Microparticle-enhanced nephelometric immunoassay of lactoferrin in human milk

A microparticle-enhanced nephelometric immunoassay was developed for lactoferrin quantitation in human milk. It is based on the nephelometric measurement of the light scattered during the competitive immunoagglutination of a microparticle-lactoferrin conjugate with an antilactoferrin antiserum. This immunoassay is sensitive (detection limit in reaction mixture, 0.2 mg/L) and can be performed in diluted milk (1/3,000 in reaction mixture), excluding any interference or sample pretreatment. It allowed the quantification of lactoferrin on a large range of concentrations (0.675–21.6 g/L) with accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation from 3% to 6%). Changes in the lactoferrin concentration of human milk during lactation were determined in 190 samples. The concentration and ratio of lactoferrin vs. total protein were found to be significantly higher in colostrum (5.9 g/l, 29%) than in transitional milk (2.9 g/L, 22%) or mature milk (2.5 g/L, 24%). J. Clin. Lab. Anal. 11:239–243, 1997. © 1997 Wiley-Liss, Inc.     

Changes in the mannan binding lectin (MBL) concentration in human milk during lactation

The mannan binding lectin (MBL) activates the complement system by the lectin pathway after the recognition of some structural motifs (saccharides) present on the surface of microorganisms. MBL has been mostly identified and quantified in human serum by ELISA or microparticle immunonephelometry assays. This article reports the MBL levels as assessed by a microparticle immunonephelometric assay in 76 human milk samples. Immunonephelometry was performed using skim-milk samples diluted 20 times over a calibration range of 0.07-4.82 mg/L. MBL is indeed present in human milk and its concentration decreases significantly during development from colostrum (0.55+/-0.09 mg/L) to transitional (0.18+/-0.02 mg/L) and mature milk (0.17+/-0.02 mg/L). This innate molecule may be involved in the primary defenses of the mammary gland and the neonate, whose immune system is immature. The high levels observed during the first days of lactation support the hypothesis that this molecule plays a key role in limiting the colonization of the newborn gut by pathogens.     

Interleukin-10 and -12 in human milk at 3 stages of lactation: A longitudinal study

This study was undertaken to analyze postpartum changes in concentrations of interleukin (IL)-10 and IL-12 through the 3 stages of lactation. A total of 87 human milk samples were collected from 29 healthy mothers during the colostrum (0–3 days), early milk (14–17 days), and mature milk (44–47 days) phases. Enzyme-linked immunosorbent assay tests were performed on the milk samples. IL-10 was detected in 7 and IL-12 in 4 of the colostrum samples. In the transitional milk samples, IL-10 was present in 4 and IL-12 in 2; however, both of these cytokines became undetectable in mature milk samples. The decrease in concentrations of IL-10 and IL-12 was statistically significant during the postpartum period (P=.001 and P=.024, respectively). IL-10 levels in the colostrum samples were higher than in the transitional samples (P=.018, with use of the post hoc test). No statistically significant differences between IL-12 levels were noted in the colostrum samples and the transitional samples (P=.068, with use of the post hoc test). A negative correlation was observed between concentrations of IL-10 in colostrum and the total number of pregnancies (R=−.401;P=.031). The findings of the present study suggest that mean concentrations of IL-10 and IL-12 are decreased in human milk as lactation continues through its 3 phases.     

Mesangial medium with IgA1 from IgA nephropathy inhibits nephrin expression in mouse podocytes

Background  IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA1, and podocyte injury plays an important role in glomerulosclerosis of the disease. Our previous study indicated that medium of mesangial cells co-incubated with aggregated IgA1 (aIgA1), isolated from IgAN patients, down-regulated nephrin expression. Yet the mechanism remains unclear.
Materials and methods  Podocytes were incubated with a medium of mesangial cells co-incubated with aIgA1, which was isolated from IgAN patients, and enalaprilat (10⁻⁵ M), valsartan (10⁻⁵ M) and anti-mouse tumour necrosis factor-α antibody (50 ng mL⁻¹) separately. Nephrin expression in podocytes was measured by real-time polymerase chain reaction and Western blot analysis.
Results  The level of angiotensinogen and angiotensin-converting enzyme mRNAs in podocytes, as well as angiotensin, was also increased by a medium of mesangial cells co-incubated with aIgA1 from IgAN patients( P  <   0·05). Enalaprilat or valsartan partly improved nephrin expression when compared with that by podocytes exposed to the mesangial medium ( P  <   0·05), while the nephrin expression of podocytes with enalaprilat or valsartan was lower than that of podocytes exposed to medium of mesangial cells stimulated by aIgA1 from healthy control ( P  <   0·05). However, anti-mouse tumour necrosis factor-α antibody did not show any improvement in nephrin expression.
Conclusion  Our findings implicate that local renin angiotensin system activation in podocytes is partly involved in down-regulation of nephrin by mesangial medium in IgA nephropathy.     

