HIV: early virus-cell interactions.

Two entry mechanisms of HIV occur in both lymphocytes and macrophages incubated with purified virus suspensions: (a) direct fusion of the viral envelope with the cell membrane and (b) receptor-mediated endocytosis via clathrin-coated pits and vesicles. Both mechanisms are shown in detail in a time-interval series of electron micrographs. The two lipid bilayers of the viral envelope and of the cellular membrane usually fuse seamlessly within 1-3 min at 37 degrees C, but occasionally membrane ruptures occur, leading to rapid cytopathic effects, i.e., vacuolization and cytolysis only a few minutes later. In the course of virus-cell fusion, gp 120 is integrated into the cell membrane; subsequent syncytia formation was observed after 1 h of incubation. The core disintegrates and releases the viral ribonucleoprotein through the opening at the fusion site into the cytoplasm.

Effects of IFN alpha on late stages of HIV-1 replication cycle.

IFN alpha causes a modest reduction of HIV-1 expression in chronically infected monocytoid U937 cells. However, the ratio between cell-associated and shed viral p24 antigen is altered, being the cell-associated fraction dose-dependently enhanced by IFN. Furthermore, a significant decrease of infectivity of both cell-associated and shed material is observed. Transmission electron microscopy of IFN-treated cells revealed virus assembly being strongly inhibited, with the production of morphologically altered (tear-drop shaped) virus particles. Proteolytic processing of gag proteins appeared to be normal in IFN-treated cultures. However, virions shed from IFN-treated cells showed a markedly reduced incorporation of virus-specific gp120 and cell-derived ICAM-1 by the virus envelope. Additionally, these particles showed a significantly decreased ability to become bound to CD4+ target cells, accounting for, at least in part, the observed decrease of infectivity. Taken together, the data suggest that, in chronically infected cells, IFN alpha can affect late stages of HIV-1 replication, by inhibiting virus assembly and release, and by reducing the infectivity of shed virions. The latter effect seems to be due, at least in part, to altered incorporation of surface glycoproteins and defective particle formation. The relationship between impaired gp120 incorporation and altered morphogenesis of HIV-1 virions is under investigation.

Distribution of undulin, tenascin, and fibronectin in the human periodontal ligament and cementum: comparative immunoelectron microscopy with ultra-thin cryosections.

We studied the ultrastructural localization of three distantly related glycoproteins of the extracellular matrix, undulin, tenascin and fibronectin, in decalcified sections of human periodontal ligament (PL) and cementum. Undulin was associated with tightly packed major collagen fibrils and not with microfibrils, indicating that this protein may be involved in the supramolecular and functional organization of collagen fibrils into flexible bundles. Tenascin was found on globular masses between less densely packed collagen fibrils, thus displaying a pattern quite distinct from that of undulin. Fibronectin was noted in bulky material between the cross-striated fibrils, often surrounding individual fibrils like garlands, and in the microfibrillar meshwork extending from cross-striated fibrils. The three glycoproteins displayed a distinct and unique pattern of distribution in PL that can be correlated with their molecular structure and potential functions.

Immunoelectron microscopic localization of collagens type I, V, VI and of procollagen type III in human periodontal ligament and cementum.

We examined the ultrastructural localization of collagens Type I, V, VI and of procollagen Type III in decalcified and prefixed specimens of the periodontal ligament and cementum, by immunoelectron microscopy using ultra-thin cryostat sections. Immunostaining for collagen Type I was pronounced on the major cross-striated fibrils entering cementum and in cementum proper, whereas staining for procollagen Type III was almost exclusively observed on the major fibrils in the periodontal ligament situated more remote from cementum. Reactivity for collagen Type V was limited to aggregated, unbanded filamentous material of about 12 nm diameter that was found mainly in larger spaces between bundles of cross-striated collagen fibrils and occasionally on single microfibrils that apparently originated from the ends of the major collagen fibrils, which may support the concept of this collagen as a component of core fibrils. Collagen Type VI was present as microfilaments appearing to interconnect single cross-striated fibrils. In the densely packed fibril bundles of the periodontal ligament, no collagen type VI was detected. Neither Type V or Type VI collagen was observed in cementum.

