Ultrastructural localization of HTLV-I gag proteins p19 and p24 by single and double immunogold labeling

We developed a post-embedding immunogold labeling procedure for the ultrastructural localization of the HTLV-I gag proteins p19 and p24 by the use of monoclonal antibodies (MAb). Both antigens were shown to withstand fixation with 1% glutaraldehyde. In addition, p19 antigenicity was found not to be affected by post-fixation with 1% osmium tetroxide. The choice of resin played a decisive role in the retention of antigenicity. P19 was preserved in Lowicryl K4M as well as in LR White, whereas p24 was preserved only in Lowicryl. Both p19 and p24 were found to be localized on the HTLV-I virions themselves, whereas no positive immunostaining could be observed on the infected cells. In Lowicryl-embedded samples, in which both antigens had been preserved, a double immunogold labeling procedure was performed that allowed the co-localization of p19 and p24 on the same section. In osmicated LR White-embedded samples the quality of ultrastructural preservation of HTLV-I virions was found to be comparable to results obtained with the traditional glutaraldehyde-osmium tetroxide-epoxy resin processing.     

HIV—1核蛋白p24在昆虫细胞中的表达

将完整的ＨＩＶ－１ ｐ２４基因克隆到杆状病毒转移质粒中,使用重组转移质粒与野生型杆状病毒ＤＮＡ共转染Ｓｆ９昆虫细胞,经筛选获得带有编码ｐ２４基因的重组杆状病毒。重组杆状病毒感染Ｓｆ９细胞后在细胞中表达了ＨＩＶ核蛋白ｐ２４。其重组蛋白的分子量为２４ｋＤ。此重组糖蛋白在免疫荧光,免疫印染和酶联免疫实验中都能被人ＨＩＶ－１阳性血清和单克隆抗体所识别。     

Immunodetection of low concentrations of ovalbumin in cryoprocessed quail oviduct cells by semi-automatic quantitative analysis

Summary— In a previous paper (Quintana et al, Biol Cell 72, (1991) 167–180) we reported the anti-ovalbumin antibody immunolabeling in the cytoplasm of tubular gland cells from cryofixed, cryosubstituted and acrylic-resin embedded quail oviduct. To confirm these preliminary visual observations, we have carried out a semi-automatic quantitative study of immunogold labeling. The quantitative results confirm the previous data. In addition, we found a significantly higher immunolabeling with low temperature Lowicryl K11M embedding procedure as compared with high temperature LR White embedding.     

Post embedding immunoelectron microscopy of human breast cancer: a comparison of three acrylic resins

Summary The suitability of three acrylic resins for the immunoelectron microscopical localization of cell surface and cytoskeletal antigens in surgically excised, immersion fixed human breast cancer, using an immunogold system, has been assessed.Good localization of milk fat globule membrane was achieved with LR White, LR Gold and Lowicryl K11M, although the embedding schedule for LR White had to be modified. The best results were achieved with Lowicryl K11M. Only scanty labelling of actin and cytokeratin was seen in LR White embedded tissue, whereas there was clear localization in LR Gold and Lowicryl K11M embedded samples. Tubulin and -actinin was detected at low level in tissues in the low temperature embedding resins, but not in LR White embedded samples. The morphology of the latter was poorer, and there was greater variability in ultrastructure and labelling.Of the two low temperature embedding resins, Lowicryl K11M gave slightly better results. However, the advantages could be outweighed by the problem incurred in achieving the low temperatures, and by poorer handling properties than LR Gold.     