Increased sialylation of polymeric lambda-IgA1 in patients with IgA nephropathy

The mechanism of mesangial IgA deposition is poorly understood in IgA nephropathy (IgAN). Abnormal glycosylation of carbohydrate moieties in the hinge region of the IgA molecule has recently attracted much attention. In this report, we studied galactosylation and sialylation profiles in kappa- and lambda-IgA1 from patients with IgAN. Total serum IgA1 was isolated from patients with IgAN or healthy controls by jacalin-affinity chromatography. Six fractions of molecular weight (MW) 50-1,000 kDa were separated by fast protein liquid chromatography (FPLC). Four lectin-binding assays were used to study the sialylation and the presence of terminal galactose or N-acetylgalactosamine (GalNAc) in the O-linked carbohydrate moieties of kappa- or lambda-IgA1. Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA) lectin recognize alpha(2,3)- and alpha(2,6)-linked sialic acid, respectively. Peanut agglutinin (PNA) and Helix aspersa (HA) lectin recognize terminal galactose and GalNAc, respectively. Reduced HA was demonstrated in macromolecular kappa or lambda-IgA1 (300-825 kDa) isolated from patients with IgAN (P < 0.05 compared with healthy controls). Lambda- but not kappa-IgA1 from patients with IgAN bound less to PNA (P < 0.05). The alpha(2,3)-linked sialic acid content in lambda- but not kappa-IgA1 of MW 150-610 kDa from patients was higher than that of controls (P < 0.005). The alpha(2,6)-linked sialic acid content in lambda-IgA1 (300-825 kDa) and kappa-IgA1 (150-610 kDa) from patients was also higher than that of controls. This unusual glycosylation and sialylation pattern of the lambda-IgA1 may have important implications for the pathogenesis of IgAN, as both the masking effect of sialic acid on galactose and the reduced galactosylation will hinder the clearance of macromolecular lambda-IgA1 by asialoglycoprotein receptor of hepatocytes. The negative charge from sialic acid may also favor mesangial deposition of macromolecular lambda-IgA1 in IgAN.     

血清IgA、IgA/C3比值在IgA肾病的诊断价值

目的探讨体检发现的IgA肾病(IgAN)患者血清IgA、IgA/C3比值在病理损伤评估和诊断预测方面的价值。方法 2005年8月至2010年12月因体检异常来中山医院就诊并经肾活检确诊为IgAN患者239例,回顾性分析这些患者的临床病理资料。所有患者都检测血清IgA、C3水平,病理学分级参照Lee′s分级标准。结果 IgAN患者血清IgA、IgA/C3水平较其他非IgA肾脏病理类型患者高;IgA、IgA/C3与IgAN病理损伤程度无相关性(P>0.05),二者的ROC曲线下面积分别为0.597、0.611。结论血清IgA、IgA/C3比值在IgAN患者明显升高,但二者水平与病理病变无相关性,对体检发现的IgAN诊断预测价值较低。     

Significance of serum IgA levels and serum IgA/C3 ratio in diagnostic analysis of patients with IgA nephropathy

Diagnostic analysis of clinical markers including serum IgA levels and serum IgA/C3 ratio in patients with IgA nephropathy is described. One hundred patients with IgA nephropathy (IgA nephropathy group) and 100 patients with other primary glomerular diseases (non-IgA nephropathy group) were examined. The analysis was performed to distinguish between these two groups using four clinical markers: 1) more than five red blood cells in urinary sediments, 2) persistent proteinuria (urinary protein of more than 0.3 g/day), 3) serum IgA levels of more than 315 mg/dl, and 4) a serum IgA/C3 ratio of more than 3.01. Patients with three or four clinical markers were easily diagnosed as having IgA nephropathy in this study. Furthermore, there was a significant difference in these clinical markers between the good prognosis and relatively good prognosis groups (Groups I and II) and the relatively poor prognosis and poor prognosis groups (Groups III and IV) of IgA nephropathy patients. It appears that the presence of microscopic hematuria and/or persistent proteinuria, high serum IgA levels, and the serum IgA/C3 ratio are useful for distinguishing IgA nephropathy from other primary renal diseases. It is postulated that these clinical markers are also useful for diagnosis of IgA nephropathy without renal biopsy.     

