Arg group);1%食盐摄入组(NS group),持续干预12周.干预期间定期观察大鼠体重、血压、尿量、尿钠及尿蛋白的变化.12周末进行有创血压测量,放射免疫法检测肾皮质中Ang Ⅱ、NO含量.进行肾脏组织HE及天狼猩红染色,观察病理变化并计算胶原容积分数.结果 12周干预结束后,CIS组与CIS L-Arg组血压升高趋势明显低于NS组.CIS组与CIS L-Arg组尿蛋白在干预期间没有发生明显改变,且较NS组低.与NS组相比,CIS组与CIS L-Arg组SHR肾小球及肾小管周胶原沉积量少,肾脏损害较轻,肾皮质Ang Ⅱ含量较低,NO含量较高;HS组SHR肾小球及肾小管周则胶原沉积明显增多,肾脏损害较重,肾皮质Ang Ⅱ含量较高,而NO含量较低(all P＜0.01).结论复合离子盐与普通食盐比较,长期同等量摄入时可改变肾皮质中Ang Ⅱ、NO含量,改善肾功能,延缓SHR血压的升高,减轻肾脏功能结构损害,L-Arg的加入可能有协同复合离子盐的作用.     

复合离子盐对高血压大鼠肾脏功能及结构的影响

目的研究复合离子盐对自发性高血压大鼠(SHR)肾脏功能及结构的影响,并探讨其可能的机制。方法38只8周龄雄性SHR随机分为4组:8%食盐摄入组(HSgroup);1%复合离子盐摄入组(CISgroup);1%复合离子盐 2·25%L-Arg摄入组(CIS L-Arggroup);1%食盐摄入组(NSgroup),持续干预12周。干预期间定期观察大鼠体重、血压、尿量、尿钠及尿蛋白的变化。12周末进行有创血压测量,放射免疫法检测肾皮质中AngⅡ、NO含量。进行肾脏组织HE及天狼猩红染色,观察病理变化并计算胶原容积分数。结果12周干预结束后,CIS组与CIS L-Arg组血压升高趋势明显低于NS组。CIS组与CIS L-Arg组尿蛋白在干预期间没有发生明显改变,且较NS组低。与NS组相比,CIS组与CIS L-Arg组SHR肾小球及肾小管周胶原沉积量少,肾脏损害较轻,肾皮质AngⅡ含量较低,NO含量较高;HS组SHR肾小球及肾小管周则胶原沉积明显增多,肾脏损害较重,肾皮质AngⅡ含量较高,而NO含量较低(allP<0·01)。结论复合离子盐与普通食盐比较,长期同等量摄入时可改变肾皮质中AngⅡ、NO含量,改善肾功能,延缓SHR血压的升高,减轻肾脏功能结构损害,L-Arg的加入可能有协同复合离子盐的作用。     

AT1受体胞外肽段AT12181主动免疫自发性高血压大鼠的肾脏保护作用

目的研究血管紧张素Ⅱ的受体1型(AT1)胞外的肽段ATR12181主动免疫自发性高血压大鼠(SHR)后是否对肾脏具有保护作用.方法根据大鼠AT1受体胞外肽段氨基酸序列设计合成多肽片段ATR12181,并作为抗原,主动免疫SHR和WISTAR大鼠,SHR用药组给予氯沙坦(10mg·kg-1·d-1)灌胃治疗,对照组皮下注射弗氏不完全佐剂,实验期间动态监测血清抗体滴度,用尾套法监测收缩压,以光镜和电镜观察肾脏的病理变化,并用RT-PCR方法半定量检测肾脏组织中AT1受体、C-fos、C-jun基因表达.结果ATR12181免疫SHR后产生高滴度的抗体,在免疫后第16周抗体滴度峰值达7100±280,并在免疫四周后开始降低血压,20周末收缩压为(145.42±8.46)mmHg,显著低于SHR对照组(197.00±7.70)mmHg,P＜0.05,与氯沙坦组无差异(139.28±17.19)mmHg,P＞0.05;WISTAR大鼠免疫组产生高滴度血清抗体,血压正常(116.57±5.50)mmHg.光镜下,AT12181免疫SHR组及氯沙坦治疗组肾小球大部分为轻度至中度硬化,少数肾小球形态正常,出入球小动脉管壁轻度至中度增厚;肾小管间质炎性细胞浸润显著减少;电镜下,在ATR12181免疫SHR组及氯沙坦治疗组中,肾小球血管袢排列轻度不规则,基底膜无明显增厚,少许足细胞肿胀,无明显的肾小管变性坏死;与SHR对照组相比,病理改变显著减轻;WISTAR大鼠免疫组与正常对照组相比无改变.并且ATR12181免疫SHR组肾脏AT1R、C-fos、C-jun基因表达水平下调,与氯沙坦治疗组无明显差异(P＞0.05),与SHR对照组相比,有显著性差异(P＜0.05).WISTAR大鼠免疫组与正常对照组相比无改变.结论用AT1胞外肽段ATR12181主动免疫SHR,可产生高滴度血清抗体,降低血压,延缓SHR肾脏病变进程,保护肾功能,其机制和效应与氯沙坦作用类似.正常大鼠免疫后可产生高滴度抗体,但血压和肾脏无异常改变.     

