Identification and partial characterisation of hyaluronidases in Lonomia obliqua venom.

By studying Lonomia obliqua (caterpillar) venom we were able to detect a lytic activity on purified hyaluronic acid. The venom hydrolyses purified chondroitin sulphate, but was unable to degrade either heparan sulphate or dermatan sulphate. Moreover, through purified hyaluronic acid-degrading kinetic assays, we observed that this lytic activity was caused by a hydrolase rather than lyase enzyme. In addition, by using the Reissig colorimetric reaction, we detected this hyaluronic acid hydrolase action as a beta-endohexosaminidase enzyme originating terminal N-acetylglucosamine residues rather than beta-endoglucuronidase, which may originate glucuronic acid residues. Zymogram analysis of the venom detected 49 and 53 kDa molecules with hyaluronic acid lytic activity. An examination of these hyaluronic acid degrading activities as a function of pH showed that these hydrolases had no apparent activities at a pH below 5.0 and higher than 8.0 and displayed their optimal activities at pH ranging from 6.0 to 7.0. Finally, through a fluorescence reaction to hyaluronic acid and confocal microscopy, we confirmed this cleaving action upon hyaluronic acid organised on the extracellular matrix of the dermis of rabbit. The data provide experimental evidence of the presence of hyaluronidases in the L. obliqua venom, probably involved in the harmful effects of the venom.     

Proteases from Lonomia obliqua venomous secretions: comparison of procoagulant, fibrin(ogen)olytic and amidolytic activities.

The hemorrhagic syndrome caused by Lonomia obliqua caterpillars is an increasing problem in Southern Brazil. The clinical profile is characterized by both hemorrhagic and pro-coagulant symptoms, constituting a paradoxical action of the venom. The effects upon blood coagulation and fibrin(ogen)olysis have been shown to result from the combined action of several active principles found mostly in the bristle extract. The present study reports quali-quantitative differences among L. obliqua secretions: Cryosecretion, hemolymph, bristle extract and tegument extract. Cryosecretion and hemolymph displayed strong amidolytic activity upon several substrates, presented moderated procoagulant activity and high fibrinogen degrading ability. Bristle and tegument extracts presented low amidolytic activity, but bristle extract showed the most potent procoagulant activity and both extracts presented low fibrinogen degrading ability. The differential involvement of these secretions during the accidents with L. obliqua can elucidate the different symptoms presented after envenomation.     

Efficacy of two different doses of antilonomic serum in the resolution of hemorrhagic syndrome resulting from envenoming by Lonomia obliqua caterpillars: a randomized controlled trial.

The hemorrhagic syndrome caused by contact with Lonomia obliqua caterpillars has reached epidemic proportions in southern Brazil. This study aimed at assessing the efficacy of two different dosages of an antilonomic serum (SALon) in the treatment of patients who had accidental contact with L. obliqua caterpillars. A randomized, prospective controlled trial was conducted at S?o Vicente de Paulo Hospital, a tertiary hospital in Passo Fundo, Brazil. From January 2000 to April 2002, 44 patients with grade I or II hemorrhagic syndrome were randomly assigned to either group A: 22 patients treated with 10.5 mg total dose (three vials of SALon) or group B: 22 patients treated with 17.5 mg total dose (five vials of SALon). Treatment efficacy was assessed according to time necessary for blood coagulation to go back to normal, incidence of adverse reactions, and hospitalization time. There were no differences in clinical picture and laboratory findings between the two groups. Mean time for coagulation to go back to normal was 15.3+/-6.6 and 19.1+/-8 h in groups A and B, respectively. Adverse reactions and hospitalization time were similar in both groups. SALon was effective and well tolerated. This study demonstrated that three vials are as effective as five vials of SALon for the treatment of envenoming by Lonomia caterpillars.     

Nociceptive and edematogenic responses elicited by a crude bristle extract of Lonomia obliqua caterpillars.