Characterization of immunostimulating complexes (ISCOMS) of HIV-1.

HIV-1, strain HTLV-III, propagated in H9 cells and purified by sucrose gradient centrifugation, was used as native antigen source for the preparation of immunostimulating complexes, HIV-iscoms. The major antigen detected in the iscom was the cell-derived HLA-DR, which readily could be removed from the virus lysate by immunosorbent. In the iscoms the HIV structural proteins MA p17, p55 and TM gp41 were identified; SU gp120 was present in only minute amounts in the virus lysate. The iscom particles appeared well preserved after freeze drying with a round shape, approximately 35 nm in diameter, comprising morphological subunits, assembled with icosahedral symmetry. Immunization experiments in mice reflected the antigen content of the iscoms. High antibody response was induced to HLA-DR in non-depleted iscoms. Major humoral responses were observed to the viral structural proteins MA p17, CA p24, p55, and also to TM gp41. A low or negligible antibody response to SU gp120 was induced by the HIV-iscoms. The negligible response was, however, overcome by the addition of recombinant gp160 to the virus lysate prior to formation of iscoms, resulting in a preparation evoking a clear serum antibody to gp160.

Monocyte-derived dendritic cells represent a transient stage of differentiation in the myeloid lineage.

Cultivation of human peripheral blood monocytes with granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-4 facilitates generation of strongly antigen-presenting dendritic cells (DC). These monocyte-derived DC (mdDC) were used here to further delineate differentiation pathways in the myeloid lineage. Incubation of mdDC with TNF or soluble CD40L led to enhanced MHC and accessory surface antigen expression with significantly elevated T cell stimulatory activity, indicative of DC maturation. In contrast, after cytokine withdrawal or incubation with M-CSF, mdDC differentiated to macrophages. Cells became adherent, monocyte/macrophage surface markers were upregulated, and MHC and accessory surface proteins were downregulated. Furthermore, the multilaminar MHC class II compartments (MIIC) were lost and the T cell stimulating capacity largely diminished. Thus, mdDC show a high developmental plasticity by retaining their ability to become macrophages or to continue their differentiation towards mature DC.

Diagnostic electron microscopy is still a timely and rewarding method.

BACKGROUND: Parallel to its technical development starting in the 1930s, electron microscopy (EM) became an important tool in basic and clinical virology. First utilized in the rapid diagnosis of smallpox, it developed to a diagnostic routine in the early 1960s using the negative staining technique. EM was applied to infected cell-cultures and also to 'dirty' specimens including urine, feces, vesicle fluid, liquor. With the implementation of molecular biological and genetic techniques, the use of diagnostic EM decreased. OBJECTIVES: (1) To give a perspective on future indications and possible uses by discussing the past and the present of diagnostic EM, (2) To describe the system of External Quality Assessment on EM virus diagnosis (EQA-EMV) established in 1994 by our laboratory and its achievements. STUDY DESIGN: EQA-EMV is run to evaluate, to confirm and to improve the quality of diagnostic EM. Two different types of specimen are sent out: (1) prepared grids to assess and train the diagnostic skills of the participants, (2) stabilized virus particle suspensions to assess preparation efficiency. RESULTS: Diagnostic EM differs from other diagnostic tests in its rapidity and its undirected 'open view'. To emphasize these advantages, the indications for diagnostic EM are discussed, fundamental for a continuing future adaptation. Besides appropriate techniques, quality control measures are required to achieve and keep high diagnostic standards. The results from 6 years of EQA-EMV are presented. CONCLUSIONS: In the history of diagnostic EM in virology, a change in use has been seen. Starting in the 1990s and coincident with the broad introduction of 'modern' diagnostic techniques, the number of EM diagnostic labs has decreased considerably--in spite of the obvious advantages of this technique. To guarantee the continuing performance of diagnostic EM in the future. EQA runs have to be performed as with other techniques in the diagnostic armament. The growing number of participants and participating countries indicates an interest in as well as a need for this program.

