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Biomedical subjects

H R Gelderblom

Publications and source records attributed to H R Gelderblom.

At least 37 records · Page 2Linked to original sources

Bacteriophage diversity in the North Sea.

In recent years interest in bacteriophages in aquatic environments has increased. Electron microscopy studies have revealed high numbers of phage particles (10(4) to 10(7) particles per ml) in the marine environment. However, the ecological role of these bacteriophages is still unknown, and the role of the phages in the control of bacterioplankton by lysis and the potential for gene transfer are disputed. Even the basic questions of the genetic relationships of the phages and the diversity of phage-host systems in aquatic environments have not been answered. We investigated the diversity of 22 phage-host systems after 85 phages were collected at one station near a German island, Helgoland, located in the North Sea. The relationships among the phages were determined by electron microscopy, DNA-DNA hybridization, and host range studies. On the basis of morphology, 11 phages were assigned to the virus family Myoviridae, 7 phages were assigned to the family Siphoviridae, and 4 phages were assigned to the family Podoviridae. DNA-DNA hybridization confirmed that there was no DNA homology between phages belonging to different families. We found that the 22 marine bacteriophages belonged to 13 different species. The host bacteria were differentiated by morphological and physiological tests and by 16S ribosomal DNA sequencing. All of the bacteria were gram negative, facultatively anaerobic, motile, and coccoid. The 16S rRNA sequences of the bacteria exhibited high levels of similarity (98 to 99%) with the sequences of organisms belonging to the genus Pseudoalteromonas, which belongs to the gamma subdivision of the class Proteobacteria.

Bacteria↗

Cell membrane vesicles are a major contaminant of gradient-enriched human immunodeficiency virus type-1 preparations.

During preliminary experiments to establish the proportion of virus-coded p24 protein to virus membrane-associated HLA-DR in gradient-enriched HIV-1 preparations, we became aware of a large variability between experiments. In order to determine whether HLA-DR-containing cellular material was contaminating the virus preparations, we carried out enrichment by gradient centrifugation of clarified supernatants from noninfected cells and tested this material for HLA-DR content. We found that, independently of the cell type used, gradient enrichment resulted in the isolation of large quantities of HLA-DR-containing material which banded at a density overlapping that of infectious HIV. Electron microscopy of gradient-enriched preparations from supernatants of virus-infected cells revealed an excess of vesicles with a size range of about 50-500 nm, as opposed to a minor population of virus particles of about 100 nm. Electron micrographs of infected cells showed polarized vesiculation of the cell membrane, and virus budding was frequently colocalized with nonviral membrane vesiculation. Analysis of the cellular molecules present in the fractions containing virus or exclusively cellular material demonstrated that virus and cellular vesicles share several cellular antigens, with the exception of CD43 and CD63, found mainly at the virus surface, and HLA-DQ, which was found only in the cellular vesicles.

Antigens, CD↗

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.

Antigen Presentation↗

Mosaic hepatitis B virus core particles allow insertion of extended foreign protein segments.

Because of its particular immunological properties, the core protein of hepatitis B virus (HBcAg) has become one of the favoured 'virus-like particles' for use as a carrier of foreign epitopes. A new strategy to construct core particles presenting extended foreign protein segments was established based on the introduction of a linker containing a translational stop codon between sequences encoding a C-terminally truncated HBcAg (HBcAg delta) and a foreign protein sequence. Expression in an Escherichia coli suppressor strain allowed the simultaneous synthesis of both HBcAg delta and a read-through fusion protein containing a part of the hantavirus nucleocapsid protein. After purification, the presence of core-like mosaic particles with HBc and hantavirus antigenicity was demonstrated by electron microscopy and immunological tests. This strategy of partial stop codon suppression should improve the use of HBcAg as a carrier of foreign epitopes by allowing insertion of long foreign sequences into particle-forming proteins. The resulting mosaic particles should be of general interest for further vaccine developments.

Amino Acid Sequence↗

Transmission of HIV-1 from productively infected mature Langerhans cells to primary CD4+ T lymphocytes results in altered T cell responses with enhanced production of IFN-gamma and IL-10.

