Immunoblot analysis of the serological response in Hantavirus infections

Sera from patients with nephropathia epidemica (NE) or Korean hemorrhagic fever (KHF) were tested for specific antibody response to antigens of H?lln?s virus and Hantaan virus strain 76-118. A Vero E6 derived cell line persistently infected with H?lln?s virus strain B1, and Vero E6 cells freshly infected with Hantaan virus type strain 76-118 were used as antigens in the immunofluorescence assay (IFA) and the immunoblot. Blots were prepared from whole cell lysates. The convalescent-phase sera of NE patients tested in this study regularly revealed a marked reaction with a 52 kilodalton (Kd) protein of H?lln?s virus and a 50 Kd protein of Hantaan virus. A convalescent serum from a patient with Korean hemorrhagic fever and a rat antiserum against Hantaan virus could recognize the 50 Kd band of Hantaan virus but showed no apparent reactivity with the 52 Kd component of H?lln?s virus in the standard dilutions. Some sera could additionally identify minor bands in the 55 Kd and/or 67 Kd region of the blots. A one-way cross reactivity between Hantaan and H?lln?s viruses was also evident from the results of the immunofluorescence assays in that NE convalescent sera reacted with both viruses, whereas KHF convalescent or anti-Hantaan sera gave strongly positive results with Hantaan virus but only faint reaction with H?lln?s virus.     

7832例孕妇HCMV-IgM的检测分析

目的为了解孕妇人巨细胞病毒（HCMV）近期感染状况。方法采用酶联免疫吸附试验（ELISA）对7832例孕妇的静脉血标本进行了HCMV-IgM检测。结果在7832例孕妇中检测出HCMV-IgM阳性标本67例,阳性率为0.86%。结论在孕妇中巨细胞病毒有一定的近期感染率。     

Naturally occurring human IgA autoantibodies against IgE-DES myeloma protein

The prevalence and specificity of naturally occurring human IgA anti-IgE autoantibodies (a-E Ab) were studied by ELISA with anti-IgA monoclonal antibodies (mAb) and a purified myeloma IgE as solid-phase protein, i.e., IgE-DES(κ). Such detected IgA a-E Ab were common among adults, and significantly increased geometric means (GM) were found in patients with atopy (P= 0.006; n= 41; GM = 79.3 arbitrary units (AU)/ml) and filariasis (P= 0.02; n= 41; GM = 75.9 AU/ml), as compared with nonatopic controls (n= 42; GM = 48.8 AU/ml). No such difference was observed between age-matched nonatopic (n= 22; GM = 36.7 AU/ml) and atopic (n= 22; GM = 38.6 AU/ml) children. Children had significantly (P= 0.001) lower IgA a-E Ab concentrations than adults, probably as a result of age, because IgA a-E Ab concentrations and age of children were significantly correlated (n= 44; P<0.05; r_s= 0.30). IgA a-E Ab concentrations were very low in cord serum (n= 32; median <0.1 AU/ml). Sex did not influence IgA a-E Ab concentrations in any study group. The specificity of IgA a-E Ab in nine sera was studied by ELISA inhibition assay using IgE-DES myeloma as solid-phase protein and inhibitory proteins of the IgG, IgM, IgD, and IgE classes, including five different IgE myeloma proteins, as well as three enzymatic fragments of IgE-DES. The inhibitions indicated that all IgA a-E Ab tested reacted in a low-affinity reaction with determinants restricted to IgE-DES, i.e., the solid-phase protein. These epitopes were heat-resistant (2 h; 56°C) and located in the Fab_?-DES fragment. No isotype-specific IgA a-E Ab were found because none of the four other IgE proteins were inhibitory. Subclass typing indicated that most IgA a-E Ab belonged to the IgA 1 subclass. It is unlikely, for reasons of restricted specificity, low affinity, and common prevalence, that such IgA 1 a-E Ab are connected with IgE-mediated disorders. The study also raises questions on the definition of anti-IgE antibodies.     

Agreement between CBNAAT,liquid culture and line probe assay for detection of Mycobacterium tuberculosis and anti-tubercular drug resistance in extrapulmonary samples