Treatment of BK virus-associated hemorrhagic cystitis and simultaneous CMV reactivation with cidofovir.

Hemorrhagic cystitis (HC) is a common complication following high-dose chemotherapy and bone marrow transplantation, and the treatment of virus-associated HC remains to be optimized. This is the first report on the successful use of cidofovir in a patient with HC and polyoma viruria concomitant with CMV reactivation after allogeneic BMT. Treatment led to a significant decrease in viruria and to sustained suppression of CMV reactivation. Administered with probenecid and hydration, cidofovir was well tolerated, and there were no side-effects.

Comparative immunoelectron microscopy with monoclonal antibodies on yellow fever virus-infected cells: pre-embedding labelling versus immunocryoultramicrotomy.

To study the intra- and extracellular distribution of yellow fever virus 17D (YFV)-specific antigens, pre-embedding immunoelectron microscopy (IEM) and IEM on ultrathin frozen sections were carried out comparatively using monoclonal antibodies (MAB) and YFV-infected cells. In addition, three electron-dense marker systems (IgG-ferritin and IgG-gold and protein A-gold) were compared for their efficiency in detecting bound MAB. Pre-embedding immuno-labelling was performed in microtest plates followed by in situ embedding and immunocryoultramicrotomy was performed using pellets of sucrose-infused cells. In both procedures, cells were prefixed with different concentrations of glutaraldehyde (GA). In pre-embedding IEM virus-specific antigens could be detected on the envelopes of extracellular virions with YFV-neutralizing MAB. Using immunocryoultramicrotomy, neutralizing MAB bound to intracellular mature virions as well as to viral antigens incorporated into cytoplasmic membranes. A concentration of 1% GA destroyed antigenicity entirely, while with 0.25% and 0.1% GA immunoreactivity was retained for more than 3 mth. Some highly reactive MAB labelled antigen significantly in pre-embedding IEM, when used at concentrations of 1 ng/ml. Immunocryoultramicrotomy was 10-100 times less sensitive. On cryosections colloidal gold was the marker of choice, due to the fact that it showed less nonspecific sticking to intracellular components and that it was easily detectable on highly contrasted cryosections. Owing to their higher sensitivity, IgG-ferritin conjugates were preferred in pre-embedding IEM.

Detection of HIV envelope specific antibodies by immunoelectron microscopy and correlation with antibody titer and virus neutralizing activity.

Human antisera positive for HIV were evaluated on HTLV-IIIB producing cells by two different immunoelectron microscopic (IEM) techniques. In preembedding immunoferritin IEM a heavy label was observed with early budding HIV. Under the same conditions cell released 'mature' particles were almost negative, which could be explained by the direct observation that most of the surface glycoprotein knobs are lost spontaneously during virus maturation. Using freshly infected cultures after agarose embedding, immunogold labelling of ultrathin cryosections allowed us to detect and differentiate internal core as well as virus envelope antigens. A good qualitative correlation between neutralization titers and IEM labelling intensity was observed. This type of immunocryoultramicrotomy appears to be useful for the detection of antigens in and on the virion. It might turn out valuable for the characterization of the env gp120 epitopes of HIV.

Determination of the size of HIV using adenovirus type 2 as an internal length marker.

The size of a virion is a key criterion to its proper classification and may have implications in many practical aspects. Size determinations by thin section electron microscopy often result in length aberrations of more than 10% because of a number of preparative and instrumental inaccuracies, e.g. specimen shrinkage or swelling and unreliable calibration. Using adenovirus type 2 as an isometric size marker for internal calibration, we have determined the diameters of mature and immature HIV-1 to be 110 to 128 and 132 to 146 nm, respectively. The marker had been used either as a purified particle suspension added to the HIV producing culture, or adenovirus had been propagated together with HIV by infecting HIV producing cells. Using well characterized isometric markers, e.g. an icosahedral virus, in thin section electron microscopy appears to be a suitable technique for viral size determinations.