Lyophilized Lonomia obliqua crude bristle extract (LOCBE) diluted in physiological saline (15, 35 and 50 microg of protein/paw) was injected in the plantar surface of the hind paw of the rat, causing a nociceptive response which lasted from 30 to a maximum of 50 min, peaking in the first 5 min. The animals also presented hematuria and nasal bleeding. Nociception was inhibited by indomethacin pretreatment (2.5 mg/kg, i.p., 60 min before), but not by guanethidine (30 mg/kg/day, s.c., for 3 days) or loratadine (5 mg/kg, p.o., 60 min before). LOCBE injection also produced paw edema peaking 1 h after injection and lasting for 6 h. Loratadine pretreatment, but neither guanethidine nor indomethacin, reduced edema. After the period of overt nociception, a nociceptive aftersensation response could be evoked up to 6 h after by immersing the paw into cold water (15 degrees C) for 10 s. Capsaicin (1.6 microg), formalin (0.5%) or prostaglandin E(2) (500 ng) did not produce the same aftersensation phenomenon. These results suggest that LOCBE-induced nociception is largely facilitated by prostaglandin production, and edematogenic response seems to be facilitated by prostanoids and histamine. Finally, LOCBE induced a state of sensitization to cold, which seemed to be specific as it was not caused by other noxious chemicals.     

双氯芬酸钾致急性肾衰竭

1名52岁女性类风湿性关节炎患者,给予口服双氯芬酸钾50mg,3次/d,枸橼酸铋钾110mg,3次/d,静脉滴注锝[99mTc]-亚甲基二膦酸盐{[99mTc]-MDP}注射液200mg,1次/d。1周后,患者出现恶心、呕吐、咳嗽、咽痛等症状,Cr由95μmol/L升至542μmol/L,BUN由6.76mmol/L升至18.9mmol/L。遂停药,给予泼尼松龙、左氧氟沙星、血浆置换等治疗,症状无明显缓解,Cr为638μmol/L,BUN为25.8mmol/L。肾穿刺活检结果显示肾淀粉样变性、急性肾小管坏死。经血液透析及对症治疗后,患者病情稳定,Cr降至379～442μmol/L,BUN降至12.3～20.76mmol/L。随访1年,患者肾功能仍未恢复正常。     

Lonomia obliqua caterpillar venom increases permeability of the blood-brain barrier in rats.

Human envenoming by caterpillars of the saturniid moth Lonomia obliqua in southern Brazil produces a mild local response (erythema, some edema, and pain) and systemic effects that include incoagulable blood, renal failure and in severe accidents intracerebral hemorrhage. In this work, we used light and electron microscopy to investigate the morphological alterations in the brain and blood-brain barrier of rats injected intravenously with venom from L. obliqua spicules (200 microg/kg). Five semi-purified fractions of venom (200 microg/kg each) were also assayed. Quantitative morphological and ultrastructural analyses were done 6, 18, 24 and 72 h after the i.v. injection of venom and its fractions. Light microscopy showed that 6h after envenoming there was cerebellar edema, which decreased by 72 h. Intracerebral hemorrhage occurred in only one rat 24h after the injection of venom. Blood-brain barrier (BBB) breakdown, assessed by transmission electron microscopy based on the passage of an extracellular tracer (lanthanum nitrate) between brain capillary endothelial cells, was observed in the cerebellum and hippocampus 18 h after venom injection. At this time, the cerebellum was more sensitive to the venom than the hippocampus, as shown by the greater number of leaky vessels. The number of capillaries showing breakdown was lower after 72 h than after 18 h. None of the semi-purified fractions significantly increased the number of leaky vessels. These results indicate that L. obliqua caterpillar venom has a deleterious action on the rat BBB. The lack of effect of the venom fractions when administered alone suggested that a synergistic action of venom components may be responsible for the damage seen in the central nervous system, but this was not confirmed when three combinations of the fractions were tested.     

Kallikrein-kinin system activation by Lonomia obliqua caterpillar bristles: involvement in edema and hypotension responses to envenomation.

Lonomia obliqua envenomation induces an intense burning sensation at the site of contact and severe hemorrhage followed by edema and hypotension, and after few days death can occur usually due to acute renal failure. In order to understand more about the envenomation syndrome, the present study investigates the role played by kallikrein-kinin system (KKS) in edematogenic and hypotensive responses to the envenomation by L. obliqua. The incubation of L. obliqua caterpillar bristles extract (LOCBE) with plasma results in kallikrein activation, measured by cromogenic assay using the kallikrein synthetic substrate S-2302 (H-D-Pro-Phe-Arg-pNA). It was also showed that LOCBE was able to release kinins from low-molecular weight kininogen (LMWK). Moreover, it was demonstrated that previous administration of a kallikrein inhibitor (aprotinin) or bradykinin B2 receptor antagonist (HOE-140) significantly reduces the edema and hypotension in response to LOCBE, using mouse paw edema bioassay and mean arterial blood pressure analysis, respectively. The results demonstrate a direct involvement of the KKS in the edema formation and in the fall of arterial pressure that occur in the L. obliqua envenomation syndrome.     

