Detection and identification ofMycobacterium avium in the blood of AIDS patients by the polymerase chain reaction

One hundred fifty-three blood samples from patients positive for the human immunodeficiency virus (HIV) were analyzed by polymerase chain reaction (PCR) to detect the presence ofMycobacterium avium. Samples were collected from patients who also had blood cultures performed by a radiometric method. Blood samples were centrifuged on a Ficoll-Hypaque gradient to purify peripheral blood mononuclear cells. The purified cells were washed and incubated with a resin, boiled to release mycobacterial DNA, and then amplified. Polymerase chain reaction products were detected by a nonisotopic method. A 123 base-pair (bp) insertion sequence, namely IS6110, fromMycobacterium tuberculosis complex was also included in the reaction as an internal control ofTaq polymerase activity to exclude the presence of enzyme inhibitors. This IS6110 fragment can be distinguished from the 383 bp target product on ethidium bromide-stained agarose gel and may also be used in a colorimetric assay. Such results were compared with the results of culture and indicated that the assay is as sensitive as bacteriological methods, though faster.     

Monoclonal antibodies against transferrin. Precipitating mixtures and lack of inter-species cross-reactivity

Five stable hybridoma lines were prepared using the myeloma cell line P3-X63-Ag.653 and spleen cells of mice hyperimmunized by pig transferrin. All hybridomas grew well in mouse peritoneal cavity and produced antibodies of the IgG₁ subclass. Antibody preparations obtained from ascitic fluids tested for their capacity of antigen precipitation. No precipitation was obtained with single antibodies and with pairs of antibodies. Three out of 10 possible triads gave clear and sharp precipitation zones and rings in immunodiffusion tests performed in agar gel. All 5 antibodies were shown by quantitative enzyme-immunoassay to be specific for pig transferrin: no cross-reaction was obtained with mouse, human, horse and sheep transferrins.     

Biochemical and biophysical analysis of heptad repeat regions from the fusion protein of Menangle virus,a newly emergent paramyxovirus

E. coli in vitro expression system. The GST-removed purified 2-Helix protein could form a stable trimer in vitro judging by gel-filtration and chemical cross-linking. CD spectra showed that the 2-Helix protein had a high percentage of α-helix and was very thermo-stable. Crystals of the 2-Helix protein preparations have been obtained in many conditions with hanging-drop diffusion method. These results indicated that Menangle virus has the common features of the fusion protein for other paramyxoviruses and should adopt a similar fusion mechanism to other members. As the HR regions of Menangle virus F protein could form stable six-helix bundle coiled coil structure, they should be used as drug target for the design of fusion inhibitors, as successfully used for other parmyxoviruses. This is especially relevant to such a newly emergent virus with zoonotic potentials. Received January 23, 2003; accepted February 28, 2003 Published online April 28, 2003     

Defective Treg response in acute kidney injury was caused by a reduction in TIM-3+ Treg cells

Background: Despite years of research, the treatment of acute kidney injury (AKI) remains a significant challenge. Animal studies presented causal links between elevated regulatory T cell (Treg) response and better prognosis in AKI. Previous studies in mice and humans showed that TIM-3+ Treg cells were more potent than TIM-3- Treg cells. In this study, we investigated the role of TIM-3 in Treg in AKI patients.

Methods: Peripheral blood from AKI patients and healthy controls were gathered, and TIM-3+ Treg subset was examined.

Results: Compared to healthy controls, the AKI patients presented a significant upregulation in the frequency of circulating CD4+CD25+ T cells; however, the majority of this increase was from the CD4+CD25+TIM-3- subset, and the frequency of CD4+CD25+TIM-3+ T cells was downregulated in AKI patients. In both healthy controls and AKI patients, the CD4+CD25+TIM-3+ T cells expressed higher levels of Foxp3, and were more potent at expressing LFA-1, LAG-3, CTLA-4, IL-10 and TGF-β. In addition, the CD4+CD25+TIM-3+ T cells from both healthy controls and AKI patients presented higher capacity to suppress CD4+CD25- T cell proliferation than the CD4+CD25+TIM-3- T cells. Interestingly, the total CD4+CD25+ T cells from AKI patients presented significantly lower inhibitory capacity than those from healthy controls, indicating that the low frequency of CD4+CD25+TIM-3+ T cells was restricting the efficacy of the Treg responses in AKI patients.

Conclusions: We demonstrated that TIM-3 downregulation impaired the function of Treg cells in AKI. The therapeutic potential of CD4+CD25+TIM-3+ T cells in AKI should be investigated in future studies.     