Serum levels of interleukin-2 receptor and disease activity in patients with IgA nephropathy

The levels of serum interleukin-2 receptor (IL-2R) were studied by enzyme-linked immunosorbent assay (ELISA) in patients with IgA nephropathy. The aim of the present study was to determine if levels of serum IL-2R might correlate with disease activity in patients with IgA nephropathy. Twenty-eight patients with IgA nephropathy were examined. This study showed a significant correlation between the levels of serum IL-2R and disease activities, i.e., levels of urinary protein, blood urea nitrogen (BUN) and uric acid, in patients with IgA nephropathy. The levels of serum IL-2R in patients with moderate or advanced stage of IgA nephropathy were significantly higher than those in patients with the minimal or slight stage of this disease or in healthy adults. It was suggested that the measurement of serum IL-2R is useful in evaluating the degree of disease activity and/or prognosis in patients with IgA nephropathy.     

Association between Delayed Lactogenesis Ⅱ and Early Milk Volume among Mothers of Preterm Infants

PurposeThis study aimed to evaluate the effect of delayed lactogenesis Ⅱ on early milk volume in mothers expressing milk for their preterm infants.Methods142 mothers with preterm infants participated in a longitudinal cohort study, the milk volumes over 14 days postpartum between mothers with delayed lactogenesis Ⅱ (≥ 72 hours) and mothers with non-delayed lactogenesis Ⅱ(< 72 hours) were compared using Wilcoxon's rank sum tests.ResultsThe prevalence of delayed lactogenesisⅡ among mothers of preterm infants was 36.0% (36/100). There existed negative correlations between the onset of lactogenesis Ⅱ and the daily milk volumes( r_s = −0.525∼−0.354, p = .002 ∼ p < .001). The milk volumes in every 24-hour of the 14 days postpartum in delayed group were significantly less than that in non-delayed group (p = .002 ∼ p < .001). After controlling for the covariates, pregnancy-induced hypertension syndrome, delayed expression initiation, shorter daily sleeping time were found to be the risk factors for delayed lactogenesis Ⅱ.ConclusionDelayed lactogenesis Ⅱ was associated with lower milk volume in early postpartum period. Women who were at risk for delayed lactogenesis Ⅱ need targeted interventions and additional support during pregnancy and postpartum.     

Vilidation of IgA1 and IgA2 measurements by a solid-phase immunoradiometric assay in serum and secretions

Summary We describe specific, sensitive and reproducible immunoradiometric assays to measure total IgA and IgA subclass levels in biological fluids, which take into account the problem that polymeric forms are differently recognized in immunoassays. Sera from subjects totally deficient in one of the IgA subclasses allowed us to ensure the specificity of the subclass assays and to define the proportions of IgA1 (84%) and IgA2 (16%) in the normal pooled serum (from 30 blood donors) used as standard. With purified milk 11-S secretory IgA1 and 11-S secretory IgA2, we determined a correction factor for the corresponding polymeric forms using, respectively, monomeric IgA1 and monomeric IgA2 from pooled serum as standards. With the monoclonal antibodies used, purified 11-S secretory IgA1 was similarly recognized by both the total IgA assay and the IgA1 assay; both total IgA and IgA1 concentrations were underestimated compared with monomeric IgA or monomeric igA1. In contrast, 11-S secretory IgA2 was better recognized by the IgA2 assay than by the total IgA assay and the values were thus overestimates. Considering this problem of recognition, we fractionated saliva and lung secretions by sucrose density gradient ultracentrifugation before measuring their IgA1 and IgA2 levels.     