Acute renal failure alters the kinetics of pralidoxime in rats

There is a trend towards increasing doses of pralidoxime to treat human organophosphate poisonings that may have relevance in subpopulations. Indeed, pralidoxime is eliminated unchanged by the renal route. This study assesses the effect of renal failure on the kinetics of pralidoxime in a rat model of acute renal failure induced by potassium dichromate administration. On the first day, Sprague–Dawley rats received subcutaneously potassium dichromate (study) or saline (control). Forty-eight hours post-injection, animals received pralidoxime methylsulfate (50 mg/kg of pralidoxime base) intramuscularly. Blood specimens were sampled during 180 min after the injection. Urine was collected daily during the 3 days of the study. Plasma pralidoxime concentrations were measured by liquid chromatography with electrochemical detection. There was a 2-fold increase in mean elimination half-life and a 2.5-fold increase in mean area under the curve in the study compared to the control group. The mean total body clearance was halved in the study compared to the control group. Our study showed acute renal failure does not modify the distribution of pralidoxime but significantly alters its elimination from plasma. These results suggest that dosages of pralidoxime should be adjusted in organophosphate-poisoned humans with renal failure when using high dosage regimen of pralidoxime.     

Intravascular hemolysis induced by Lonomia obliqua caterpillar bristle extract: an experimental model of envenomation in rats.

Hemostatic disturbances are frequent findings in human accidents caused by Lonomia obliqua caterpillars in the southern region of Brazil. In severe envenomation, patients may present life-threatening bleedings. Such disturbances may be mimicked in rats, which also develop intravascular hemolysis. The scope of this study was to investigate the time-course and intensity of intravascular hemolysis induced by i.d. injection of 750 microg/kg crude L. obliqua bristle extract in rats. Total blood cell count, reticulocyte count, plasma hemoglobin and haptoglobin assays were performed in control and envenomed rats at 1, 6, 24 and 48 h after envenomation. Rats presented a drastic drop of haptoglobin levels at 1 and 6h with increased plasma hemoglobin levels, a decrease in packed cell volume values at 6, 24 and 48 h, and increased reticulocyte counts throughout after envenomation. Such observations indicated that intravascular hemolysis occurred as early as 1h following envenomation, and lasted for more than 6h. Intravascular hemolysis is probably induced by phospholipase A(2) and other proteins with direct hemolytic activity present in crude caterpillar bristle extract.     

卡培他滨引起急性肾衰竭

1名94岁男性直肠腺癌患者接受卡培他滨化疗。在第3周期给予卡培他滨1500mg,2次/d,用药12d后,患者出现嗜睡、心慌、发热,血Cr由145μmol/L上升到173μmol/L,BUN由10.9mmol/L上升到27.4mmol/L,立刻停药。6d后,SCr310μmol/L,BUN62.5mmol/L。给予复方α-酮酸、利尿剂、低蛋白饮食等治疗,SCr及BUN逐渐下降。4个月后复查,SCr及BUN恢复正常。     

Effects of Lonomia obliqua (lepidoptera, saturniidae) toxin on clotting, inflammatory and antibody responsiveness in genetically selected lines of mice.

Lines of mice genetically selected for high (H) or low (L) antibody response and for maximal (AIRMAX) or minimal (AIRMIN) acute inflammatory reaction, in which the opposite extreme potentialities have been clearly defined, offer an appropriate model for investigating the environmental and genetic factors acting on innate and adaptative immunobiological functions. This model has been successfully employed to study the resistance or susceptibility against pathogens and/or toxins. It had been demonstrated that the skin contact with Lonomia obliqua caterpillar bristles induces local inflammation and may elicit severe hemorrhagic disorders. In the present study, blood coagulation time, and the acute inflammatory reaction were scored 24 h after injection of the Lonomia bristles crude extract in a subcutaneous dorsal air pouch. The acute inflammation was determined by the leukocyte concentration in the local exudates. The highest interline differences were observed between the AIRMAX (10(6) cells/ml) and AIRMIN (2 x 10(5) cells/ml) and this distinct expression involves the number of monocytes, eosinophils and mainly neutrophils. Regarding coagulation, the highest interline difference was observed between the HIII and LIII mice, and the F1)[LIII x HIII] hybrids showed the overdominance of the fast clotting character. The adaptative immune response was evaluated by comparing the anti-Lonomia bristle extract IgG titer among the lines: the antibody titers were higher in the H lines than in the L ones and equivalent in the AIRMAX and AIRMIN mice, in accordance to the phenotype profiles generated by the distinct selective processes. The genetically selected mice lines-AIRMAX, AIRMIN, HI, HIII, HG, LIII and LG-showed an almost continuous distributions for inflammation, coagulation time and IgG antibody titers, being the interline variances always higher than the intraline ones for the individually measured phenotypes. Altogether, these results suggest the independent polygenic regulation of these traits, being indicative of the genetic control to Lonomia toxin innate and adaptative sensitivity in humans.     