PurposeCartridge based nucleic acid amplification test (CBNAAT) has been endorsed by the WHO as the screening test for diagnosing extrapulmonary tuberculosis (EPTB). In the present study we report the agreement between CBNAAT (Xpert MTB/RIF), liquid culture (LC) and line probe assay (LPA) for diagnosis of Mycobacterium tuberculosis and detection of drug resistance among EPTB cases.MethodsThe EP samples were subjected to CBNAAT (Xpert MTB/RIF, Cepheid, USA) and wherever possible, to LC (MGIT 960, Becton Dickinson, USA) followed sequentially by first line and second line-LPA (FL-LPA, SL-LPA, Hain Lifescience, Germany) on the isolates.ResultsTotal 566/4080 (13.9%) EP samples were detected positive for M. tuberculosis on CBNAAT. Aspirates from lymph nodes were most often positive (11/30; 36.6%), followed by pus (240/873; 27.5%) and CSF samples (166/104; 15.8%). The detection of M. tuberculosis was more in adults than children except in tissue biopsy samples. Rifampicin resistance was also higher among adults except CSF in which resistance was more in children. Total 185 of 566 (32.7%) CBNAAT positive and 770 of 3510 (21.9%) CBNAAT negative samples could be cultured of which 110/185 (59.4%) and 33/770 (4.3%) respectively turned positive. FL-LPA and SL-LPA of 143 culture isolates showed that 27 isolates had drug resistance, of which 3 (2.1%) were XDR, 11 (7.7%) were Pre-XDR (FQ) and 13 (9.1%) were MDR. Of these 27 resistant isolates, 12 were negative by CBNAAT and two were mislabeled as Rifampicin sensitive or indeterminate based on the unique RpoB gene mutation patterns on LPA. The positive and negative agreements between LC and CBNAAT for detection of M. tuberculosis were 67.1% and 92.7% respectively and between LPA and CBNAAT for rifampicin resistance detection were 98.9% and 92.9% respectively.ConclusionsFor EPTB, CBNAAT should be accompanied with LC wherever possible irrespective of the CBNAAT result.     

Histochemical demonstration of endogenous estrogen in breast carcinomas: Biochemical and clinical correlation

Summary In a study of 277 patients with breast carcinomas, the PAP immunoperoxidase method for demonstrating endogenous estrogen was correlated with the sucrose density gradient (SDG) assay and with histologic and clinical features. The results from the PAP method and SDG assay agreed in 59 of 84 patients (82.1%) on whom both methods were performed. Histologically, the PAP method was positive in 7 of 7 patients with non-invasive carcinomas, in 164 of 233 patients (70.4%) with common invasive ductal carcinoma, and in 21 of 22 of those with special histological types of invasive carcinomas not including Paget's disease, medullary or apocrine carcinoma, where only 5 of 14 were positive. Clinically, 15 of 18 patients with positive endogenous estrogen showed a response to endocrine therapy as opposed to 1 of 9 patients with a negative endogenous estrogen. The mean survival was 31.2 and 15.6 months, respectively for patients with positive and negative endogenous estrogen. Remission for longer than 2 years was seen more often in patients with positive endogenous estrogen. These results suggest a clinical utility of the present PAP method which, therefore, deserves a further trial as an alternative to histochemical methods aiming at the estrogen receptors.This work was supported by Grants-in Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (No. 56480119).This paper was presented at the 72nd Annual Meeding of International Academy of Pathology (United States-Canadian Division), Atlanta, Georgia, March 1, 1983.     

The adaptation of the plaque reduction assay for measuring specific cytotoxic cells in limiting dilution cultures

The use of the original haemolytic plaque reduction technique to measure cytotoxic T lymphocytes (CTL) has been developed further as a rapid screening assay, particularly suitable for limiting dilution analyses. Using hybridoma cells as targets, the cytotoxicity has been measured by the loss of haemolytic plaque formation and by the reduction of the amount of haemolytic monoclonal antibody secreted from viable target cells into the assay supernatants. The assessment of large numbers of cytotoxic samples has been greatly facilitated by quantitating the amount of haemoglobin released in the assay with an automated microELISA multiscanner and by scoring visually using a modification of the spot test. Using these new techniques, relatively high frequency estimates of cytotoxic cell precursors in an allogeneic response (1 in 462 spleen cells) and an anti-fluorescein response (1 in 3970 spleen cells) were obtained.     

Meiotic differentiation during colony maturation in Saccharomyces cerevisiae

As yeast colonies ceased growth, cells at the edge of these colonies transited from the cell division cycle into meiosis at high efficiency. This transition occurred remarkably synchronously and only at late stages of colony maturation. The transition occurred on medium containing acetate or low concentrations of glucose, but not on medium containing high glucose. The repression by high glucose was overcome when IME1 was overexpressed from a plasmid. Experiments with different growth media imply that meiosis in colonies is triggered by changes in the nutrient environment as colonies mature. HAP2 is required to sporulate in any carbon source, whereas GRR1 is required for glucose repression of sporulation. CLN3 is required to repress meiosis in colonies but not in liquid cultures, indicating that the regulators that mediate the transition to meiosis in colonies are not identical to the regulators that mediate this transition in liquid cultures.     