Mature Langerhans cells (mLC), the ex vivo correlates of interdigitating dendritic cells (IDC), are susceptible to infection with HIV-1. As IDC are important activators of T helper (Th) cells in vivo, we examined the interaction of HIV-1-infected mLC with CD4+ T lymphocytes. HIV-1-infected mLC readily formed clusters with the T cells and efficiently transmitted HIV-1 to the CD4+ Th cells. Formation of syncytia between mLC and T cells was initiated by HIV-1-infected mLC. In the clusters of HIV-1-infected mLC and activated T cells a massive HIV-1 production was observed leading to the subsequent elimination of the activated and infected T helper cells. Examination of the cytokine pattern produced during interaction of infected mLC with CD4+ T cells revealed an enhanced production of IFN-gamma and IL-10 in the cocultures. These results suggest that during antigen presentation-driven T cell activation by IDC in the lymphoid tissues, HIV-1-infected IDC might efficiently transmit the virus to Th cells, leading to altered Th cell responses.

CD4-Positive T-Lymphocytes↗

Intracellular compartmentalization of the glycoprotein B of herpesvirus Simian agent 8 expressed with a baculovirus vector in insect cells.

The intracellular localization of the glycoprotein B of herpesvirus simian agent 8 expressed with a baculovirus system in insect cells was studied. Cell fractionation and immunoprecipitation revealed that gB is present in microsomal as well as in nuclear membranes. Both fractions contain oligomers, probably dimers, of gB with endoglycosidase-H sensitive, mannose-rich carbohydrates. Nuclear transport of gB was further analysed by immuno electron microscopy of recombinant baculovirus-infected cells. The glycoprotein is present both in the outer and the inner nuclear membrane as well as in cytoplasmic structures and at the cell surface. This study precludes the possibility that glycosylation and/or oligomerisation of SA8 gB are responsible for nuclear targeting.

Animals↗

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.

Candidiasis, Oral↗

Spontaneous apoptosis of dendritic cells is efficiently inhibited by TRAP (CD40-ligand) and TNF-alpha, but strongly enhanced by interleukin-10.

In the lymphoid tissues, adaptive immune responses are initiated by the interaction of interdigitating dendritic cells (IDC) with naive T cells. To understand this interplay better, we used mature Langerhans cells (mLC), migrating from human epidermis, as the correlate of IDC ex vivo to evaluate the different effects of tumor necrosis factor (TNF)-alpha. TNF-related activation protein (TRAP; CD40-ligand) and interleukin-10 (IL-10) on induction or prevention of apoptotic cell death in these cells. Spontaneous decrease of mLC viability in culture was due to apoptosis, as determined by the appearance of typical morphological changes such as dilatation of the endoplasmic reticulum (ER), chromatin condensation and membrane blebbing. IL-10 strongly reduced mLC viability, whereas TRAP and TNF-alpha facilitated the survival of mLC. Spontaneous DNA fragmentation was detectable after 24 h in culture. IL-10 led to an earlier onset of DNA fragmentation, whereas TRAP and TNF-alpha delayed internucleosomal DNA cleavage. We found that IL-10-treated mLC were readily ingested and removed by macrophages. TNF-alpha and TRAP, in contrast, reduced engulfment of mLC by macrophages. Interestingly, IL-10, even at low concentrations, reverted the effects of TNF-alpha and TRAP in inhibiting mLC apoptosis. Furthermore, IL-10 led to the down-regulation of various surface antigens, especially of CD86 and CD54, whereas TNF-alpha and TRAP enhanced the expression of MHC class I and II antigens and of the accessory molecules CD40, CD54, CD80 and CD86. Taken together, these results show that mLC spontaneously undergo apoptosis in culture and that the progression of mLC to apoptosis is inhibited by TRAP and TNF-alpha, but accelerated by IL-10.

Apoptosis↗

Replication pattern of human immunodeficiency virus type 1 in mature Langerhans cells.