腹膜透析对横纹肌溶解综合征致急性肾衰治疗的优势

目的 讨论腹膜透析对横纹肌溶解综合征 ( ) RM 致急性肾衰治疗的效果和优势。方法 对 6例横纹肌溶解综合征致急性肾衰的病人施行腹膜透析 ( CAPD 治疗,观察治疗效果及治疗前后白介素 1β( -1β) ) IL、肿瘤坏死因子α( TNF -α)CPK LDH 、G OT 的变化。结果 6例病人于治疗 2～5周后肾功能全部恢复正常,上述观察指标在治疗 3周 、 、后显著下降。结论 腹膜透析可以清除 IL -1βTNF -α等炎症介质,对横纹肌溶解综合征致急性肾衰治疗效果满意,且 、有更为突出的优势。     

复方利福平胶囊致急性肾衰竭

1例29岁男性患者,因肺结核服用复方利福平胶囊（每粒含利福平600mg,异烟肼600mg）,1粒/次,隔日1次。治疗3个月后症状好转自行停药。1年后患者再次出现咳嗽、盗汗等症状,遂按原治疗剂量自行服用复方利福平胶囊。服药8d后,出现恶心、呕吐,腰痛,颜面及双下肢水肿,少尿、茶色尿。实验室检查：BUN21.2mmol/L,SCr877.8μmol/L,尿蛋白（＋）,尿潜血（＋＋＋）;ECT检查示左肾和右肾GFR值分别为21.11ml/min和20.98ml/min。停用复方利福平胶囊,行血液透析,同时给予对症、支持治疗,25d后症状好转。     

In vitro hemolytic activity of Lonomia obliqua caterpillar bristle extract on human and Wistar rat erythrocytes.

Human accidental envenomation caused by skin contact with the bristles of Lonomia obliqua caterpillar causes coagulation and fibrinolysis disorders. Alterations of hematologic parameters are observed only in severe cases of envenomation, but with no clinical evidence of intravascular hemolysis. However, since we have observed intravascular hemolysis in preliminary studies using Wistar rats as an experimental model for investigating L. obliqua envenomation, the objective of the present study was to investigate the in vitro hemolytic activity of the bristle extract of L. obliqua caterpillars on human and rat erythrocytes.Our results showed that the bristle extract has indirect and direct hemolytic activity on human and rat erythrocytes, although direct hemolytic activity was only observed at higher bristle extract concentrations. We also observed that the bristle extract has a proteolytic activity on band 3 of human and rat erythrocyte membranes. Thus, crude L. obliqua bristle extract was found to contain at least two components with hemolytic activity on erythrocytes, a phospholipase enzyme and another protein with a direct activity on the erythrocyte membrane.     

阿昔洛韦过量致急性肾衰竭

例19岁男性传染性单核细胞增多症患者拟给予阿昔洛韦600 mg加入10%葡萄糖注射液250 ml中静脉滴注.但因用药错误将阿昔洛韦3.0 g加入10%葡萄糖注射液250 ml中静脉滴注.输注完毕后9 h,患者出现明显腰痛,尿量减少,尿蛋白(+ +),尿潜血(+ +).随后出现24 h无尿.第3天肾功能检查:血清肌酐557 μmol/L,尿素9.2 mmol/L.肾脏超声示双肾弥漫性病变.行血液净化,并给予甲泼尼龙40 mg/d静脉滴注,治疗2 d.肾功能逐渐好转,尿量增多.第15天患者肾功能及尿常规检查均无异常,痊愈出院.     

大面积脑梗死并急性肾功能衰竭对预后的影响

目的：探讨大面积脑梗死高死亡率原因及防治对策。方法：对７２例大面积脑梗死临床资料进行分析。结果：梗死灶越大病死率越高，急性肾功能衰竭（ＡＲＦ）发生率也越高；有并发症者病死率显著高于无并发症者（Ｐ〈０．０１）。ＡＲＦ居并发症首位，有４４．４％患者并发ＡＲＦ，死亡病例中有６０．５％并发ＡＲＦ；并发ＡＲＦ者病死率（７５％）明显高于无ＡＲＦ者（３５％）（Ｐ〈０．０１）。结论：大面积脑梗死死亡原因主要为脑疝     

Efficacy of serum therapy on the treatment of rats experimentally envenomed by bristle extract of the caterpillar Lonomia obliqua: comparison with epsilon-aminocaproic acid therapy.

