Benzo(a)pyrene-induced anemia and splenomegaly in NZB/WF1 mice.

Benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon, is a known immunomodulator. At high doses, BaP is immunosuppressive but at low doses it can enhance the immune response. Studies were conducted to determine if BaP would exacerbate the development of autoimmune disease in genetically prone NZB/WF1 mice. Five week old female NZBW/F1 mice were exposed dermally to 5, 20 and 40 mg/kg BaP for 30 days. Vehicle mice were exposed to an acetone:olive oil mixture for 30 days. BaP did not increase total IgG, anti-DNP-HSA or anti-dsDNA antibody levels. However, hematological evaluation revealed a decrease in erythrocyte number, hemoglobin and hematocrit and an increase in mean corpuscular volume and red cell distribution width in the 20 and 40 mg/kg dose groups. Liver and spleen weights were increased in the high dose groups; however, an increase in spleen cell number was not observed. Histopathological evaluation revealed splenic red pulp expansion in a mouse treated with 40 mg/kg BaP. An increase in splenic CFU-e production was observed in mice treated with 20 and 40 mg/kg BaP. A decrease in splenic total B cells, total T cells, CD4(+) and CD8(+) T cells was observed in mice treated with 20 and 40 mg/kg BaP. An increase in splenic null cells (non-T, non-B cells) was also observed in the high dose groups, consistent with extramedullary hematopoiesis. Coombs' tests, flow cytometry and an immune-mediated hemolysis assay indicated that the anemia was not autoimmune-mediated. Although no change was observed in the percentage of reticulocytes in these animals, further bone marrow analysis is needed to determine if the anemia is due to bone marrow suppression, possibly caused by BaP exposure, or chemical-induced hemolysis, perhaps contributed to by erythrocyte fragility inherited from a parent strain, NZB, which spontaneously develops autoimmune hemolytic anemia and subsequent splenomegaly.     

Combined repeated dose and reproductive/developmental toxicity screening test of 4-methoxy-2-nitroaniline in rats

4-Methoxy-2-nitroaniline (4M2NA) is widely used as an intermediate for the synthesis of dyes, pigments and other chemical compounds. Since 4M2NA has amino-group and nitro-group on the benzene ring, it was expected that it induced obvious hemolytic anemia. We conducted a combined repeated dose and reproductive/developmental toxicity screening test according to Organisation for Economic Co-operation and Development (OECD) Test Guideline No. 422 (OECD TG 422) to enrich the toxic information and ensure the safety of 4M2NA. 4M2NA was administered to Crl:CD(SD) male and female rats by gavage at 0, 12.5, 75 or 450?mg/kg/day for 42 to maximum of 54 days through pre-mating, mating, pregnancy and lactation periods. An extramedullary hematopoiesis and congestion in spleen, and higher reticulocyte ratio were noted in only females at 450?mg/kg/day without decreased anemic parameters in the hematological examination. Hypertrophy of centrilobular hepatocytes in both sexes was observed with increased relative liver weight at 450?mg/kg/day. Furthermore, the diffuse follicular cell hypertrophy of the thyroid was observed in females at 450?mg/kg/day. No abnormalities were detected in the reproductive indices of copulation, delivery or fetal viability. We concluded the no-observed-adverse-effect level (NOAEL) for repeated-dose toxicity was 75?mg/kg/day based on the trace evidences of hemolytic anemia, and the NOAEL for reproductive/developmental toxicity as 450?mg/kg/day based on no toxicological concerns for reproductive endpoints. The hemolytic anemia was much milder than expected. Thus, we discussed the reason of this much less hemolytic effect from the point of view of the structural characteristics of 4M2NA.     

