IgA肾病患者血液、尿液及咽拭子标本中穿通支原体的分离检出

目的探讨IgA肾病患者血液、尿液及咽拭子标本中穿通支原体（Mycoplasma penetrans,Mp）的分离检出率以及与病理型别相关性。方法采用分离培养法,共计从26例IgA肾病患者血液、尿液及咽拭子标本及38例正常对照相应标本中进行穿通支原体分离检测,对培养阳性标本用穿通支原体套式PCR进行证实。结果在11例（42．3％）患者血液与尿液或（和）咽拭子中同时分离到穿通支原体,单独尿液或咽拭子标本阳性分别为1例（3．8％）与7例（26．9％）。26例IgA肾病患者血液、尿液及咽拭子穿通支原体的分离检出率分别为42．3％、23．1％与57．7％;与38例正常对照组血液、尿液及咽拭子检出0例、2例（5．3％）与7例（18．4％）相比较,差异有非常显著性（P〈0．01）,在正常对照组中无2种以上标本同时检出穿通支原体。结论穿通支原体在ISA肾病患者的血液、尿液与咽拭子标本中均有较高的检出率且与病理型别有一定的相关性。     

Effects of Mycoplasmal LAMPs on Receptor Responses to Steroid Hormones in Mammalian Cells

Many individuals are chronically infected or parasitically colonized with mycoplasmas in their respiratory or urogenital tracts without apparent clinical significance. However, prolonged close interaction between prokaryotic agents and eukaryotic host cells may gradually and significantly alter normal biological or physiological properties of infected hosts. Steroid hormones are associated with rates of cancer formation in human. The purpose of this study is to establish a sensitive reporting system to examine whether mycoplasmal infections affect biological responses to steroid hormones in mammalian cells. We established pMTV-CAT stably transfected cell lines to test the effect of mycoplasmal lipid-associated membrane proteins (LAMPs). Results showed that LAMPs (1 μg/ml) from seven different species of human mycoplasmas—M. penetrans, M. fermentans, M. genitalium, M. salivarium, M. pneumoniae, M. orale, and M. hominis—had an inhibitory effect on androgen receptor (AR) response to 5α-dihydrotestosterone (DHT) in the E82 transfectants. The inhibitory effect of mycoplasmal LAMPs appeared to be dose dependent. LAMPs from M. penetrans, M. genitalium, M. salivarium, M. pneumoniae, and M. orale also had an inhibitory effect on glucocorticoid receptor (GR) response to hormone dexamethasone (Dex) in TSU transfectants. In contrast, LAMPs from M. fermentans and M. hominis showed a stimulatory effect on the GR response to Dex in these TSU cells. The results suggest that colonization or chronic infection by mycoplasmas may significantly affect the responses of mammalian host cells to various steroid hormones, potentially affecting rates of cancer formation. Received: 2 January 2001/Accepted: 26 January 2001     

Evaluation of IgG, IgM, and IgA antibodies to mycoplasma penetrans detected by ELISA and immunoblot in HIV-1-infected and STD patients, in São Paulo, Prazil

The aim of the present study was to determine the frequency of IgG, IgA, and IgM antibodies to Mycoplasma penetrans in HIV-1-infected patients and in patients with sexually transmitted diseases. We tested serum samples from 106 HIV-1-positive patients and 110 individuals with clinical symptoms of urethritis. ELISA and the immunoblot test were performed using M. penetrans lipid associated membrane proteins as antigen. By ELISA, we found a higher frequency (P < 0.05) of IgG against M. penetrans in HIV-1-infected and STD patients (25.5 and 17.3%) than in controls (1.2%), as well as a higher frequency of IgA (P < 0.05) (15.1 and 17.3% compared to 1.2%). For IgM, no differences were observed (P >/= 0.05) (3.8, 9.1, and 5. 8%, respectively). When the frequencies of IgG, IgM, and IgA antibodies of the HIV-1-infected patients were compared taking into account the CD4/CD8 cell ratios < 0.3 and >/= 0.3, no significant differences were observed between the two groups (13.3, 10, and 20%, compared to 20, 0, and 5%, respectively) (P > 0.05), possibly due to the low number of samples on which we could perform T-cell counts (53/106). The M. penetrans peptide of 38 kDa, considered immunodominant, was recognized in immunoblot by 51.8% of positive sera by ELISA for IgG, 50.0% for IgM, and 75% for IgA in the AIDS patients group, and by 47.4, 60.0, and 75.0%, respectively, in the sexually transmitted disease group. Cross-reactions in immunoblot for IgG were observed in sera from individuals infected with Mycoplasma pneumoniae and Mycoplasma hominis, and cross-reactions in immunoblot for IgA were observed in sera from individuals infected with M. hominis; all of them were ELISA negative to M. penetrans.     

