Induction of Hepatitis B Antibody in Experimental Animals by Immunization with A-2 Plaque Virus

A sero-conversion for hepatitis B antibody has resulted from immunization of patas monkeys, guinea pigs, and rabbits with the A-2 plaque virus. This agent was isolated in tissue culture from patient sera that were positive for hepatitis B antigen. The immune response was assayed with techniques of direct and indirect counter-immunoelectrophoresis, and immune electron microscopy.     

Ultrasensitive DNA hybridization biosensor based on polyaniline

Ultrasensitive DNA hybridization biosensor based on polyaniline (PANI) electrochemically deposited onto Pt disc electrode has been fabricated using biotin-avidin as indirect coupling agent to immobilize single-stranded 5'-biotin end-labeled polydeoxycytidine (BdC) probes and 5'-biotin end-labeled 35 base-long oligonucleotide probe (BdE) to detect complementary target, using both direct electrochemical oxidation of guanine and redox electroactive indicator methylene blue (MB), respectively. These polyaniline-based disc electrodes have been characterized using differential pulse voltammetry (DPV), Fourier transform infrared spectroscopy (FT-IR), impedance measurements and scanning electron microscopy (SEM) techniques, respectively. Compared to direct electrochemical oxidation of guanine, hybridization detection using MB results in the enhanced detection limit by about 100 times. These DNA immobilized PANI electrodes have hybridization response time of about 60 s.     

Negative staining in the detection of viruses in clinical specimens

Viruses have unique morphology and are therefore good candidates for negative staining. Negative staining with phosphotungstic acid (PTA) or uranyl acetate has facilitated the detection of many viruses in clinical specimens. Enhancement procedures have included the use of centrifugation and agar diffusion for concentrating virus particles, the use of solid phase capture reagents to trap virus particles and the use of secondary antibodies and electron dense markers to help visualize them. Techniques currently in use and employing negative staining include direct EM, immune electron microscopy (IEM), solid phase immune electron microscopy (SPIEM), colloidal gold-labeled protein A (PAG), solid phase IEM employing a second decorator antibody (SPIEMDAT), and solid phase IEM using colloided gold-labeled secondary antibodies (SPEIMDAGT). IEM methods assist with the detection of small viruses or viruses present in low numbers while PAG offers increased sensitivity over direct EM and IEM. In our experience the serum-in-agar (SIA) method is the most sensitive of the PAG IEM techniques for detection of rotavirus particles in clinical specimens. SPIEMDAT enhances the detection of small viruses which are often missed by other techniques due to background staining in specimens. SPEIMDAGT employing colloidal gold-labeled secondary antibody has increased sensitivity and offers the advantage of detecting viral antigen when whole virus particles are not visible. IEM techniques have recently been used for typing viruses using either monospecific antisera or monoclonal antibodies and colloidal gold-labeled secondary antibody.     

Use of protein A in the serum-in-agar diffusion method in immune electron microscopy for detection of virus particles in cell culture

A modified technique using protein A in the serum-in-agar (SIA) method for immune electron microscopy (IEM) was presented. Grids coated with staphylococcal protein A were floated on samples mounted on agar containing 2% antiserum and incubated at 37 C, for 60 min. After washing and staining, the grids were observed in an electron microscope. The effects of protein A on virus detection were evaluated using poliovirus and bovine rotavirus infected cell culture fluids. The results showed that the technique using protein A (PA-SIA) had at least 10-fold higher sensitivity for virus detection than the original SIA. The optimal concentration of protein A was 1 to 10 micrograms/ml for coating the grids to trap virus particles. The PA-SIA method was also compared with immunosorbent electron microscopy (ISEM). The former showed higher or at least the same sensitivity and some advantages in detecting antigen-antibody reaction than the latter method. These results indicate that our PA-SIA method may be superior to other IEM techniques presented previously for the detection and identification of viruses.     