Signaling through Toll-like receptors triggers HIV-1 replication in latently infected mast cells

Evidence that human progenitor mast cells are susceptible to infection with CCR5-tropic strains of HIV-1 and that circulating HIV-1-infected FcepsilonRIalpha(+) cells with a similar progenitor phenotype have been isolated from AIDS patients has led to speculation that mast cells may serve as a potential reservoir for infectious HIV-1. In this study, progenitor mast cells, developed in vitro from CD34(+) cord blood stem cells, were experimentally infected with the CCR5-tropic strain HIV-1Bal after 28 days in culture as they reached their HIV-1-susceptible progenitor stage. HIV-1 p24 Ag levels were readily detectable by day 7 postinfection (PI), peaked at 2-3 wk PI as mature (tryptase/chymase-positive) HIV-1 infection-resistant mast cells emerged, and then steadily declined to below detectable limits by 10 wk PI, at which point integrated HIV-1 proviral DNA was confirmed by PCR quantitation in ( approximately 34% of) latently infected mast cells. Stimulation by ligands for Toll-like receptor (TLR) 2, TLR4, or TLR9 significantly enhanced viral replication in a dose- and time-dependent manner in both HIV-1-infected progenitor and latently infected mature mast cells, without promoting degranulation, apoptosis, cellular proliferation, or dysregulation of TLR agonist-induced cytokine production in infected mast cells. Limiting dilution analysis of TLR activated, latently infected mature mast cells indicated that one in four was capable of establishing productive infections in A301 sentinel cells. Taken together, these results indicate that mast cells may serve both as a viral reservoir and as a model for studying mechanisms of postintegration latency in HIV infection.     

Immunocytochemistry of contractile and cytoskeletal proteins in smooth muscle: Lowicryl,LR White,and polyvinylalcohol compared

Summary Three embedding media have been compared with respect to post-embedding immunolabeling of contractile and cytoskeletal antigens in aldehyde-fixed smooth muscle tissue: the methacrylate derivates lowicryl K4M (cured at –35 or 60°C) and LR White (cured at 0 or 60°C) and the water soluble resin, polyvinylalcohol (dried at 60°C). Measurements of intensity of labeling of ultrathin sections in the fluorescence microscope showed that five antigens (actin, myosin light chain, tropomyosin, filamin and vinculin) reacted more or less equally with their respective antibodies in all the embedding media, including those cured at 60°C. One antibody (anti-light meromyosin) reacted well only with polyvinylalcohol-embedded tissue. In contrast to the relative invariance of antibody reactivity between media clear differences in the preservation of ultrastructural integrity were observed. Embedding in polyvinylalcohol (dried at 60°C) and in Lowicryl (cured at –35°C) resulted in superior preservation as compared to Lowicryl or LR White cured at 60°C. Examples of uitrastructural immunocytochemistry with the antibodies against filamin and myosin light chain, using the immunogold staining procedure are presented: the sites of localization by these antibodies were the same with all the media tried. The relative merits of the different methods are discussed.Abbreviations EGTA Ethyleneglycol-bis(-amino ethyl ether)N,N,N,N-tetra acetic acid - PIPES 1,4-Piperazinediethanesulfonic acid - LR London Resin     

Human immunodeficiency virus type 1 neutralization measured by flow cytometric quantitation of single-round infection of primary human T cells

There is currently intensive research on the design of novel human immunodeficiency virus type 1 (HIV-1) vaccine immunogens that can elicit potent neutralizing antibodies. A prerequisite for comparing and optimizing these strategies is the ability to precisely measure neutralizing antibody responses. To this end, we sought to develop an assay that directly quantifies single-round HIV-1 infection of peripheral blood mononuclear cells (PBMC). Initial experiments demonstrated that essentially all productively infected PBMC could be identified by flow cytometric detection of intracellular p24 antigen (p24-Ag). After infection of PBMC with HIV-1, p24(+) lymphocytes could be distinguished beginning 1 day postinfection, and the majority of CD8(-) T cells were p24-Ag positive by 3 to 4 days postinfection. To directly quantify first-round infection, we included a protease inhibitor in PBMC cultures. The resulting 2-day assay was highly sensitive and specific for the detection of HIV-1-infected PBMC. Serial dilutions of virus stocks demonstrated that the number of target cells infected was directly related to the amount of infectious virus input into the assay. In neutralization assays, the flow cytometric enumeration of first-round infection of PBMC provided quantitative data on the number of target cells infected and on the inactivation of infectious virus due to reaction with antibody. We also used this single-round assay to compare the percentage of cells expressing p24-Ag to the number of copies of HIV-1 gag per 100 PBMC. The precision and reproducibility of this assay will facilitate the measurement of HIV-1 neutralization, particularly incrementally improved neutralizing antibody responses generated by new candidate vaccines.     