Langerhans cells (LC), the dendritic antigen presenting cells of the skin, mature into potent immunostimulatory cells during migration to regional lymph nodes, where they are identified as interdigitating cells (IDC). Since mature Langerhans cells (mLC) resemble IDC in phenotype and immunostimulatory capacity, we examined whether these cells were susceptible to infection with macrophagetropic and lymphotropic strains of human immunodeficiency virus type 1 (HIV-1). Highly purified cell preparations of mLC migrating from human epidermis expressed high amounts of major histocompatibility complex (MHC) class I and II antigens and of the accessory molecules CD40, CD80 and CD86, indicative of the phenotype of potent immunostimulatory cells. CD4 expression was upregulated on mLC during cultivation, independent of the presence of tumour necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the culture medium. The macrophagetropic HIV-1 strain SF162 replicated to higher titres in mLC than the lymphotropic strain IIIB. Both strains induced syncytia, with SF162 showing a more rapid cytopathic effect. Addition of TNF-alpha enhanced virus production, due to better cell viability under TNF-alpha treatment, whereas GM-CSF did not significantly influence viability of cells and replication pattern of the virus. These findings suggest that in the infected individual IDC in lymph nodes may function as target cells for HIV-1.

Antibodies, Monoclonal↗

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.

Adenoviruses, Human↗

Inhibition of infectious human immunodeficiency virus type 1 particle formation by Gag protein-derived peptides.

Sequential overlapping Gag protein-derived oligopeptides of human immunodeficiency virus type 1 (HIV-1) 22 to 24 amino acids long, were synthesized and tested in vitro for antiviral activity. Two synthetic peptides, one derived from the matrix protein p17 (NPGLLETSEGCRQ, amino acids 47 to 59) and one located in the capsid protein p24 (PAATLEEMMTA, amino acids 339 to 349) inhibited the production of infectious virus when added to HIV-1-infected cultures when used in the range of 20 to 200 micrograms/ml. As shown by thin section electron microscopy, peptide treatment resulted in the release of immature, deformed virus particles suggesting that the two peptides interfered with assembly and maturation. Other Gag protein-derived oligopeptides had little or no influence on virus production. To characterize further the functionally active regions we synthesized peptide derivatives with three consecutive amino acids substituted by alanine; they did not cause inhibition. Therefore the regions responsible for inhibition were located between amino acids 50 to 61 in p17, and 342 to 350 in p24. These observations might lead to the development of a new antiviral strategy affecting the late stage of virus replication.

Amino Acid Sequence↗

Analysis of protein expression and virus-like particle formation in mammalian cell lines stably expressing HIV-1 gag and env gene products with or without active HIV proteinase.

Cell lines stably releasing noninfectious virus-like particles containing wild type or mutant gene products represent useful tools for a biochemical, immunological, and structural analysis of virus assembly. Human immunodeficiency virus (HIV) type 1 gag and env gene products were transiently and stably expressed in mammalian cells and the formation of virus-like particles incorporating viral glycoproteins was analyzed. Transient cotransfection of plasmids directing the synthesis of gag and env gene products yielded efficient release of particles but specific incorporation of HIV glycoproteins was not detected. A stable cell line expressing wild type HIV-1 glycoproteins was generated and transient transfection of this cell line with gag-encoding constructs led to the release of virus-like particles incorporating HIV surface and transmembrane glycoproteins. Attempts to establish stable cell lines expressing wild type HIV gag and pol genes were unsuccessful and only highly unstable lines primarily expressing uncleaved precursor polyproteins were obtained. This result appears to be caused by the cytotoxic effects of the viral proteinase since stable lines were readily selected after transfection of constructs either encoding an inactive mutant of the proteinase or a mutated frameshift signal which prevented expression of the pol reading frame. Stable coexpression of uncleaved Gag polyprotein and wild type env gene products yielded efficient release of immature virus-like particles incorporating HIV glycoproteins. Electron micrographs revealed lentiviral budding structures with the typical surface projections of viral glycoprotein oligomers.

Animals↗

Assembly of the matrix protein of simian immunodeficiency virus into virus-like particles.