Prolonged jaundice in newborns is associated with low antioxidant capacity in breast milk

Abstract

In breastfeeding newborns who are otherwise healthy, the mechanism of prolonged jaundice remains unclear. The aim of this study was to investigate relations between prolonged jaundice and oxidative parameters in breast milk. Full-term, otherwise healthy newborns with jaundice lasting more than 2 weeks were enrolled prospectively in the study. As a control group, newborns in the same age group but without prolonged jaundice were selected. All newborns in the study were exclusively breastfed. In the newborns with prolonged jaundice, investigations of the etiology of the jaundice included complete blood count, peripheral blood smear, blood typing, direct Coombs test, measurement of serum levels of total and direct bilirubin, tests for liver and thyroid function (TSH, free T4, total T4), urine culture and measurement of urine reducing substances, and determination of glucose 6 phosphate dehydrogenase enzyme levels. Breast milk was collected from the mothers of the newborns in both groups. The antioxidant status of the breast milk was assessed via determination of total antioxidant capacity (TAC). Oxidative stress was also assessed in breast milk by measurement of total oxidation status (TOS) and calculation of the oxidative stress index (OSI). The prolonged jaundice group differed significantly from the control group in terms of mean TAC and OSI (p < 0.001), but not in terms of TOS. In conclusion, in the breast milk of mothers of newborns with prolonged jaundice, oxidative stress was found to be increased, and protective antioxidant capacity was found to be decreased.     

IgA肾病合并急性间质性肾炎2例报告

正急性间质性肾炎(AIN)是导致急性肾损伤(AKI)的重要原因之一。AIN的主要病理表现为肾间质炎细胞浸润,通常不伴肾小球病变。在慢性肾小球肾炎基础上发生的AIN的临床病理表现与单纯AIN、慢性肾小球肾炎继发间质损害有一定区别。本科室近期连续诊治2例IgA肾病合并急性间质性肾炎患者,现报告如下。     

280例IgA肾病患者不同年龄组病理分析

目的：回顾性分析不同年龄组IgA。肾病患者的病理改变特点,以利于早期诊断和治疗。方法：将我院280例（2005-2007年）经皮肾活检确诊为原发性IgA。肾病的患者按不同年龄分为儿童组、青少年组和成人组,逐项分析病例组织光镜和免疫荧光检查的相关资料。结果：在280例病理资料中,儿童组患者病理学分级以I～Ⅱ级为主,免疫荧光检查以单纯IgA和或C3沉积多见;青少年组患者病理学分级以I～Ⅲ级为主,免疫荧光检查也是以单纯IgA和或C3沉积多见;成人组病理学改变从I～Ⅳ级均可见到,主要以Ⅲ～Ⅳ级为主,免疫荧光检查以单纯IgA、IgA＋IgM和或C3沉积较多。结论：IgA肾病的病理改变有随着年龄增大,病理改变越明显肾脏损害越严重的趋势,早期进行经皮肾活检有利于明确诊断及尽早治疗。     

IgA缺乏症献血者酶联免疫试验筛查方法的建立

目的建立适合用于大规模IgA缺乏症献血者筛选的ELISA检测方法。方法采用间接酶联免疫法,在微孔板中包被羊抗人IgA、用辣根过氧化物酶标记羊抗人IgA抗体作为酶标二抗。结果建立的ELISA测定法灵敏度为0.1μg/mL,IgA浓度为0.1、100μg/mL的批间变异系数(CV)是1.74%、3.49%,批间CV中位数为3.48%(范围:1.83%~6.96%)。检测过程约80min。结论成功建立了IgA缺乏症筛查的ELISA测定法,其灵敏度高、特异度强、省时、操作简易,可用于大规模筛选IgA缺乏症献血者及建立IgA缺乏献血者库。     

IgA肾病患者外周血Th1、Th2、Th3细胞水平的变化及意义

目的观察IgA肾病（IgA nephropathy,IgAN）患者外周血中Th1、Th2、Th3细胞亚群的表达,探讨其在IgAN免疫发病机制中的作用。方法采用流式细胞仪检测23例IgAN患者（IgAN组）及20例健康体检者（正常对照组）外周血Th1、Th2、Th3细胞比例,并用Spearman或Pearson相关分析法对其分布变化与IgAN各项临床指标进行相关性分析。结果 IgAN组外周血中Th1细胞比例较正常对照组减少,差异无统计学意义（P〉0.05）;IgAN组外周血中Th2、Th3细胞比例均显著高于正...     