Heterogeneity of HLA-A2 in Asia-Oceanic populations

HLA-A2 is one of the most common yet most diversified HLA antigens with 17 subtypes so far identified at the molecular level. A2 subtyping may have significant impact on clinical medicines. We developed a PCR/SSO-based comprehensive typing protocol for HLA-A and investigated the distribution of A2 alleles in regional ethnic groups. A2 was detected with high frequencies in most study populations. A total of 480 A2+ samples were identified and subtyped. The gene frequencies of A2 ranged from 34% in Chinese, 29% in Australian Caucasoids, 21% in Polynesians, 14% in Javanese and 13% in Australian Aborigines. However, in Melanesians and Micronesians A2 was absent. Six A2 alleles were found in the present experiments including A^*0201, 0203, 0205, 0206, 0207 and 0210. In Aborigines all the A2+ donors were typed as 0201. In Caucasoids A^*0201 accounted for 95% of A2+ samples though other three subtypes A^*0203, 0205 and 0207 were also detected. Extraordinary A2 heterogeneity was observed in Asia-Pacific populations where A^*0201 has become a minority. In Chinese all the six A2 alleles were discovered with A^*0201, 0203, 0206 and 0207 as the four major ones. In Javanese A2 was equally divided into A^*0201, 0203 and 0206 while in Polynesians A2 is overwhelmingly dominated by the oriental A^*0206 (71%). Our study also showed that comprehensive DNA matching for A2 would eliminate most A mismatches in the unrelated-donor transplantation in study populations.     

人肿瘤细胞与正常细胞内微管的PAP免疫酶细胞化学观察

我们利用兔抗微管蛋白抗体和兔抗辣根过氧化物酶(HRP)抗血清制备的HRP—抗HRP(PAP)复合物,建立了微管的PAP免疫酶细胞化学方法。应用此法观察到人食管癌ECa 109、胃癌SGC 7901,乳腺癌MCF 7和成骨肉瘤OS 732细胞间期胞质微管减少或消失,只有大量弥散分布的微管蛋白棕色反应产物,在微管组织中心(MTOC)附近十分密集,而正常成纤维细胞和胎儿胃粘膜上皮细胞间期,都有发达的胞质微管结构(CMTC)。在分裂期,这些肿瘤细胞都显示纺锤体微管,与正常细胞比较无明显差异。本研究应用PAP方法进一步证明,以前用免疫荧光细胞化学方法观察到的人肿瘤细胞间期胞质微管缺陷的特征。除去低温(4℃)或秋水仙酰胺处理后,解聚的CMTC又可恢复,表明本方法与免疫荧光染色法,同样具有很高的特异性。本工作在细胞固定及免疫反应的某些步骤上有所改进。     

Peripheral nerve regeneration by microbraided poly(L-lactide-co-glycolide) biodegradable polymer fibers

Tiny tubes with fiber architecture were developed by a novel method of fabrication upon introducing some modification to the microbraiding technique, to function as nerve guide conduit and the feasibility of in vivo nerve regeneration was investigated through several of these conduits. Poly(L-lactide-co-glycolide) (10:90) polymer fibers being biocompatible and biodegradable were used for the fabrication of the conduits. The microbraided nerve guide conduits (MNGCs) were characterized using scanning electron microscopy to study the surface morphology and fiber arrangement. Degradation tests were performed and the micrographs of the conduit showed that the degradation of the conduit is by fiber breakage indicating bulk hydrolysis of the polymer. Biological performances of the conduits were examined in the rat sciatic nerve model with a 12-mm gap. After implantation of the MNGC to the right sciatic nerve of the rat, there was no inflammatory response. One week after implantation, a thin tissue capsule was formed on the outer surface of the conduit, indicating good biological response of the conduit. Fibrin matrix cable formation was seen inside the MNGC after 1 week implantation. One month after implantation, 9 of 10 rats showed successful nerve regeneration. None of the implanted tubes showed tube breakage. The MNGCs were flexible, permeable, and showed no swelling apart from its other advantages. Thus, these new poly(L-lactide-co-glycolide) microbraided conduits can be effective aids for nerve regeneration and repair and may lead to clinical applications.     

Autoimmunity to Polymorphonuclears: Functional Consequences of the Binding of Antibodies to Membrane and Cytoplasmic Target Antigens of Polymorphonuclear Leukocytes

Antineutrophil autoantibodies reacting with cytoplasmic antigens are associated with various types of vasculitides, whereas antibodies reacting with neutrophil membrane antigens are mostly related to autoimmune neutropenias. The aim of this study was the investigation of the effect of monoclonal antibodies (MoAbs) reacting with surface and cytoplasmic antigens of polymorphonuclear leukocytes (PMN) known to be targets for autoantibodies in human diseases. Blood of healthy volunteers was tested for several phagocytic functions in the presence of MoAbs against surface (CD16, GD11b, CD18, NB1) and cytoplasmic (proteinase 3; PR3) molecules. Candidacidal activity was significantly inhibited in the presence of all MoAbs but isotypic control. Phagocytic activity was inhibited by anti-CD11b and/or anti-CD18 MoAbs. Zymosan-induced chemiluminescence was reduced by MoAbs anti-CD16, CD18, and NB1, enhanced by anti-PR3 MoAb, and less enhanced by anti-CD11b. In conclusion, antimembrane antibodies diminished phagocytic functions at multiple steps; in contrast, anticytoplasmic MoAb promoted activation of oxidative burst in addition to impairment of microbicidal activity. This fact may be related to different pathogenic aspects of diseases associated with antimembrane and anticytoplasmic antibodies.