Influence of embedding media in prolactin labelling with immunogold techniques

Summary Immunogold labelling of prolactin in three different embedding media was compared. The polymeric prolactin in secretory granules was labelled in the three media, however, acrylic monomers (Lowicryl K4M and LR White) provided a more intense labelling with higher dilutions of the primary antibody, compared to the labelling in the epoxy resin (araldite). An intense labelling of monomeric prolactin in Golgi complex was detected only in acrylic embedments, and the labelling on the rough endoplasmic reticulum was significant only in LR White embedded tissues.     

Demonstration of anionic sites on the luminal and abluminal fronts of endothelial cells with poly-L-lysine-gold complex

An attempt was made to demonstrate the anionic sites on the endothelial cell (EC) surfaces of mouse brain micro-blood vessels (MBVs) after embedding of tissue samples in hydrophilic media: Lowicryl K4M, LR White, and Polyamph-10. As a cationic probe, poly-L-lysine-gold complex (PLG), prepared according to the procedure of Skutelsky and Roth (J Histochem Cytochem 34:693, 1986), was used. In ultra-thin sections of brain samples embedded in Lowicryl K4M and LR White, the anionic sites were demonstrated in the entire cross-section of the vessel wall. After embedding in Polyamph-10, however, the anionic sites could not be detected. Brain capillaries, representing blood-brain barrier type MBVs, showed polar distribution of anionic sites, evidenced by more intense labeling of luminal than of abluminal plasma membrane of the EC. Some differences in labeling of ECs and of basement membrane in arterioles and venules were also noted. The use of cationic gold and the ultra-thin sections of tissue samples embedded in hydrophilic media (Lowicryl K4M and LR White) seems to be a promising new method for detection of anionic constituents located on both luminal and abluminal surfaces of the EC, in the basement membrane, and in other components of the vessel wall.     

Ultrastructural and nuclear antigen preservation after high-pressure freezing/freeze-substitution and low-temperature LR White embedding of HeLa cells

A protocol for high-pressure freezing and LR White embedding of mammalian cells suitable for fine ultrastructural studies in combination with immunogold labelling is presented. HeLa S3 cells enclosed in low-temperature gelling agarose were high-pressure frozen, freeze-substituted in acetone, and embedded in LR White at 0°C. The morphology of such cells and the preservation of nuclear antigens were excellent in comparison with chemically fixed cells embedded in the same resin. The immunolabelling signal for different nuclear antigens was 4-to-13 times higher in high-pressure frozen than in chemically fixed cells. We conclude that one can successfully use high-pressure freezing/freeze-substitution and LR White embedding as an alternative of Lowicryl resins.     

Applicability of using acrylic resins in post-embedding ultrastructural immunolabelling of human neutrophil granule proteins