The relationship of free and conjugated catecholamines in plasma and cerebrospinal fluid in cerebral and meningeal disease

Summary The concentration of free and conjugated norepinephrine (NE), epinephrine (E) and dopamine (DA) were measured by a modified radioenzymatic assay in the plasma and in the cerebrospinal fluid (CSF) of 45 patients with normal and in 21 patients with disturbed blood-CSF barriers. In patients with an undisturbed blood-CSF barrier the free NE and E in CSF were 128±45 ng/l and 27±20 ng/l (mean values±S.E.), respectively, and represented about 50 % of the average plasma values. Mean DA was not different in plasma (47±22 ng/l) and in CSF (41±19 ng/l). Both in plasma and in CSF, considerable higher free catecholamine (CA) levels were measured in patients with dysfunction of the blood-CSF barrier. In one patient with bacterial meningitis twofold higher concentrations of free NE and DA in CSF as compared with plasma were detectable. Sulfate conjugates of catecholamines are predominant in plasma and CSF.The contribution of conjugated CA to total CA in plasma from patients with normal blood-CSF barrier averaged 69.7 %, 63.1 % and 98.1 % for NE, E and DA, respectively and was significantly lower in the CSF (p<0.001). In patients with disturbed blood-CSF barrier, the increases of conjugated CA were more pronounced in CSF than in plasma. Further, the contribution of conjugated NE and E to total NE and E in CSF was not only increased in patients with bacterial meningitis, but also in patients with renal insufficiency compared to the control patients (p<0.02 and p<0.001 resp.). Free and conjugated NE, E and DA in the plasma and CSF were related significantly (p<0.01 resp.) with stronger correlation for conjugated CA (p<0.001 resp.). These results together with findings in the literature, suggest that there is little or no rostral-caudal gradient in CSF CA conjugate concentrations and that even in patients with intact blood-CSF barrier plasma conjugated CA concentrations influence those in CSF. Thus only free CA levels in CSF may reflect the central adrenergic activity.     

Huntington's disease: biochemical prediction by determination of GABA synthesis of cultured fibroblasts

Summary GABA synthesis in skin fibroblasts from patients with Huntington's chorea was compared with that in a control group by means of the highly specific ³H-muscimol radioreceptor assay. A significantly increased rate of GABA synthesis was found in the group with Huntington's chorea in an early cell passage. The possible use of this method for early diagnosis of Huntington's chorea is considered.

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Zusammenfassung Die GABA-Synthese in Hautifbroblasten von Chorea Huntington-Patienten im Vergleich zu einer gesunden Kontrollgruppe wurde mittels des hochspezifischen ³H-Muscimol-Radiorezeptoren-Assays untersucht. Wir fanden eine signifikante 8fache Erhöhung der GABA-Synthese bei Chorea Huntington in einer frühen Zellpassage. Es wird erwogen, diese Methode zur Frühdiagnostik von Chorea Huntington einzusetzen.

     

ATP生物荧光检测在气管镜清洗消毒质量监测中的应用研究

目的探讨三磷酸腺苷(ATP)生物荧光检测对气管镜清洗消毒合格参考值范围和检测方法。方法选取360条患者使用后的气管镜随机分为A、B、C三组,每组120条。A组床旁预处理后分为A1、A2两组(每组60条气管镜),B组气管镜在人工清洗后分为B1、B2两组,C组气管镜在高水平消毒后分为C1、C2两组,A1、B1、C1组采用细菌采样,A2、B2、C2组进行ATP生物荧光采样。两种采样方法对气管镜表面采样为全部长度涂抹采样,管腔的采样方法均为冲刷法。同时选取临床使用后的60条气管镜经过人工清洗后,对管腔运用冲洗法,对冲洗液进行ATP生物荧光检测,比较冲洗法和冲刷法对管腔采样的有效性。结果气管镜在人工清洗后,对管腔采样的冲洗法和冲刷法,两组ATP总量的相对发光值(RLU)差异有统计学意义(P=0.040)。气管镜在A1、B1组的细菌采样中,管腔和表面菌落数差异均有统计学意义(P均<0.001),高水平消毒后表面和管腔菌落数均为0;气管镜在A2、B2、C2三组的ATP生物荧光检测采样中,表面ATP总量RLU值A2与B2组间比较差异有统计学意义(P<0.001),B2与C2组间比较差异有统计学意义(P=0.040);管腔ATP总量RLU值A2与B2组间比较差异有统计学意义(P<0.001),B2、C2两组差异无统计学意义(P=0.450)。结论 ATP生物荧光采样冲刷法对气管镜管腔采样更有效;人工清洗环节非常重要,人工清洗后气管镜表面和管腔ATP总量建议低于10 000和3 400。