High prevalence of Mycoplasma infections among European chronic fatigue syndrome patients. Examination of four Mycoplasma species in blood of chronic fatigue syndrome patients

Prevalence of Mycoplasma species infections in chronic fatigue syndrome (CFS) has been extensively reported in the scientific literature. However, all previous reports highlighted the presence of Mycoplasmas in American patients. In this prospective study, the presence of Mycoplasma fermentans, M. penetrans, M. pneumoniae and M. hominis in the blood of 261 European CFS patients and 36 healthy volunteers was examined using forensic polymerase chain reaction. One hundred and seventy-nine (68.6%) patients were infected by at least one species of Mycoplasma, compared to two out of 36 (5.6%) in the control sample (P<0.001). Among Mycoplasma-infected patients, M. hominis was the most frequently observed infection (n=96; 36.8% of the overall sample), followed by M. pneumoniae and M. fermentans infections (equal frequencies; n=67; 25.7%). M. penetrans infections were not found. Multiple mycoplasmal infections were detected in 45 patients (17.2%). Compared to American CFS patients (M. pneumoniae>M. hominis>M. penetrans), a slightly different pattern of mycoplasmal infections was found in European CFS patients (M. hominis>M. pneumoniae, M. fermentansz.Gt;M. penetrans).     

生殖支原体脂质相关膜蛋白激活核因子κB诱导人单核细胞表达前炎症细胞因子及凋亡

为了解生殖支原体(Mg)潜在的致病性及其脂质相关膜蛋白(LAMPs)诱导人单核细胞(THP-1)凋亡及表达前炎症细胞因子(CKs)的分子机制,用Mg提取的LAMPs刺激THP-1细胞,以ELISA法和RT-PCR方法分析CKs产生和其mRNA的表达。不同试实验组的细胞经AnnexinV联合PI染色后通过流式细胞仪检测细胞凋亡。采用EMSA方法检测LAMPs处理的THP-1细胞中核转录因子kappaB(NF-κB)的激活,并分析NF-κB抑制剂二硫代氨基甲酸吡咯烷(pyrrolidine dithiocoarbamate,PDTC)对LAMPs处理的THP-1细胞产生CKs的量和其mRNA表达及细胞凋亡的影响。LAMPs能以时间和剂量依赖方式刺激THP-1细胞产生TNF-α、IL-1β和IL-6,且能激活NF-κB诱导THP-1细胞表达CKs的mRNA及发生凋亡,PDTC能显著抑制CKs的mRNA表达水平和细胞凋亡。由于LAMPs能激活NF-κB诱导THP-1细胞表达CKs及产生细胞凋亡,因而可能是一个重要的致病因素。     

Electrophoretic and immunologic comparative analysis of Mycoplasma pneumoniae and Mycoplasma genitalium proteins

The Mycoplasma pneumoniae FH strain routinely used in our laboratory for over 25 years as antigen in serological tests, 2 reference M. pneumoniae strains from ATCC (29342 and M129) and 3 isolates of M. pneumoniae obtained in 1995 from pneumonia patients were compared by SDS-PAGE, complement fixation test (CFT) and by Western-immunoblotting against human and rabbit serum samples with high level of mycoplasmal antibodies. On SDS-PAGE all M. pneumoniae strains showed the same number of 23 polypeptides on the gel with identical molecular weights. The same strains on immunoblotting against human and rabbit serum samples showed six bands: 170, 89, 75, 55, 38 and 33 kDa with the strongest antibody staining in 170-(P1 protein) and 89-kDa bands. Because of its known antigenic relationships Mycoplasma genitalium was used for comparison. The pattern of M. genitalium proteins on SDS-PAGE was similar to pattern of M. pneumoniae but distinguishable. On immunoblotting six proteins of M. genitalium (135, 127, 110, 95, 75 and 45 kDa) reacted with human and rabbits immunoglobulins for M. pneumoniae antigens. Furthermore in complement fixation test both antigens, prepared from M. pneumoniae and M. genitalium, reacted as well with human and rabbit immunoglobulins for M. pneumoniae and with rabbit immunoglobulins for M. genitalium. These cross-reactions observed in serological techniques could give false positive results in routine diagnosis of M. pneumoniae infections. In such situations showing on immunoblott of presence in tested serum sample of antibodies to 170- and 89 kDa proteins could confirm M. pneumoniae infection.     