Summary In this study, quantitative assessments were carried out, (1) by light microscopy during tissue preparation for electron microscopy and (2) by electron microscopy after on-grid immunogold staining, to determine the suitability of using LR White and Lowicryl K4M thin sections to identify lactoferrin and elastase in the granules of human neutrophil leucocytes. Quantitative assessment of the effect of fixation, dehydration and embedding on the preservation of antigenicity during tissue preparation for electron microscopy, using light microscopic peroxidase anti-peroxidase immunocytochemistry, enabled the selection of preparation conditions that adequately preserved both antigenicity and ultrastructure. OsO₄ post-fixation, following primary aldehyde fixation, improved the retention of antigenicity during dehydration and embedding and the preservation of fine structure. Partial rather than complete dehydration retained more of the antigenicity. The efficiency, sensitivity and resolution of immunolabelling and the ultrastructure and quality of sections achieved after embedding in LR White were superior to those obtained after embedding in Lowicryl K4M. Consequently room temperature embedding in LR White following double fixation and partial dehydration is a better and more reliable preparation technique than low-temperature embedding in Lowicryl K4M following single fixation and partial dehydration for localizing lactoferrin and elastase to the specific and primary granules respectively in human neutrophilic granulocytes by the on-grid immunogold staining method.     

Interleukin 1 induces HIV-1 expression in chronically infected U1 cells: blockade by interleukin 1 receptor antagonist and tumor necrosis factor binding protein type 1.

BACKGROUND: Cytokines and cytokine antagonists modulate human immunodeficiency virus (HIV) replication in vitro and may be involved in HIV disease pathogenesis. An understanding of these cytokine networks may suggest novel treatment strategies for HIV-seropositive persons. MATERIALS AND METHODS: U1 cells, a chronically infected promonocytic cell line, were stimulated with interleukin 1 alpha (IL-1 alpha), IL-1 beta or tumor necrosis factor (TNF) for 24 hr. The effects of these cytokines, and of anti-IL-1 receptor type 1 and type 2 (IL-1RI and II) antibody, IL-1 receptor antagonist (IL-1Ra), and recombinant human TNF binding protein type 1 (rhTBP-1, a form of TNF receptor p55), on HIV-1 replication, as measured by ELISA for HIV-1 p24 antigen, were determined. The effects of IL-1 and IL-1Ra on nuclear factor-kappa B (NF-kappa B) DNA binding activity, as measured by electrophoretic mobility shift assays, were also determined. RESULTS: IL-1 alpha and IL-1 beta increased p24 antigen production in a concentration-dependent manner. IL-1Ra completely, and rhTBP-1 partially, suppressed IL-1-induced p24 antigen production. IL-1 increased NF-kappa B DNA binding activity and IL-1Ra blocked this effect. Since IL-1Ra blocks IL-1 from binding to both the IL-1RI and Il-1RII, monoclonal antibodies directed against each receptor were used to ascertain which IL-1R mediates IL-1-induced HIV-1 expression. Antibody to the IL-1RI reduced IL-1-induced p24 antigen production. Although anti-IL-1RII antibody blocked the binding of 125IL-1-1 alpha to U1 cells by 99%, this antibody did not affect IL-1-induced p24 antigen production. IL-1 beta enhanced TNF alpha-induced HIV expression when added before or simultaneously with TNF alpha. CONCLUSIONS: IL-1 induces HIV-1 expression (via the IL-1RI) and NF-kappa B activity in U1 cells. These effects are blocked by IL-1Ra and partially mediated by TNF. IL-1 enhances TNF alpha-induced HIV replication in U1 cells.     

Applications of plastic embedding to electron microscopic immunocytochemistry and in situ hybridization in observations of production and secretion of peptide hormones.

Plastic embedding has been used to localize various antigens in conjunction with immunohistochemistry. Peptide hormones have been among the antigens that have been studied extensively. Recent application of water-soluble plastics such as LR White and Lowicryl has extended the ranges of detectable antigens and enabled the observation of antigen-antigen or mRNA-antigen combinations. This review article deals with technical aspects, procedures, and applications in endocrine cells.     

A monoclonal antibody (12G5) directed against CXCR-4 inhibits infection with the dual-tropic human immunodeficiency virus type 1 isolate HIV-1(89.6) but not the T-tropic isolate HIV-1(HxB).