New trends in fluorescence in situ hybridization for identification and functional analyses of microbes

Fluorescence in situ hybridization (FISH) has become an indispensable tool for rapid and direct single-cell identification of microbes by detecting signature regions in their rRNA molecules. Recent advances in this field include new web-based tools for assisting probe design and optimization of experimental conditions, easy-to-implement signal amplification strategies, innovative multiplexing approaches, and the combination of FISH with transmission electron microscopy or extracellular staining techniques. Further emerging developments focus on sorting FISH-identified cells for subsequent single-cell genomics and on the direct detection of specific genes within single microbial cells by advanced FISH techniques employing various strategies for massive signal amplification.     

Microstructural analysis of bile: relevance to cholesterol gallstone pathogenesis

The study of physical-chemical factors and pathways leading to cholesterol crystallization in bile has important clinical relevance. The major processes in cholesterol gallstone formation can be subdivided into nucleation, formation and precipitation of solid crystals (crystallization), crystal growth, crystal agglomeration and stone growth. A clear understanding of the microstructural events occurring during the earliest stages of these processes in bile is crucial for the identification of factors possibly delaying or preventing precipitation of cholesterol crystals and, therefore, gallstone formation in bile. Detection and characterization of microstructures in native and model biles can be achieved by both direct and indirect techniques. Direct imaging techniques provide more readily interpretable information, but sample preparation problems, particularly for electron microscopy, are a source of artifacts. Moreover, microscopic techniques provide only qualitative data without the possibility to quantitate or to analyse the composition of microstructures. Several indirect techniques have been used to obtain additional microstructural information about nucleating bile. These techniques have the disadvantage of often being model dependent in addition to constraints specific for each method. The systematic, judicious use of a combination of complementary direct and indirect techniques have led to a comprehensive understanding of the various microstructural processes and interactions occurring during bile secretion, flow in the biliary tract and storage in the gallbladder. This forms the basis for our current understanding of cholesterol nucleation, crystallization and gallstone formation.     

Direct and Indirect Bioleaching of Cobalt from Low Grade Laterite and Pyritic Ores by Aspergillus niger

Abstract

The bioleaching efficiency and mechanism of recovery of cobalt (Co) and nickel from laterites and pyritic ores by Aspergillus niger were investigated. Recoveries of Co from laterites and pyritic ores by direct bioleaching were 65.9?±?1.8% and 4.9?±?2.7%, respectively, while 30.9?±?0.6% and 10.9?±?6.2% recovery of Ni were obtained from laterites and pyritic ores, respectively. Recovery of Co via indirect bioleaching in the absence of the fungal biomass from laterite was significantly lower when compared with Co released by direct bioleaching. In the latter, hyphal penetration and colonization of the laterites were clearly observed by scanning electron microscopy (SEM). X-ray powder diffraction (XRPD) analysis of mineral phases before and after bioleaching indicated that cobalt-bearing goethite was the main phase bioleached in the laterites. No significant difference was found between Co recoveries from synthesized cobalt-bearing goethite by both direct and indirect bioleaching. Therefore, we propose that two processes are involved in bioleaching from laterites: (1) cobalt-bearing goethite was exposed via direct interactions between the fungus and the minerals and (2) cobalt-bearing goethite was dissolved by released metabolites of A. niger, such as organic acids. An incongruent pattern of Co and Fe bioleaching from the laterites was also a feature of the metal recovery process.     

Endocytosis and intracellular localisation of type 1 ribosome-inactivating protein saporin-s6