New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge.

Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV 'mosaic particles' in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.

Shammah-induced oral leukoplakia-like lesions.

A Shammah-induced oral leukoplakia-like lesion is described in a 44-year-old Algerian patient, who used this specific chewing tobacco since 33 years. The extended white lesion was located to the right mandibular vestibule and had a homogeneous appearance. Shammah is a chewing tobacco consisting of powdered tobacco leaves with carbonate of lime and other substances. It has been associated with oral cancer in Saudi Arabia. Histologically, acanthosis, hyperortho- and parakeratosis were seen. The spinous cell layer showed large pale staining epithelial cells with pycnotic nuclei. Atypia was not observed, however, an increase in mitotic activity was apparent. The subepithelial infiltrate was mild. Electron microscopy showed changes in the basal membrane with interruptions, duplications and triplications. Follow-up of the patient for 2 years revealed that, whenever, the patient changed the location of application, the white lesion regressed or disappeared within 4-6 weeks. Due to the composition of Shammah, the lesion induced has features of a mucosal burn. In contrast to other smokeless tobacco variants, Shammah seems to cause changes which, according to the small number of reports, may transform into oral cancer. As such, Shammah-induced oral leukoplakia-like lesions may be considered precancerous.

Spatial visualization of progressive states of maturing lentivirus.

Progressive states of maturing lentivirus: maedia visna virus (MVV) and human immunodeficiency virus (HIV), respectively, have been visualized by 2-D electron microscopy and by 3-D electron microscopic tomography. A major fraction of MVV and a low percentage of HIV appear as immature particles 4 to 5 days post virus infection. Upon budding the gag-precursor material is densely packed inside the external envelope. After virus release the major portion of precursors is assembled within an approximately 25 nm thick layer directly attached to the envelope. Structural maturation of the core is different for the two viruses. Pleomorphic cores are observed in mature MVV in contrast to structurally defined cores of HIV. The latter are principally cone-shaped, spanning the entire diameter of the virion with a 40 to 60 nm wide free end and an approximately 20 nm narrow end attached to the envelope with a core-envelope-link.

Electron spectroscopic imaging (ESI) of viruses using thin-section and immunolabelling preparations.

Electron spectroscopic imaging (ESI) and conventional bright-field transmission electron microscopy (TEM) were applied comparatively for the analysis of the fine structure and the antigenic make-up of human immunodeficiency virus and two herpes viruses. In addition to the information obtained in conventional bright-field TEM, ESI leads to high-contrast imaging of ultrathin sections and improves the resolution of thin and thick sections, and allows a better detectability of the immunolabelling markers.

Osteosarcoma of the maxilla. Case report and ultrastructural study.

An osteosarcoma, occurring in the maxillary alveolar ridge of a 17-year-old girl is presented. The tumor consisted predominantly of pleomorphic, mainly osteoblast-like and anaplastic cells. At the ultrastructural level the osteoblast-like cells were characterised by excentrically situated lobed nuclei, extensively swollen, dilated and rough endoplasmic reticulum (RER), polymorphism of mitochondria and sparse Golgi-systems. The undifferentiated cells were characterized by large, electron-lucent nuclei and paucity of non-dilated RER and mitochondria. The intercellular matrix contained collagen fibers with areas of focal collections of calcification.

Oral manifestations in a patient with idiopathic CD4+ lymphocytopenia.

A 56-year-old patient with idiopathic CD4+ lymphocytopenia (ICL) is described. In addition to a complex medical history and clinical course, he presented with oral manifestations including episodic erythematous candidiasis, persistent angular cheilitis, lingua exfoliativa areata, and teleangiectasia of facial skin and buccal mucosa. Light microscopy and transmission electron microscopy (TEM) revealed vascular structures similar to findings in clinically uninvolved oral mucosa of patients with HIV infection. Further observations of patients with ICL are warranted to clarify the significance of oral findings made in the present case.