重复给予恒河猴rhKGF后其免疫原性及抗体中和性质的检测

目的测定恒河猴经静脉重复给予重组人角质细胞生长因子(rhKGF)后血清中特异性抗体的水平及抗体中和性质。方法采用间接ELISA法检测猴血清中的抗体水平,同时采用32D-KGFR细胞检测抗体的中和活性。结果确定了抗原包被浓度为10μg·mL-1,抗体从1.6×103开始稀释,二抗稀释倍数为1∶5×103的间接ELISA法实验体系;14～56 d均可检测到抗体的存在;生物学活性测定的结果显示其为非中和性抗体。结论重复给予恒河猴rhKGF的毒性试验中,部分猴的血清中检测到抗体,但不是中和抗体。     

Role of metabolites in propanil-induced hemolytic anemia

Hemolytic anemia and methemoglobinemia induced by exposure to certain arylamines, such as aniline and dapsone, are known to be mediated by their N-hydroxylamine metabolites. The arylamide propanil (3,4-dichloropropionanilide), a herbicide used extensively in rice fields, is also thought to induce methemoglobinemia through the action of metabolites. However, the hemolytic potential of this compound has not previously been reported. The present studies were undertaken to determine the hemolytic potential of propanil, and, if positive, the role of metabolites in this hemotoxicity. The survival of previously administered 51Cr-labeled erythrocytes in rats was reduced in a dose-dependent manner by ip administration of both propanil and its deacylated metabolite, 3,4-dichloroaniline (ED50 for both ca. 1.8 mmol/kg). When labeled erythrocytes were exposed in vitro to propanil or 3,4-dichloroaniline and then readministered to rats, no decrease in erythrocyte survival was observed, which indicated that these compounds were not direct-acting hemolytic agents. In contrast, erythrocyte survival was markedly reduced by ip administration or in vitro exposure to N-hydroxy-3,4-dichloroaniline. In addition, N-hydroxy-3,4-dichloroaniline was detected in the blood of propanil-treated rats in amounts sufficient to account for the hemolytic activity of the parent compound. These data indicate that N-hydroxy-3,4-dichloroaniline mediates propanil-induced hemolytic anemia, and that occupational exposure to propanil may result in an increased risk of hemolytic episodes.     

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.     

Immunological properties of S-1090, cefmatilen hydrochloride hydrate

S-1090, a cefmatilen hydrochloride hydrate, is being developed as a cephalosporin antibiotic for oral use. Immunogenicity, hypersensitivity-eliciting antigenicity and immunological cross-reactivity with other antibiotics were evaluated by active systemic anaphylaxis (ASA) test, passive cutaneous anaphylaxis (PCA) test and enzyme-linked immunosorbent assay (ELISA) using guinea pigs and mice/rats. In addition, in vitro direct Coombs' test was also performed to examine the possibility of hemolytic anemia in clinical use. Immunogenicity of S-1090 was not observed in guinea pigs after repeated immunization with S-1090 by ASA or PCA tests. Even in ELISA, only weak antibody production against S-1090 was found in some guinea pigs from the intraperitoneal groups showing the antibody titers only 10(1) to 10(2). When the sera collected from C3H/He mice and C57BL/6J mice immunized with S-1090 were tested for immunogenicity, rat PCA was elicited in a C3H/He mouse serum by S-1090 and antibodies against S-1090 were detected in a C57BL/6J mouse serum by ELISA. When adjuvant was used in mice and guinea pigs, the production of antibody against S-1090 was less frequent in comparison with other antibiotics such as cefmetazole (CMZ) and cefotiam (CTM). When hypersensitivity-eliciting antigenicity of S-1090 was examined using S-1090 as an eliciting antigen in ASA and PCA tests, positive ASA and PCA were observed in guinea pigs and positive PCA in a C3H/He mouse. Hypersensitivity-eliciting antigenicity was also observed in other reference antibiotics, i.e. cephalothin (CET), CMZ and CTM. Immunological cross-reactivity among S-1090, penicillin G (PCG), CET, CMZ and CTM was tested by ASA and PCA tests. S-1090 was found to immunologically cross-react only with CET in guinea pigs. In the present study, immunological cross-reactivities were also noted between PCG and CET, PCG and CMZ, PCG and CTM, and between CET and CMZ. In in vitro direct Coombs' test using human red blood cells, S-1090.Na, PCG and CET gave positive reactions at the final concentrations of 40 mg/mL, 20 to 40 mg/mL and 2.5 to 10 mg/mL, respectively.     