Saporin-S6 is a single-chain ribosome-inactivating protein (RIP) that has low toxicity in cells and animals. When the protein is bound to a carrier that facilitates cellular uptake, the protein becomes highly and selectively toxic to the cellular target of the carrier. Thus, saporin-S6 is one of the most widely used RIPs in the preparation of immunoconjugates for anti-cancer therapy. The endocytosis of saporin-S6 by the neoplastic HeLa cells and the subsequent intracellular trafficking were investigated by confocal microscopy that utilises indirect immunofluorescence analysis and transmission electron microscopy that utilises a direct assay with gold-conjugated saporin-S6 and an indirect immunoelectron microscopy assay. Our results indicate that saporin-S6 was taken up by cells mainly through receptor-independent endocytosis. Confocal microscopy analysis showed around 30% co-localisation of saporin-S6 with the endosomal compartment and less than 10% co-localisation with the Golgi apparatus. The pathway identified by the immunofluorescence assay and transmission electron microscopy displayed a progressive accumulation of saporin-S6 in perinuclear vesicular structures. The main findings of this work are the following: i) the nuclear localisation of saporin-S6 and ii) the presence of DNA gaps resulting from basic sites in HeLa nuclei after intoxication with saporin-S6.     

Subcellular localization of mycobacteria in tissues and detection of lipid antigens in organelles using cryo-techniques for light and electron microscopy

The survival of intracellular pathogens within a host is determined by microbial evasion, which can be partially attributed to their subcellular trafficking strategies. Microscopic techniques have become increasingly important in understanding the cell biology of microbial infections. These recently developed techniques can be used for the subcellular localization of antigens not only in cultured cells but also within tissues such as Mycobacterium tuberculosis in lung and Mycobacterium leprae in skin. High-resolution immunofluorescence microscopy can be used in combination with cryo-immunogold electron microscopy using consecutive cryo-sections on the same tissue block forming a direct connection between the two microscopy techniques. The detection of mycobacterial lipid antigens in situ at an ultrastructural level is currently a challenge, but new modifications can be used to address this. These methods might be of interest to microbiologists and cell biologists who study host-pathogen interactions.     

Ex vivo confocal laser scanning microscopy: An innovative method for direct immunofluorescence of cutaneous vasculitis

Ex vivo confocal laser scanning microscopy (ex vivo CLSM) offers an innovative diagnostic approach through vertical scanning of skin samples with a resolution close to conventional histology. In addition, it enables fluorescence detection in tissues. We aimed to assess the applicability of ex vivo CLSM in the detection of vascular immune complexes in cutaneous vasculitis and to compare its diagnostic accuracy with direct immunofluorescence (DIF) microscopy. Eighty‐two sections of 49 vasculitis patients with relevant DIF microscopy findings were examined using ex vivo CLSM following staining with fluorescent‐labeled IgG, IgM, IgA, C3 and fibrinogen antibodies. DIF microscopy showed immunoreactivity of vessels with IgG, IgM, IgA, C3 and Fibrinogen in 2.0%, 49.9%, 12.2%, 59.2% and 44.9% of the patients, respectively. Ex vivo CLSM detected positive vessels with the same antibodies in 2.0%, 38.8%, 8.2%, 42.9% and 36.7% of the patients, respectively. The detection rate of positive superficial dermal vessels was significantly higher in DIF microscopy as compared to ex vivo CLSM (P < .05). Whereas, ex vivo CLSM identified positive deep dermal vessels more frequently compared to DIF microscopy. In conclusion, ex vivo CLSM could identify specific binding of the antibodies in vessels and showed a comparable performance to conventional DIF microscopy in diagnosing vasculitis.     

Direct and indirect estimation of the sex ratio of mutation frequencies in hemophilia A.

Carrier detection tests were carried out in 119 families with hemophilia A by using the data obtained with current DNA techniques (e.g., RFLP analysis and direct identification of mutations), conventional carrier detection tests (e.g., factor VIII:C and von Willebrand factor antigen), and pedigree information. On the basis of this data, we estimated the sex ratio of mutation frequencies with three completely different methods and compared the results. Since the classical indirect method derived from Haldane is substantially influenced by reproductive fitness (f), the sex ratio of mutation frequencies was estimated for both f = .3 and f = .5, resulting in a male:female mutation ratio of 5.37 (95% confidence interval 2.16-13.02) and 3.26 (95% confidence interval 0.97-8.73), respectively. According to the equilibrium-independent indirect method formulated by Rosendaal et al., the male:female ratio was estimated to be 3.4 (95% confidence interval 1.18-8.81). Since current DNA techniques provide information on the grandparental origin of the patient''s X chromosome, we were twice able to estimate directly the male:female mutation ratio as 15:1, by using the quotients of mutation origin (maternal grandfather/maternal grandmother and maternal grandfather/patient''s mother, respectively). Application of the Fisher test shows that the male mutation rate is higher than the female rate (P = 8.55 x 10(-7). Since all three completely different approaches unequivocally showed a higher male than female mutation frequency, this should be considered to be an established fact in calculating the risk in hemophilia A families.     