Detection of antibody to M protein of measles virus in patients with subacute sclerosing panencephalitis: a comparative study on immunoprecipitation

Consistent results have not been obtained yet on the presence of antibody to the M protein of measles virus in the sera of patients with subacute sclerosing panencephalitis (SSPE). We performed a comparative study on various immunoprecipitation systems which appeared in the literature and found that the difference in the composition of the solubilizing buffer produced a large variety of results on the immunoprecipitation. [35S]Methionine-labeled Vero cells infected with the Edmonston strain of measles virus were solubilized by 10 different buffers and reacted with hyperimmune rabbit serum to whole virus, monospecific antisera to H, NP, and M proteins of the virus, normal adults' sera, and the sera from 16 SSPE patients. The immune complex was absorbed by protein A and both solubilization and precipitation rates were compared with each viral protein. Although viral proteins were solubilized by all buffers, the solubilization rate varied considerably. M protein was solubilized and was not coprecipitated nonspecifically with any of the other viral proteins. Purified protein A conjugated to Sepharose was preferable to Staphylococcus aureus for absorption of the immune complex since the latter absorbed both viral and host proteins nonspecifically. The precipitation rates of the viral proteins also varied according to the buffers. Better solubilization of the viral proteins seemed to reduce their rate of precipitation for which the presence of SDS may be responsible, and the presence of the protease inhibitors may also affect the results of immunoprecipitation. Detection of M protein in the immunoprecipitates was largely influenced by the kind of buffer used: some buffers could detect it clearly, but others could not defect it at all. Among the solubilizing buffers tested, Saleh's buffer (Virology 93: 369-376 (1979)),, which contains 0.5% DOC and 0.5% Triton X-100, was most reliable for detection of the anti-M antibody in the rabbit serum, because it showed a high solubilization and high precipitation rates of viral proteins without nonspecific absorption by protein A or coprecipitation of M proteins with any of the other proteins. Using this buffer, we could definitely detect M proteins in the immunoprecipitates from the sera of all six healthy adults and 15 out of 16 patients with SSPE. It was found, however, that the amount of M proteins in SSPE patients was lower than that in healthy adults and varied considerably.     

用改进的ELISA法检测腺病毒抗原

用改进的ELISA法检测94份咽拭子标本中的腺病毒抗原,标本先接种人肾细胞,使病毒有一定程度的增殖,同时与传统的病毒分离作对比,结果表明,腺病毒分离阳性的18例,ELISA法检测全部阳性,76例分离阴性(盲传3代)者ELISA法也全都阴性,两者完全相符,ELISA法检测增殖24、48、72小时的细胞培养物腺病毒抗原的阳性率分别为16.7％,72.2％和83.3％,本法特异、可靠、判断客观、可用来检测腺病毒抗原。     

Monoclonal antibody to rat brain actin antigenically enhanced with HVJ (Sendai Virus) M protein

Summary Monoclonal antibody to rat brain actin was easily produced using HVJ (Sendai Virus) M protein to enhance the antigenicity of the actin. This monoclonal antibody was determined to be IgM with a kappa light chain. By immunoblot analysis the antibody was also shown to react with rat brain actin but not with HVJ M protein on nitrocellulose sheets. Utilizing the antibody, neuronal cytoplasm in the cerebral cortex, the anterior and posterior horns in the spinal cord, the spinal ganglion and astrocytes showed positive immunohistochemical staining by light microscopy. However, Purkinje cells showed variable staining, some staining intensely, while others were negative. All of neurons in specific anatomical locations showed always positive staining but variable intensities. Vascular walls were stained only faintly. By electron microscopy, neuronal cytoplasm showed diffuse positive staining. Other areas showed a positive reaction, including dendrites, the postsynaptic densities, and a few capillary endothelial cells and arterial smooth muscle cells. The results suggest that the HVJ M protein was effective for producing monoclonal antibody to brain actin, and that the antibody could be utilized for the immunohistochemical study of neuronal elements in both normal and pathological conditions.     