Lipids versus proteins as major targets of pro-oxidant, direct-acting hemolytic agents.

Lipid peroxidation and the accompanying translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the lipid bilayer have recently been identified as key components of a signaling pathway for phagocytosis of apoptotic cells by macrophages. Drug-induced hemolytic anemia has long been known to be caused by an accelerated uptake of damaged (but intact) erythrocytes by macrophages in the spleen, and this process has been associated with enhanced formation of reactive oxygen species (ROS). However, the role of lipid peroxidation in hemolytic injury has remained unclear, and the effect of hemolytic agents on the distribution of PS in the erythrocyte membrane is unknown. The present studies were undertaken to determine whether lipid peroxidation and PS translocation could be detected in rat and human erythrocytes by three types of direct-acting hemolytic agents--dapsone hydroxylamine, divicine hydroquinone, and phenylhydrazine. 2',7'-Dichlorodihydrofluorescein diacetate was employed as a probe for intracellular ROS formation; lipid peroxidation was assessed by GC/MS analysis of F2-isoprostanes; and PS externalization was measured by annexin V labeling and the prothrombinase assay. The data confirmed that all three hemolytic agents generate ROS within erythrocytes under hemolytic conditions; however, no evidence for lipid peroxidation or PS translocation was detected. Instead, ROS production by these hemolytic agents was associated with extensive binding of oxidized and denatured hemoglobin to the membrane cytoskeleton. The data suggest that the transmembrane signal for macrophage recognition of hemolytic injury may be derived from oxidative alterations to erythrocyte proteins rather than to membrane lipids.     

红花注射液溶血与凝聚试验的探讨

目的建立红花注射液溶血与凝聚试验的检查法。方法采用《中国药典））2010年版溶血与凝聚检查法和分光光度法进行试验。试验组分别用0．1～1．1mL各制备3个平行样。阴性对照组用0．9％氯化钠溶液、阳性对照用蒸馏水。每个试验管内加2％的兔红细胞混悬液。结果在两种方法检测后,红花注射液不对兔红细胞产生溶血与凝聚现象。结论红花注射液可按《中国药典》规定的方法进行溶血与凝聚试验。     

微柱凝胶直接抗人球蛋白试验对自身免疫性溶血性贫血诊断的应用

目的 分析微柱凝胶直接抗人球蛋白试验对自身免疫性溶血性贫血诊断的效果,以便为临床提供参考.方法 选择208例交叉配血次侧阳性的标本为观察组,采用微柱凝胶直接抗人球蛋白试验;将受检红细胞分成洗涤组和非洗涤组,检测红细胞洗涤对微柱凝胶直接抗人球蛋白试验的影响;同时对此检测方法阳性结果的186例红细胞进行IgG和C3d分型.对照组(208名)采用试管法直接抗人球蛋白试验.结果 观察组与对照组阳性率比较差异有统计学意义[ 89.4％( 186/208)与53.8％ (112/208),P＜0.05].微柱凝胶直接抗人球蛋白试验生理盐水洗涤和非洗涤组受检红细胞的208份血液样本中同时检出阳性反应红细胞186份.观察组检测阳性的红细胞IgG、C3d、IgG+ C3d分型分别为46.8％ (87/186)、3.2％(6/186)、50.0％(93/186).结论 微柱凝胶直接抗人球蛋白试验对自身免疫性溶血性贫血诊断具有较高的临床应用价值.     

A toxicogenomic approach revealed hepatic gene expression changes mechanistically linked to drug-induced hemolytic anemia.

A variety of pharmaceutical compounds causes hemolytic anemia as a significant adverse effect and this toxicity restricts the clinical utility of these drugs. In this study, we applied microarray technology to investigate hepatic gene expression changes associated with drug-induced hemolytic anemia and to identify potential biomarker genes for this hematotoxicity. We treated female Sprague-Dawley rats with two hemolytic anemia-inducing compounds: phenylhydrazine and phenacetin. Hepatic gene expression profiles were obtained using a whole-genome oligonucleotide microarray with pooled RNA samples from individual rats within each dose group and analyzed in comparison with hepatic histopathology, hematology, and blood chemistry data. We identified a small subset of genes that were commonly deregulated in all the severe hemolytic conditions, some of which were considered to be involved in hepatic events characteristic of hemolytic anemia, such as hemoglobin biosynthesis, heme metabolism, and phagocytosis. Among them, we selected six upregulated genes as putative biomarkers, and their expression changes from microarray measurements were confirmed by quantitative real-time PCR using RNAs from individual animals. They were Alas2, beta-glo, Eraf, Hmox1, Lgals3, and Rhced. Expression patterns of all these genes showed high negative and positive correlation against erythrocyte counts and total bilirubin levels in circulation, respectively, suggesting that these genes may be the potential biomarkers for hemolytic anemia. These findings indicate that drug-induced hemolytic anemia may be detected based on hepatic changes in the expression of a subset of genes that are mechanistically linked to the hematotoxicity.     

A cost-effective particle agglutination assay to detect viral antibodies in dried blood spots--a simple solution to HIV and HCV screening

OBJECTIVES: To conduct a serological survey of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in Gabon and Ga-Rankuwa, South Africa. A secondary objective was to test a novel, simple, inexpensive agglutination assay for anti-HIV IgG and anti-HCV IgG from blood samples stored as spots dried onto filter paper. DESIGN: Blood from heel pricks was dried onto filter paper and stored. Blood was eluted from the spots and serum antibody was then assayed using a modified agglutination assay--blood was added to gelatin agglutination beads that had been sensitised with viral antigen. A positive result showed as an agglutination pattern while a negative result appeared as a tight bead. SUBJECT: This was a hospital-based study involving 271 neonates at Ga-Rankuwa Hospital, South Africa, and 856 patients ranging in age from three months to over 50 years who attended clinics in Gabon. RESULTS: Seroprevalence to HIV was determined in Ga-Rankuwa to be just under 14% (13.8%). Antibodies to HCV were not detected. In Gabon, the prevalence to HIV was just under 1% (0.82%) with a relatively high incidence of HCV, nearing 4% (3.79%). CONCLUSION: The sensitivity of the agglutination assay compared favourably to enzyme immune assay (EIA) with respect to sensitivity, simplicity and cost. This assay may be useful in sero-epidemilogical assays in developing countries.     

以自身免疫性溶血性贫血为首发表现的小儿再生障碍性贫血1例并文献复习

目的 提高对以自身免疫性溶血性贫血(Autoimmune hemolytic anemia, AIHA) 为首发症状的儿童再生障碍性贫血(aplastic anemia, AA)的认识.方法 回顾分析1 例以AIHA 就诊、疗效欠佳、后经骨髓细胞学复查确诊为AA患儿的诊治过程.结果 初诊时进行性面黄、乏力、纳差,血象示三系减少,骨髓细胞学检查示增生活跃,细胞形态正常,间接抗人球蛋白试验阳性; 考虑自身免疫性溶血性贫血.给予激素治疗后血象无明显改善.多部位骨髓检查确诊为再生障碍性贫血.结论 以AIHA 为首发症状的儿童再生障碍性贫血非常少见,二者有相似临床表现,而且部分再生障碍性贫血骨髓有散在增生灶,易误诊.     

Severe immune hemolytic anemia due to pemetrexed in a patient with non-small cell lung cancer

Pemetrexed-induced immune hemolytic anemia is a rare acute complication and a potentially life-threatening syndrome. Here we report a case of severe hemolytic anemia induced by pemetrexed in a 67-year-old man with lung adenocarcinoma. Hemoglobin concentration reached a nadir of 6.8 g/dl after 6 days of pemetrexed initiation. Pemetrexed was discontinued and the patient was given a transfusion of red blood cells. The direct antiglobulin test was positive (anti-IgG = 4+, anti-C3d = 2+). The patient's plasma reacted with pemetrexed-treated red blood cells by the immune complex test.     

Delayed response of the red blood system (erythron) to carboplatin administration during pregnancy

Pregnant rats exhibited regenerative anemia development 3 months upon a single intravenous injection of carboplatin in a maximum tolerated dose; the effect increased by the end of pregnancy. The character of changes in parameters of the peripheral and central erythron parts (increased level of erythrocyte hemolysis, decrease in the amount of erythrocytes, increase in the number of reticulocytes, development of erythropoietic hyperplasia in bone marrow and spleen) was indicative of the hemolytic type of anemia.     

婴儿自身免疫性溶血的临床分析

目的探讨婴儿自身免疫性溶血性贫血（AIHA）的病因、诱因及治疗方法。方法对32例婴儿AIHA的临床资料进行回顾性分析。结果婴儿AIHA女性多于男性;32例中病因明确者22例;免疫分型以IgG＋C3型多见,且其溶血、贫血程度最重;患者采用肾上腺皮质激素治疗有效率为84．4％。结论自身免疫性溶血性贫血抗体分型与临床特点相关;基础疾病的追查及治疗有重要意义;应重视临床表现及实验室检查特征;皮质激素加免疫抑制剂或加用大剂量免疫球蛋白的治疗效果好。     

The role of N-hydroxyphenetidine in phenacetin-induced hemolytic anemia

Phenacetin is well known to cause hemolytic anemia and methemoglobinemia in humans. Early mechanistic studies clearly established a causal role for active/reactive drug metabolites in the process but did not unequivocally identify these metabolite(s) or resolve the question of whether these two hemotoxicities are mechanistically linked. As part of ongoing studies on the mechanism underlying arylamine-induced hemotoxicities, we have recently shown that the arylhydroxylamine metabolites of aniline and dapsone mediate the hemolytic activity of aniline and dapsone, respectively. The present study was undertaken to determine if N-hydroxyphenetidine (PNOH), the known arylhydroxylamine metabolite of phenacetin, is responsible for phenacetin-induced hemolytic anemia. As measured by decreased survival of 51Cr-labeled erythrocytes in rats, phenacetin, p-phenetidine, and PNOH were all hemolytic in vivo, with PNOH being significantly the most potent of the three. In vitro exposure of 51Cr-tagged erythrocytes to PNOH, followed by transfusion into isologous rats, resulted in a concentration-dependent reduction in erythrocyte survival, indicating that PNOH is a direct-acting hemolytic agent. Phenacetin and p-phenetidine were inactive. Phenacetin, p-phenetidine, and PNOH all produced dose-dependent methemoglobinemia in rats. In parallel in vitro studies, PNOH elevated methemoglobin levels, p-phenetidine and phenacetin did not. However, attempts to identify PNOH in the blood of phenacetin- and p-phenetidine-treated rats were unsuccessful, despite the use of a highly sensitive analytical method. Hemotoxic concentrations of PNOH were found to be highly unstable in the presence of red cells, though relatively stable in the buffer vehicle alone. Inhibitors of acetylation (p-aminobenzoic acid [PABA]) and deacetylation (bis-[p-nitrophenyl]phosphate [BNPP]), used to alter the cyclic interconversion of phenacetin and p-phenetidine, caused changes in phenacetin hemotoxicity that indicated the hemotoxin was a deacetylated metabolite distal to p-phenetidine. These data are consistent with the hypothesis that PNOH, formed during the metabolic clearance of phenacetin, mediates phenacetin-induced hemolytic anemia and methemoglobinemia through direct toxic actions in the erythrocyte.     