Atrial natriuretic polypeptide-like immunoreactivity in the rat pituitary: light and electron microscopic studies

Immunoreactive atrial natriuretic polypeptide (ANP) was investigated in the pituitary of rats by light and electron microscopy using the indirect immunofluorescence and peroxidase-antiperoxidase techniques. ANP-like immunoreactivity was present in 30-35% of anterior pituitary cells. These cells have two types of secretory granules being characteristic of rat gonadotrophin-storing granules, and were usually adjacent to the capillary endothelium. The results of this study suggest the co-occurrence of ANP and gonadotrophins in the anterior pituitary cells.     

Activation of Guinea Pig Herpesvirus Antigen in Leukemic Lymphoblasts of Guinea Pig

Herpesvirus (GPHV) antigen is either present in very small amounts, or absent in leukemic lymphoblasts taken directly from strain 2 guinea pigs. However, after maintenance in tissue culture for 72 hr, almost 100% of these lymphoblasts contained GPHV antigen. The expression of GPHV antigen could be demonstrated by indirect immunofluorescent technique as well as by the direct (125)I labeled antibody technique. However, infectious virus or virus capsids could not be detected in these cells either by infectivity tests or electron microscopy.     

Attachment of Azospirillum to isolated plant cells

Abstract Azospirillum brasilense cells were shown to attach to isolated plant cells. A rapid semi-quantitative assay to measure plant cell attachment, as was described previously for Agrobacterium , was found to work especially well for Azospirillum . Attachment was measured in an indirect way and confirmed by direct observations through scanning electron microscopy.     

Preparation of chromosome spreads for electron (TEM,SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactionsAbbreviations CLSM confocal laser scanning microscope - EM electron microscopy - kV kilovolt(s) - LM light microscope - SEM scanning electron microscope - STEM scanning-transmission electron microscope - TEM transmission electron microscope     

Electron spectroscopic imaging for high-resolution immunocytochemistry: use of boronated protein A

In the present study we adapted electron spectroscopic imaging (ESI) for high-resolution immunocytochemistry. To accomplish this, we applied boronated protein A (B-pA) for indirect detection of specific antigenic sites using pre-embedding and post-embedding protocols. Isolated acinar cells were exposed to wheat germ agglutinin (WGA) and anti-WGA, followed by B-pA, to reveal WGA binding sites at the level of the plasma membrane. The cells were then embedded in Epon and unstained ultra-thin sections were examined by electron microscopy using the ESI mode. For post-embedding, ultra-thin sections of glutaraldehyde-fixed, Lowicryl-embedded pancreatic tissue were exposed to specific antibodies (anti-insulin or anti-amylase), followed by B-pA. The unstained sections were examined using the ESI mode. In both cases, boron was detected with high resolution either at the level of the plasma membrane of acinar cells, demonstrating WGA binding sites, or over secretory granules in pancreatic insulin-secreting cells or acinar cells, demonstrating insulin and amylase, respectively. These findings were compared to those obtained with the protein A-gold technique, and have demonstrated the analogy of both types of labeling. In addition, several control experiments assessed this novel approach. They have demonstrated the specificity of labeling and the high reactivity of B-pA, as well as its antibody-binding properties. Finally, electron energy loss spectral analysis confirmed the presence of boron in the tissue sections at sites where immunolabeling was detected. These results demonstrate that ESI is an appropriate approach for cytochemistry. Since the technique is based on detection of elements, spatial resolution is considered to be in the magnitude of 0.5 nm, which represents a major improvement in resolution over actual electron microscopic cytochemical techniques.     