DNA repair after DNA fragmentation in mouse small intestinal epithelial cells

In our earlier work, we found that, in mice, i.p. injection of anti-CD3 monoclonal antibody activated intraepithelial lymphocytes (iIEL), leading to DNA fragmentation in villous epithelial cells of the duodenum and jejunum within 30 min. By 2 h after injection, nearly half of the enterocytes had detached from the villi, and DNA fragmentation could barely be detected in the remaining villous epithelium. We hypothesized that DNA had been repaired in enterocytes in which DNA fragmentation had previously been induced. In this study, enterocytes became negative for TUNEL staining at 60 min after anti-CD3 treatment, prior to detachment. The remaining villous epithelial cells, after DNA fragmentation and detachment, were found to be positive for 5-bromo-2-deoxyuridine labeling. To confirm whether fragmented DNA had been repaired in situ, we investigated the appearance and/or mobilization of DNA-repair-related proteins. Focus formation, a typical staining pattern of repair-related proteins including phosphorylated H2AX, phospo-ATM substrate, and Nbs1, was observed 30 min after anti-CD3 injection, with the kinetics virtually identical to that of DNA fragmentation. The co-localization of γ-H2AX and phospo-ATM substrate was also confirmed. The disappearance of a positive reaction for TUNEL staining in previously fragmented DNA, the appearance of representative DNA-repair-related proteins, the coincidence of the kinetics of DNA fragmentation and this appearance of DNA-repair-related proteins, and the co-localization of two of the repair-related proteins strongly indicated that enterocyte DNA could be repaired after it had been fragmented in vivo. Thus, DNA fragmentation per se may not necessarily be an immediate sign of cell death. This work was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (16590132 to T.M., 16390045 to T.I., and 20590181 to M.O.).     

Differential expression of ABH histo-blood group antigens and LAMPs in infantile hemangioma

Summary Although infantile hemangioma (IH) are the most common tumors of infancy, the mechanism of their proliferation and involution remains vague. Proliferation, differentiation and death of endothelial cells are the basic processes involved in their pathobiology. Here we hypothesize that the glycoconjugates ABH histo-blood group antigens (HBGA) and lysosome-associated membrane proteins (LAMPs) might be implied in both the differentiation and death of endothelial cells during vascular remodeling in IH. Proliferating and involuting IH were examined immunohistochemically for HGBA and LAMP expression together with vWF and CD31. Proliferative and apoptotic indices were determined. LAMPs were found in immature endothelium of proliferating IH. In involution an increased number of immunopositive cells stained with higher intensity was detected. The enhanced expression might be associated with augmented autophagy required for tissue remodeling during tumor involution. HBGA presented an opposite pattern of expression – they stained intensely the endothelium of mature capillaries, while the immature ones were positive for vWF. The presence of HBGA in endothelial cells of IH may be related to the differentiation process only, as well as to endothelial adhesion and angiogenesis. Novel evidence for differential expression of HBGA and LAMPs in proliferative and involutive phases of IH is presented.     

Monoclonal antibody to rat brain actin antigenically enhanced with HVJ (Sendai virus) M protein

Monoclonal antibody to rat brain actin was easily produced using HVJ (Sendai Virus) M protein to enhance the antigenicity of the actin. This monoclonal antibody was determined to be IgM with a kappa light chain. By immunoblot analysis the antibody was also shown to react with rat brain actin but not with HVJ M protein on nitrocellulose sheets. Utilizing the antibody, neuronal cytoplasm in the cerebral cortex, the anterior and posterior horns in the spinal cord, the spinal ganglion and astrocytes showed positive immunohistochemical staining by light microscopy. However, Purkinje cells showed variable staining, some staining intensely, while others were negative. All of neurons in specific anatomical locations showed always positive staining but variable intensities. Vascular walls were stained only faintly. By electron microscopy, neuronal cytoplasm showed diffuse positive staining. Other areas showed a positive reaction, including dendrites, the postsynaptic densities, and a few capillary endothelial cells and arterial smooth muscle cells. The results suggest that the HVJ M protein was effective for producing monoclonal antibody to brain actin, and that the antibody could be utilized for the immunohistochemical study of neuronal elements in both normal and pathological conditions.     