缬沙坦治疗对自发性高血压大鼠心肌纤维化的影响

目的　观察缬沙坦对自发性高血压大鼠 (SHR)心肌纤维化的影响。方法　 3 0只 12周龄雄性SHR大鼠 ,随机分成3组 :(1)大剂量缬沙坦组 (n =10缬沙坦 3 0mg/kg·d) ;(2 )小剂量缬沙坦组 (n =10缬沙坦 10mg/kg·d) ;(3 )SHR空白对照组 (n =10 ) ;(4)同龄雄性正常血压WKY大鼠对照组 (n =10 )。给药 4周 ;天狼星红染色法使胶原特殊染色 ,计算机图象分析测量心肌切片的胶原容积分数和心肌小动脉周围胶原面积与小动脉面积比表示心肌纤维化程度。结果　与SHR组相比大小剂量缬沙坦组 ,均能有效降低SHR血压 (P <0 0 5)。并能改善SHR大鼠左心室肥厚 (P <0 0 5)并使心室内、外膜及心肌小动脉周围的胶原减少 ,其中大剂量组各指标均较SHR有显著差异 (P <0 0 5)。结论　缬沙坦对SHR大鼠的心肌纤维有显著的抑制作用     

主动免疫AT1受体对自发性高血压大鼠血管重构的影响

目的观察血管紧张素Ⅱ的Ⅰ型(AT1)受体细胞外不同肽段主动免疫自发性高血压大鼠(SHR)对血压及血管重构的影响。方法2月龄SHR在血压升高后,以合成的AT1受体细胞外的多肽片段ATR12185、ATR10014和ATR12181作为抗原,分别主动免疫,用药组给予氯沙坦(10mg/(kg·d))灌胃。同龄Wistar大鼠免疫干预组同SHR,对照组只用免疫佐剂。ELISA法检测抗体滴度,大鼠尾动脉血压计测量血压。试验结束前,测定肠系膜三级动脉中层及内径,观察肠系膜动脉病理改变,以及胸主动脉超微病理结构改变。结果SHR于2月龄血压开始升高;所有免疫组动物均产生了针对特定短肽的抗体。ATR12181免疫组SHR收缩压于免疫后1月降低,并持续至实验结束[(145.4±8.5)mmHg,n=7],与SHR对照组[(197.0±7.7)mmHg,n=7,P<0.05]相比,收缩压降低,与氯沙坦治疗组[(139.3±17.2)mmHg,n=7,P>0.05]相比,无显著性差异,而ATR12185和ATR10014免疫组SHR收缩压无明显变化。Wistar大鼠免疫前后收缩压无明显变化。ATR12181免疫组SHR肠系膜三级动脉中膜厚度/管腔半径(1.10±0.18)及中膜面积/管腔面积(3.21±0.98),分别较SHR对照组(1.39±0.21)及(4.62±1.21)降低。结论用ATR12181主动免疫SHR,可降低SHR血压,降低肠系膜三级动脉中膜厚度/管腔半径及中膜面积/管腔面积,逆转主动脉超微结构的损害。     

心脏肥大细胞在自发性高血压大鼠心肌重构中的作用

目的探讨心脏肥大细胞在自发性高血压大鼠(SHR)心肌重构中的作用.方法应用病理检查、计算机分析结合逆转录-聚合酶链式反应等方法,观察SHR及Wistar-Kyoto大鼠(WKY)收缩压、左室重量指数、心肌细胞直径、肥大细胞密度、心肌胶原容积分数(CVF)、心肌血管周围胶原面积比(PV-CA)和心肌Ⅰ、Ⅲ型胶原mRNA表达水平的变化.肥大细胞密度与左室重量指数、CVF及PVCA之间的关系采用相关分析.结果与WKY比较,SHR收缩压为(206±18)比(108±10)mm Hg(P<0.01);SHR组左室重量指数为(4.6±0.4)比(3.3±0.3)mg/g,(P<0.01);SHR组心肌细胞长径为(17.4±1.9)比(10.0±2.2)μm(P<0.01);SHR组心肌细胞短径为(9.0±2.0)比(5.8±1.7)μm(P<0.01);SHR组肥大细胞密度为(7.4±3.2)比(1.9±1.2)个/mm2(P<0.01),SHR组肥大细胞密度为WKY组的3.9倍.SHR组CVF、PVCA分别为46.4%±7.8%和1.9±0.9,WKY组分别为24.4%±10.7%和0.4±0.1,SHR组明显升高(P<0.01).SHR组心肌Ⅰ、Ⅲ型胶原mRNA表达相对含量也均明显高于WKY(P<0.01),心脏肥大细胞密度与左室重量指数、CVF及PVCA存在明显的正相关(相关系数分别为0.67、0.87和0.95,P<0.01).结论心脏肥大细胞密度增加可能是促进SHR心肌重构的重要原因.     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.