Simultaneous detection of multiple targets for ultrastructural immunocytochemistry

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.     

Immune deposits formed in situ by a monoclonal antibody recognizing a new intrinsic rat mesangial matrix antigen

We describe a unique mesangial matrix component of the rat glomerulus identified by a murine monoclonal antibody. The antigen is present exclusively in the glomerular mesangium and cannot be detected in other rat tissues by indirect immunofluorescence techniques or following pretreatment of tissue sections with acid urea or other nonionic detergents. Specific immunoprecipitation of the solubilized antigen yields a single peptide with an apparent m.w. of 81,000 when analyzed by discontinuous SDS-PAGE. This mesangial matrix component is collagenase resistant and trypsin sensitive. Perfusion of an isolated kidney preparation with this antibody results in direct binding of the mouse immunoglobulin to its mesangial antigen. Passive administration of the monoclonal antibody to Lewis rats results in characteristic electron dense deposits within the mesangial matrix that can be visualized ultrastructurally as early as 3 days. The immune deposits form without the activation of rat complement and persist for longer periods than those that develop after the planting of aggregated proteins or preformed immune complexes. Experimental animals that received either a monoclonal antibody specific for laminin or a non-kidney binding preparation did not develop such immune deposits at any time during the course of the autologous phase of the immune process. The results obtained in this study indicate that electron dense immune deposits can develop in the mesangium with the participation of a unique intrinsic matrix component and specific circulating monoclonal antibodies by an in situ mechanism of immune complex formation.     

Lipid vesicle-cell interactions. III. Introduction of a new antigenic determinant into erythrocyte membranes

The introduction of a new antigenic determinant, 2,4-dinitrophenyl-aminocaproyl-phosphatidylethanolamine (DNP-Cap-PE), into the surface membranes of intact human erythrocytes is described. Fresh cells were incubated in the presence of liposomes composed of 10% DNP-Cap-PE, 5% stearylamine, 20% lysolecithin, and 65% lecithin. Such liposome-treated erythrocytes are shown to be susceptible to immune lysis by anti-DNP serum in the presence of complement. Uptake of DNP-Cap-PE by erythrocyte membranes is also demonstrated by immunofluorescence using indirect staining with rabbit anti-DNP serum followed by fluroescein-conjugated goat anti-rabbit IgG and by electron microscopy using ferritin-conjugated antibody. Antigen uptake did not occur at low temperatures or from vesicles lacking lysolecithin and stearylamine. Fluorescence microscopy shows that the antigen-antibody complexes are free to diffuse over the cell surface, eventually coalescing into a single area on the cell membrane. Electron microscopy suggests that a substantial proportion of the lipid antigen is incorporated by fusion of vesicles with the cell membrane. There are indications that vesicle treatment causes a small proportion of cells to invaginate.     

Trichomonas vaginalis detection using real-time TaqMan PCR

1978 women and 93 men, all suspected of having a Trichomonas vaginalis infection, were tested for the presence of T. vaginalis by real-time PCR using the T. vaginalis-specific 2-kb repeated sequence, and by direct microscopy and culture. 40 samples were positive by T. vaginalis real-time PCR and 27 were positive by wet mount microscopy, either direct or after culture. All samples positive by direct microscopy of culture were also positive by real-time PCR. Of the 13 samples which were real-time PCR positive but negative by direct microscopy and culture 11 were confirmed by another T. vaginalis real-time PCR based on the beta tubulin gene. Only 2 samples (0.1%) showed inhibition in the PCR. The prevalence of T. vaginalis infection in the female patients was 1.8%. The sensitivity, specificity, positive and negative predictive values of the real-time PCR were 100%, 99.9%, 95% and 100%, respectively. The same test characteristics for the combined conventional T. vaginalis detection methods (microscopy+culture) were 71%, 100%, 100% and 99%, respectively. Therefore, real-time PCR is the method of choice for the diagnosis of T. vaginalis infection.