Large number of accidents caused by contact with Lonomia obliqua caterpillars, with hemorrhagic complications, have occurred in southern Brazil. Based on Venezuelan expertise to treat Lonomia achelous envenomation, the use of the antifibrinolytic drug epsilon-aminocaproic acid (EACA) has been indicated to treat L. obliqua envenomation, although no evidence has been presented to justify its use. Specific antivenom (antilonomic serum (ALS)) that neutralizes toxins that cause envenomation was developed. To compare the effectiveness of such treatments, rats were injected i.d. with the bristle extract of L. obliqua caterpillars and treated 15 min, 1 and 6 h after with saline, ALS, EACA, or with both ALS and EACA. ALS elicited fibrinogen recovery and normalization of thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT), independent of when it was administered; however, hematocrit was decreased in the group treated later. Saline or EACA-treated groups presented neither fibrinogen recovery nor normalization of hemostatic parameters. A high death rate was observed in the group treated with EACA 15 min after the envenomation. Prolongation of TT and APTT observed in the group treated with EACA and ALS indicated that this association gave no benefit in relation to the group treated solely with ALS. The results presented herein suggest that ALS is the only effective treatment for envenomation caused by contact with Lonomia obliqua caterpillars and indicate that EACA should not be administered in the initial phase of envenomation.     

Acute renal failure enhances the antidotal activity of pralidoxime towards paraoxon-induced respiratory toxicity

We recently showed in a rat model of dichromate-induced acute renal failure (ARF) that the elimination but not the distribution of pralidoxime was altered resulting in sustained plasma pralidoxime concentrations. The aim of this study was to compare the efficiency of pralidoxime in normal and acute renal failure rats against paraoxon-induced respiratory toxicity.     

万古霉素致肾衰竭死亡2例

2例精神病患者因肺部感染静脉滴汪万古霉素致肾衰竭死亡。 例1为84岁男性精神病患者，因肺部感染和呼吸衰竭行气管切开。给予患者糖皮质激素、头孢他啶及依替米星治疗，后改为万古霉素1g，2次／d，静脉滴注。患者的Cr和BUN水平用药前为分别为71μmol／L和7．3mmol／L；治疗第9天分别升至223μmol／L和27.3mmol／L。患者尿量由1930ml（治疗第6天）减少至650ml（治疗第8天）。患者出现肺栓塞、急性肾衰竭、代谢性酸中毒、心律失常及浅昏迷，最后经抢救无效死亡。例2为58岁女性精神病患者，因肺部感染给予头孢吡肟、亚胺培南-西司他丁及低分子肝素钙，后改为万古霉素1g，2次／d，静脉滴注。患者的Cr和BUN水平用药前分别为87μmol／L和7．5mmol／L，用药第3天分别升至124μmol／L和14．7mmol／L，用药第6天分别升至436μmol／L和30．1mmol／L。患者尿量自用药第3天下降至900ml，用药第6天下降至200ml，并出现腹泻。诊断为急性肾衰竭，停用万古霉素，经抢救无效，于停用万古霉素第3天死亡。     

不同血液净化方法治疗重症急性肾衰的临床疗效观察

目的观察床边急诊插管腹膜透析（PD）和连续性肾脏替代治疗（CRRT）对于重症急性肾功能衰竭（ARF）患者的疗效。方法分析33例重症急性肾衰竭患者采用PD和CRRT治疗的疗效，其中行床边急诊插管行PD者17例，CRRT者16例。观察患者血液净化前后连续5d血肌酐（Scr）、尿素氮（BUN）、钾（K^＋）、二氧化碳结合力（CO2CP）水平的变化，预后，平均每日透析费用。结果血液净化前后连续5d血BUN、Scr、K^＋、CO2CP的变化并无差异（P〉0．05）。2组患者中肾功能恢复及存活者所占比例无显著性差异（P〉0．05）。但CRRT组患者平均每日透析费用为（3220．94±95．32）元，明显高于PD组的（619．27±108．90）元（P〈0．01）。结论床边急诊插管PD与CRRT均是治疗重症急性肾衰竭的有效方法，但床边急诊插管PD是一种安全、简单、经济的方法。