We used a monoclonal antibody (12G5) directed against an extracellular domain of CXCR-4 to investigate the role of this receptor in infection of immortalized lymphoid cell lines, peripheral blood mononuclear cells (PBMCs), and primary brain microglia with a dual-tropic strain of human immunodeficiency virus (HIV-1(89.6)) and a T-tropic strain (HIV-1(IIIB)). Addition of antibody 12G5 to cells prior to and during infection with HIV-1(89.6) inhibited p24 production 100- to 10,000-fold in CEMx174 and 174-CD4 cells and about 10-fold in PBMC cultures but had no activity against infection of either monocyte-derived macrophages or brain microglia. In contrast, 12G5 had little or no effect on infection of CEMx174 cells with HIV-1(IIIB) or HIV-1(HxB). To identify the region of the HIV-1(89.6) envelope that confers sensitivity to 12G5, we used chimeric molecular clones. Chimeras containing the V3 loop region of HIV-1(89.6) were inhibited by 12G5 to the same degree as wild-type HIV-1(89.6) whereas replication of those viruses containing the V3 loop of HIV-1(HxB) was not inhibited by the antibody. A similar pattern was seen in infections of a U87 glioblastoma line that coexpresses CD4 and CXCR-4. Antibody 12G5 was also able to block fusion between HeLa-CD4 cells and CEMx174 cells chronically infected with HIV-1(89.6) but had no effect on fusion mediated by cells chronically infected with HIV-1(IIIB). Taken together, these results suggest that different strains of HIV-1 may interact with different sites on CXCR-4 or may have different binding affinities for the coreceptor.     

Post-embedding methods for immunolocalization of elastin and related components in tissues

Elastic tissue is composed of amorphous-appearing elastin and 12-nm diameter microfibrils, one component of which has recently been isolated and characterized as the 31 KD microfibril-associated glycoprotein MAGP. Monospecific antibodies to each of these components have been developed in this laboratory. The parameters that determine optimal localization of colloidal gold probes for post-embedding immunolabeling of elastic tissue components have been systematically studied in a variety of normal and developing tissues in mammals and birds. Protein A-gold probes stabilized with dextran have been shown to provide complexes that remain stable after more than 2 years. Conditions have been defined that permit precise localization within the extracellular matrix of antibodies to MAGP and to elastin, singly and together. Best results were obtained with acrylic resins (Lowicryl K4M or LR White). Fixation in glutaraldehyde or other aldehydic fixatives, with or without osmium, did not affect the immunostaining of elastic tissue with affinity-purified antibodies to tropoelastin, or to anti-[alpha-elastin] or anti-[alkali-insoluble elastin]. Immunostaining with the anti-MAGP antibody was less robust and was possible in tissues which had been fixed only lightly before embedding in Lowicryl K4M or LR White. This staining was enhanced by metaperiodate oxidation of the sections as well as by reduction of the tissues with sodium borohydride en bloc, followed by hyaluronidase digestion of the sections. The effects on immunostaining of a range of enzyme digestions have also been examined. Conditions have thus been defined that make possible detailed study of the relationship between elastic tissue, elastin-associated microfibrils, and other microfibrillar structures in normal and abnormal tissues during development and aging.     

HIV-1 Gag pl7-p24在痘苗病毒中的表达及性质研究

研究了重组痘苗病毒表达的ＨＩＶ－１核心蛋白（Ｇａｇ）ｐ１７－ｐ２４蛋白的些生物学及免疫学特点。间接免疫荧光、Ｄｏｔ 及ＬＩＳＡ及Ｗｅｓｔｅｒｎ ｂｌｏｔ结果表明,构建的两株重组病毒分别表达了ＨＩＶ－１Ｇａｐ ｐ２４及ｐ１７－ｐ２４融合蛋白。电镜观察证实,Ｇａｇ ｐ２４及ｐ１７－２４重组蛋白均可形成病毒样粒子。重组病毒可诱导小鼠产生抗ＨＩＶ－１Ｇａｐ ｐ２４抗体。重组病毒感染ＢＨＫ２１细胞后,可见由     

Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells.