Nagel Robyn, Traub Rebecca J, Kwan Marcella M S, Bielefeldt-Ohmann Helle
School of Veterinary Science, The University of Queensland, Gatton Campus, Gatton, QLD, 4343, Australia.
Toowoomba Gastroenterology Clinic, Suite 105 Medici Medical Centre, 15 Scott St, Toowoomba, QLD, 4350, Australia.
Parasit Vectors. 2015 Sep 15;8:453. doi: 10.1186/s13071-015-1069-x.
Blastocystis species are common enteric human parasites and carriage has been linked to Irritable Bowel Syndrome (IBS), particularly diarrhoea-predominant IBS. The spectrum of immune reactivity to Blastocystis proteins has been reported previously in symptomatic patients. We investigated differences in serum immunoglobulin profiles between patients with IBS, both positive and negative for Blastocystis carriage, and healthy controls (HC).
Forty diarrhoea-predominant IBS patients (26 patients positive for Blastocystis sp., 14 negative patients) and forty HC (24 positive, 16 Blastocystis-negative) were enrolled. Age, gender, ethnicity and serum immunoglobulin A (IgA) levels were recorded and faecal specimens were analysed using smear, culture and polymerase chain reaction amplification of ribosomal DNA. Sera were tested in Western blots and the reactivities compared to known targets using monoclonal antibodies Blastofluor® (Blastocystis specific antibody), MAb1D5 (cytopathicto Blastocystis cells), anti-promatrix metalloprotease-9 (anti-MMP-9) and SDS-PAGE zymograms.
Levels of serum IgA were significantly lower in Blastocystis carriers (p < 0.001) but had no relationship to symptoms. Western blots demonstrated serum IgG antibodies specific for Blastocystis proteins of 17,27,37,50,60-65, 75-90, 95-105 and 150 kDa MW. Reactivity to the 27, 50 and 75-95 kDa proteins were found more frequently in the IBS group compared to the HC's (p < 0.001) and correlation was greater for Blastocystis-positive IBS patients (p < 0.001) than for negative IBS patients (p < 0.05). MAb1D5 reacted with proteins of 27 and 100 kDa, and anti-MMP-9 with 27, 50 and 75-100 kDa proteins. Bands were seen in zymograms around 100 kDa.
Low serum IgA levels are associated with Blastocystis carriage. All IBS patients were more likely to demonstrate reactivity with Blastocystis proteins of 27 kDa (likely a cysteine protease), 50 and 75-95 kDa MW compared to HC. The presence of antibodies to these Blastocystis proteins in some Blastocystis-negative subjects suggests either prior exposure to Blastocystis organisms or antibody cross reactivities. The anti-proMMP-9 reaction at 50 and 75-100 kDa and the zymogram result suggest that metalloproteases may be important Blastocystis antigens.
Australian and New Zealand Clinical Trials registry ACTRN: 12611000918921.
芽囊原虫属是常见的人体肠道寄生虫，其携带与肠易激综合征（IBS）相关，尤其是腹泻型肠易激综合征。先前已有报道有症状患者对芽囊原虫蛋白的免疫反应谱。我们研究了芽囊原虫携带阳性和阴性的肠易激综合征患者与健康对照（HC）之间血清免疫球蛋白谱的差异。
纳入40例腹泻型肠易激综合征患者（26例芽囊原虫属阳性患者，14例阴性患者）和40例健康对照（24例阳性，16例芽囊原虫属阴性）。记录年龄、性别、种族和血清免疫球蛋白A（IgA）水平，并使用涂片、培养和核糖体DNA的聚合酶链反应扩增分析粪便标本。血清进行蛋白质印迹检测，并使用单克隆抗体Blastofluor®（芽囊原虫特异性抗体）、MAb1D5（对芽囊原虫细胞有细胞病变作用）、抗基质金属蛋白酶-9（抗-MMP-9）和SDS-聚丙烯酰胺凝胶电泳酶谱图与已知靶点比较反应性。
芽囊原虫携带者的血清IgA水平显著较低（p < 0.001），但与症状无关。蛋白质印迹显示血清IgG抗体对分子量为17、27、37、50、60 - 65、75 - 90、95 - 105和150 kDa的芽囊原虫蛋白具有特异性。与健康对照相比，肠易激综合征组中对27、50和75 - 95 kDa蛋白的反应性更常见（p < 0.001），且芽囊原虫阳性的肠易激综合征患者的相关性大于阴性肠易激综合征患者（p < 0.001）（p < 0.05）。MAb1D5与27和100 kDa的蛋白反应，抗-MMP-9与27、50和75 - 100 kDa的蛋白反应。在酶谱图中可见约100 kDa处有条带。
低血清IgA水平与芽囊原虫携带有关。与健康对照相比，所有肠易激综合征患者更有可能表现出对27 kDa（可能是一种半胱氨酸蛋白酶）、50和75 - 95 kDa分子量的芽囊原虫蛋白的反应性。一些芽囊原虫阴性受试者中存在针对这些芽囊原虫蛋白的抗体，表明要么先前接触过芽囊原虫生物体，要么存在抗体交叉反应。50和75 - 100 kDa处的抗-proMMP-9反应和酶谱图结果表明金属蛋白酶可能是重要的芽囊原虫抗原。
澳大利亚和新西兰临床试验注册中心ACTRN：12611000918921。