葛根素引起急性溶血和肾功能衰竭的致病机制探讨

目的 :探索葛根素引起急性溶血和肾功能衰竭 (肾衰 )的病理机制。方法 :作血液免疫学体外药物模拟抗球蛋白试验 (AGT)。以病人d 1,d 8及8mo的血样为标本 ,作直接抗球蛋白试验 (DAT)检测病人红细胞 (Pc)上的免疫复合物 ,行间接抗球蛋白试验 (IAT)检测O型供者红细胞 (Dc)与病人血清(Ps)、葛根素、补体孵育后在盐水介质及抗球蛋白血清 (AGS)中有否凝集或溶血。结果 :PcDAT阳性 ;IAT———孵育后的Dc在盐水介质中凝集 ,在IAT洗涤过程中部分溶血 ;在AGS中 ,Dc与抗 广谱AGS、抗 C3的凝集强于抗 IgG。单纯药物孵育的Dc不凝集 ,不加葛根素的Ps及加入葛根素的AB型供者体血清红细胞 (Ds)不凝集 ;8mo后重复Pc和Ps试验 ,凝集全部消失。结论 :Ps中含有可活化补体的IgM类抗葛根素抗体 ,此为急性血管内溶血、肾衰的病理基础     

磷酸川芎嗪注射液血管刺激性、溶血性及过敏性试验研究

目的观察磷酸川芎嗪注射液是否具有血管刺激性、溶血性和过敏作用,评价其注射用药的安全性。方法通过兔耳缘静脉刺激性试验、体外溶血性试验、豚鼠全身主动过敏性试验（ASA）和大鼠被动皮肤过敏试验（PCA）,试验观察磷酸川芎嗪注射液的安全性。结果磷酸川芎嗪注射液对家兔耳静脉注射无明显刺激性;对家兔血细胞无溶血和凝集反应;豚鼠全身主动过敏性试验亦无动物出现过敏症状;在大鼠被动皮肤过敏试验中仅低剂量组有20%动物出现过敏反应症状。结论在本次实验条件下,除磷酸川芎嗪注射液低剂量组有轻微过敏反应症状外,其注射使用是安全的。     

鼠疫疫苗在食蟹猴模型中的免疫学评价

目的 通过将鼠疫耶尔森菌的F1抗原和重组V抗原组成的鼠疫疫苗免疫食蟹猴,对疫苗的免疫效果进行评价.方法 将20只食蟹猴按简单随机法分成低剂量组、高剂量组和生理盐水对照组,分别于0和2周肌内免疫,并于1剂后2周和2剂后2周采血.用ELISA检测免疫动物血清中的总IgG抗体;另外,分离外周血淋巴细胞,用酶联免疫斑点试验检测分泌IFN-γ的外周血淋巴细胞.用t检验对结果进行比较.结果 免疫后,对照组均未产生抗体,而疫苗组产生了较强的抗体应答.2剂免疫后2周,低、高剂量组的抗F1抗原IgG抗体几何平均滴度分别为(4.71±0.32)1g和(5.09±0.21)lg(t=-2.76,P＜0.05),两组的抗重组V抗原IgG抗体几何平均滴度分别为(4.75±0.52) lg和(5.12±0.58) lg(t=-1.37,P＞0.05).经F1和V抗原体外刺激产生IFN-γ的外周血淋巴细胞,细胞数均无明显增加.F1抗原刺激后,低、高剂量组分泌IFN-γ的外周血淋巴细胞数分别为(1±1)/106和(1±2)/106(t=-0.16,P＞0.05).重组V抗原刺激后,两组分别为(7±15)/106和(6±7)/106(t=0.88,P＞0.05).结论 鼠疫疫苗在食蟹猴模型中能诱导较强的体液免疫应答,但不能诱导明显的细胞免疫应答.