To obtain a better understanding of the processes of assembly and morphogenesis of simian immunodeficiency virus (SIV), recombinant vaccinia viruses containing regions of the gag-pol open reading frame were constructed and their intracellular expression as well as the ability of the Gag polypeptides to be released into the culture medium as constituents of virus-like particles were studied. Biochemical and electron microscopy analyses of cells infected with a recombinant expressing only the SIV matrix (MA) domain of the Gag polyprotein (v-p17 gag) showed that this protein self-assembles into 100-nm virus-like particles which are released into the culture medium. Interestingly, coexpression of SIV MA and Env proteins resulted in incorporation of gp120 and gp41 proteins into the recombinant p17-made particles. In addition when a positively charged domain of SIV MA (residues 26-33), which is highly conserved among all HIV and SIV MA proteins, was mutated into an acidic region, particle release was abolished without affecting protein expression, processing, or stability. Further characterization of the phenotype of this mutant by electron microscopy indicated that this mutant was blocked at the stage of assembly. These results suggest that SIV MA protein, along with its function in myristic acid-mediated membrane targeting, has intrinsic information for self-assembly as well as incorporation of viral Env glycoproteins into particles.

Amino Acid Sequence↗

Sensitivity of HIV antibody detection in saliva.

To assess the sensitivity and specificity of HIV antibody detection in saliva we tested matched serum and saliva samples from HIV-infected and uninfected individuals. Saliva specimens were collected by two different devices of the Salivette system and stored at different temperatures. Samples were tested for HIV antibodies by two commercially available enzyme-linked immunosorbent assays (ELISAs; Wellcome, Biotest). HIV antibodies were detected in 98.5% (Wellcome) and 97.8% (Biotest) of the saliva samples (standard Salivettes) from 135 seropositive individuals. Using the Salivettes flavoured with citric acid the sensitivity was only 22.9%. No reactions in ELISA were found in saliva from HIV-seronegative individuals. Salivary HIV-specific IgA was detected in 90% of seropositive individuals. All positive saliva samples stored at room temperature were still reactive after 20 days; of those stored at 37 degrees C, 23 out of 24 were positive when retested on day 5. Sensitivity of HIV antibody detection in saliva samples dried onto filter paper was 100% when a minimum of 100 microliters of saliva was used. HIV antibody testing in saliva is an efficient tool for large scale epidemiological studies when standard Salivettes are used for sample collection. Saliva samples can be stored in Salivettes or dried onto filter paper for several days at room temperature and under tropical conditions (37 degrees C).

Enzyme-Linked Immunosorbent Assay↗

Epstein-Barr virus in lingual epithelium of liver transplant patients.

Thirty-three liver transplant patients and 20 controls were examined for the presence of EBV in epithelium of the lateral border of the tongue by negative staining electron microscopy. Five of the specimens (15%) showed particles typical of the herpes virus family, while all controls were negative for EBV. In 3 of the 5 patients moderate oral hairy leukoplakia was observed clinically. Our results indicate that EBV may be expressed at the lateral border of the tongue of liver transplant patients, who in some cases show oral hairy leukoplakia clinically.

Adult↗

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.

Adolescent↗

Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells.

Segments of the human immunodeficiency virus (HIV) type 1 gag and pol genes and mutants thereof were transiently expressed in mammalian cells. Expression was dependent on the presence of the rev responsive element in cis and the rev protein in trans and was readily detected by indirect immunofluorescence or Western blotting. Transfection of constructs encoding the entire gag and pol open reading frames yielded efficient release of particles banding at a density of 1.16 g of sucrose per milliliter and consisting mainly of processed gag proteins. In addition, these particles contained the p66/p51 heterodimer of reverse transcriptase (RT), had associated RT activity, and contained RNA. Electron micrographs revealed immature retrovirus-like particles budding primarily from the plasma membrane and extracellular particles with morphological characteristics of HIV. Particle production was independent of the pol open reading frame or an active HIV proteinase (PR) but without active PR, cell-associated and particle-associated proteins remained completely uncleaved and budding occurred primarily into intracellular vacuoles. A mutation preventing myristoylation of the viral polyproteins abolished particle release but did not interfere with polyprotein synthesis and did not prevent processing. Expression of gag and PR in the same reading frame yielded complete processing of polyproteins but no budding and led to increased cell toxicity. A mutation of the PR active site in this construct prevented cytotoxicity and restored particle release indicating that the observed phenotype was caused by the overexpression of PR. These particles were aberrant in size and morphology when analyzed on sucrose density gradients and by electron microscopy. Budding was arrested at an early stage and extracellular particles appeared to be released by a different mechanism. Only short C-terminal extensions were compatible with this release mechanism since expression of a similar mutant construct encoding the entire gag-pol open reading frame did not yield particles.

Animals↗