糖皮质激素治疗IgA肾病79例临床分析

目的　探讨糖皮质激素在IgA肾病治疗中的价值,为其临床干预治疗提供依据。方法　对应用糖皮质激素治疗的79例IgA肾病患者的临床资料进行回顾性分析。结果　79例IgA肾病患者肾组织活检后平均随访( 39. 6 ±42. 2 )个月;治疗前尿蛋白量为( 1. 57 ±2. 40 )g/24 h, 治疗后则降到(0. 43±1. 07)g/24h(P=0. 000 );治疗前平均内生肌酐清除率为( 86. 64±29. 99 )ml/min,治疗后为(92. 06±35. 90)ml/min(P>0. 05)。同时不同蛋白尿程度、有无高血压、治疗前有无肾功能受损及不同的肾脏病理表现的患者激素治疗后尿蛋白水平均显著降低。结论　应用糖皮质激素治疗IgA肾病能显著降低其患者的蛋白尿,保护肾功能。     

Prevalence of IgA antitissue transglutaminase antibodies in children with type 1 diabetes mellitus

The association of celiac disease with type 1 diabetes mellitus is known, but the evolution of celiac disease is most frequently asymptomatic, without any clinical signs. Thus, diagnosis is impossible to make in the absence of serological tests. Our study aimed to determine the prevalence and the efficiency of IgA antitissue transglutaminase antibodies in the screening of celiac disease in children with type 1 diabetes mellitus. Method: During the course of 2008–2009, we performed an analytical clinical study that included the determination of IgA antitissue transglutaminase antibodies in a group of 119 children with type 1 diabetesmellitus. Fifty‐seven percent of the subjects were male and 43% were female, with a mean age of 11±4 years. Results: By evaluating IgA antitissue transglutaminase antibodies, we obtained a prevalence of 9.2% in children with type 1 diabetes mellitus, with a sensitivity and specificity of 80 and 82.6%, respectively. Conclusions: There is an increased prevalence of IgA antitissue transglutaminase antibodies, which suggests the need to use this method as an effective first‐line test in the screening of celiac disease in children with type 1 diabetes mellitus. J. Clin. Lab. Anal. 25:156–161, 2011. © 2011 Wiley‐Liss, Inc.     

2型糖尿病合并IgA肾病与糖尿病肾病患者的临床分析

目的：分析2型糖尿病合并IgA肾病与2型糖尿病合并糖尿病肾病患者的临床特点,鉴别诊断要点。方法：回顾分析18例2型糖尿病合并IgA肾病与15例2型糖尿病合并糖尿病肾病患者的临床资料。结果：2型糖尿病合并IgA肾病患者糖尿病病史（10.72±18.66个月）明显短于糖尿病肾病患者（58.73±71.12个月）,P〈0.05。IgA肾病组在肾活检时血肌酐升高的比例（5.56%）明显低于糖尿病肾病组（46.67%）,P〈0.05,伴有血尿的患者（61.11%）明显多于糖尿病肾病组（20.00%）,P〈0.05。IgA肾病组总胆红素、丙氨酸氨基转移酶、血清免疫球蛋白A、血清免疫球蛋白G、血钙明显高于糖尿病肾病组（分别为13.28±4.14μmol.L^-1比9.95±4.87μmol.L^-1、34.22±18.11U.L^-1比19.73±16.04U.L^-1;4.00±2.16g.L^-1比2.11±0.86g.L^-1;12.47±4.76g.L^-1比9.04±2.41g.L^-1和2.37±0.17mmol.L^-1比2.22±0.20mmol.L^-1）。IgA肾病组血尿素氮、血肌酐、50%补体溶血单位、随机尿白蛋白/肌酐比值、24h尿蛋白显著低于糖尿病肾病组[分别为5.86±1.59mmol.L^-1比10.76±5.89mmol.L^-1;82.72±23.76μmol.L^-1比185.20±107.19μmol.L^-1;50.51±5.80IU.mL^-1比55.37±6.17IU.mL^-1;959.50±395.00μg.（mgCr）^-1比3193.85±2085.00μg.（mgCr）^-1和2.22±2.13g.（24h）^-1比4.69±2.92g.（24h）^-1]。两组血糖、血脂均无统计学差异。IgA肾病患者荧光眼底血管造影和肌电图检查未见异常,糖尿病肾病组有2例糖尿病视网膜病变和3例糖尿病周围神经病变。结论：2型糖尿病合并IgA肾病的患者尿检异常出现前糖尿病病史大多短于5年,血尿、血清免疫球蛋白A升高多见,尿检异常出现时无糖尿病视网膜病变或周围神经病变。