Cationization of protein antigens. V. Effect of the degree of cationization on patterns of immune responsiveness.

Preparations of bovine serum albumin (BSA) were cationized by substituting anionic side chain carboxyl groups with polycationic aminoethylamide groups. Different degrees of substitution were obtained by varying the reaction time. Mice immunized with partially cationized proteins produced early increased levels of antibody over those made by mice immunized with nBSA, followed by a period of decreased response before returning to a second period of enhanced and prolonged antibody synthesis. In contrast, fully substituted BSA gave rise to a significantly enhanced response which was delayed in its onset. Differences in isotype or in antibody specificity during the early and late periods of enhanced responsiveness could not be demonstrated. Cell transfer experiments showed that T cells harvested from mice immunized with the less cationized cBSA preparations could, in contrast to T cells from mice immunized with the fully cationized preparations, suppress antibody responses to both nBSA and cBSA in normal mice. These data are consistent with the possibility that the partially cationized proteins, in contrast to the fully cationized antigen, yield a unique pattern of responsiveness due to retention of determinants necessary for the induction of Ts while exhibiting the enhanced immunogenicity characteristic of cationized molecules.     

Sensitive enzyme immunoassay for the rapid diagnosis of influenza a virus infections in clinical specimens

Samples of nasopharyngeal secretion (NPS) from 100 infants and small children admitted for acute respiratory disease during the period from January to March 1989 were examined for the presence of influenza A virus. All samples were tested by enzyme immunoassay (EIA), fluorescent antibody (FA) technique and by isolation in cell culture 3–6 h after they were obtained from the patients. Of 24 influenza strains found by isolation, 21 were detected by EIA and 19 were FA⁺. In comparison with virus isolation, EIA gave the following values: sensitivity 88 %, specificity 100 %, positive prognostic value (PPV) 100 %, and negative prognostic value (NPV) 96 %. A rabbit anti-influenza-A serum (A-13) was used as catching antibody and a monoclonal anti-influenza-A pool against NP protein was used as detector antibody in EIA. A-13 gave bands corresponding to influenza A core proteins (NP and M1) in Western blot (WB) studies when different H3N2 strains were employed as antigens. A-13 gave only a band corresponding to the NP protein when H1N1 strains were examined by WB. The detection level by EIA for both H3N2 and H1N1 strains precipitated by polyethylene glycol from tissue culture maintenance medium was 1–2 ng.     

Immune response in subacute sclerosing panencephalitis: reduced antibody response to the matrix protein of measles virus.

Immune precipitation was used to study the humoral immune response of patients with subacute sclerosing panencephalitis (SSPE). Patients with SSPE have a progressive infection of the CNS by measles or a measles variant despite high serum antibody levels to measles virus as measured by standard serologic techniques. However, when the antibody response to individual measles virus proteins was measured, we found a striking reduction in the ability of sera from patients with SSPE to precipitate the matrix (M) protein as compared to the precipitation of the M protein by sera from normal adults who had natural measles infection in childhood, or by convalescent sera obtained 3 to 5 weeks after a naturally occurring measles infection. The decreased antibody response to the M protein in sera from patients with SSPE occurred despite a vigorous antibody response to the other viral proteins, suggesting a selective defect in the production of antibody to a single viral protein. The reduced anti-M antibody in sera from patients with SSPE was demonstrated whether immune precipitation was performed with wild-type measles virus or SSPE virus proteins. These results suggest that in SSPE only small amounts of the M protein are produced. This result may help explain how measles virus persists in the central nervous system of patients with SSPE.     

Rapid Diagnosis of Herpesvirus Hominis Infections in Superficial Lesions by Immunofluorescent Antibody Techniques

Scrapings from lesions of the skin and mucous membranes of 14 patients suspected of suffering from suspected Herpesvirus hominis infections were examined by both fluorescent antibody and routine isolation techniques. There was complete correlation between results by both methods in all 14 cases, 12 being positive. No positive fluorescence was obtained from scrapings of seven control patients with a variety of skin diseases. Thirteen patients with corneal lesions were similarly investigated. Of the 10 scrapings which showed positive fluorescence, nine were confirmed by virus isolation. It is suggested that as more antiviral agents become available the application of a fluorescent antibody technique for testing virus sensitivity in tissue culture could become a practical method.     

Conserved terminal organelle morphology and function in Mycoplasma penetrans and Mycoplasma iowae

Within the genus Mycoplasma are species whose cells have terminal organelles, polarized structures associated with cytadherence and gliding motility. Mycoplasma penetrans, found mostly in HIV-infected patients, and Mycoplasma iowae, an economically significant poultry pathogen, are members of the Mycoplasma muris phylogenetic cluster. Both species have terminal organelles that interact with host cells, yet the structures in these species, or any in the M. muris cluster, remain uncharacterized. Time-lapse microcinematography of two strains of M. penetrans, GTU-54-6A1 and HF-2, and two serovars of M. iowae, K and N, show that the terminal organelles of both species play a role in gliding motility, with differences in speed within and between the two species. The strains and serovars also differed in their hemadsorption abilities that positively correlated with differences in motility speeds. No morphological differences were observed between M. penetrans and M. iowae by scanning electron microscopy (SEM). SEM and light microscopy of M. penetrans and M. iowae showed the presence of membranous filaments connecting pairs of dividing cells. Breaking of this filament during cell division was observed for M. penetrans by microcinematography, and this suggests a role for motility during division. The Triton X-100-insoluble fractions of M. penetrans and M. iowae consisted of similar structures that were unique compared to those identified in other mycoplasma species. Like other polarized mycoplasmas, M. penetrans and M. iowae have terminal organelles with cytadherence and gliding functions. The difference in function and morphology of the terminal organelles suggests that mycoplasmas have evolved terminal organelles independently of one another.     

检测脊髓灰质炎IgM抗体捕捉法ELISA的建立及其在早期快速诊断中的应用

首次建立了用于脊髓灰质炎快速、分型诊断的血清IgM抗体捕捉法ELISA。检测了52例疑似病人,IgM阳性率为76.9％(40/52),而粪便病毒分离率仅为44.2％(23/52),前者的阳性检出率明显高于后者。做病毒分型诊断结果,与分离病毒中和试验定型的总符合率为92.86％(39/42)。检测601例来自全国各省的脊灰疑似病人血清,其阳性率波动于13％～92.3％,平均为61.1％。阳性率与收集血清的病日相关,发病0～3天收集者,阳性检出率为69.5％,4～25天者为64％,26～54天者为52％,55天以上者则未能检出。本检测方法需时1.5天,简便、敏感、特异,重复性良好,适用于脊灰的早期快速诊断。对其实用性进行了讨论。     

解脲脲原体脂质相关膜蛋白通过激活核因子κB调控诱导性一氧化氮合酶在小鼠巨噬细胞中的表达

为探讨解脲脲原体(Uu)的脂质相关膜蛋白(LAMPs)诱导小鼠巨噬细胞表达诱导性一氧化氮合酶(iNOS)的分子机制,从解脲脲原体提取的脂质相关膜蛋白,刺激小鼠巨噬细胞,以RT_PCR、Western blot等方法分析iNOS的表达及NO的产生;用细胞免疫化学、间接免疫荧光及Western blot等方法检测核因子κB(NF_κB)的激活,另外检测了NF_κB的特异性抑制剂二硫代氨基甲酸吡咯烷(PDTC)和蛋白酶抑制剂放线菌酮(CHX)对iNOS的表达及NF_κB激活的影响。结果表明,解脲脲原体的LAMPs通过激活NF_κB诱导小鼠巨噬细胞表达iNOS的mRNA和蛋白,且能以时间和剂量依赖方式刺激小鼠巨噬细胞产生NO,NF_κB的抑制剂PDTC或蛋白酶抑制剂放线菌酮(CHX),可抑制NF_κB的激活及iNOS的表达。由于解脲脲原体的脂质相关膜蛋白通过激活NF_κB诱导小鼠巨噬细胞表达iNOS和产生NO,因而可能是一个重要的致病因素。