Biomedical subjects

H Gelderblom

Publications and source records attributed to H Gelderblom.

At least 37 records · Page 2Linked to original sources

PubMed1999

In vivo rescue of a silent tax-deficient bovine leukemia virus from a tumor-derived ovine B-cell line by recombination with a retrovirally transduced wild-type tax gene.

The lack of bovine leukemia virus (BLV) expression is a consistent finding in freshly isolated ovine tumor cells and in the B-cell lines derived from these tumors. In order to gain further insight into the mechanisms of BLV silencing in these tumors, we have used the YR2 B-cell line, which was derived from the leukemic cells of a BLV-infected sheep. This cell line contains a single, monoclonally integrated, silent provirus, which cannot be reactivated either by stimulation in vitro or by in vivo injection of the tumor cells or cloned proviral DNA in sheep. Sequence analysis of the tax gene from the YR2 cell line identified two G-to-A transitions (G7924 to A7924 and G8149 to A8149) that result in E-to-K amino acid changes at positions 228 and 303 in the Tax protein. Following retroviral vector-mediated transfer of a wild-type tax gene into YR2 cells, we showed that BLV mRNA, viral proteins, and virions were produced, demonstrating that the cellular factors required for virus expression were present in the original YR2 cell line. Injection of this transduced YR2 cell line in sheep led to the rescue of replication-competent BLV proviruses. The integrated competent proviruses exhibited unique chimeric tax genes, which arose from homologous recombination between the transduced wild-type tax and the YR2-derived tax sequences. Furthermore, in one of these functional recombinant proviruses, only the A8149-to-G8149 reversion was present, providing clear evidence that the defect underlying the silent phenotype in YR2 cells results from a single C-terminal E303-to-K303 amino acid substitution in the BLV Tax protein. Our observations suggest that a single strategically located mutation in tax provides a mechanism for BLV inactivation in B-cell tumors.

Animals↗

PubMed1999

Disposition of [G-(3)H]paclitaxel and cremophor EL in a patient with severely impaired renal function.

In the present work, we studied the pharmacokinetics and metabolic disposition of [G-(3)H]paclitaxel in a female patient with recurrent ovarian cancer and severe renal impairment (creatinine clearance: approximately 20 ml/min) due to chronic hypertension and prior cisplatin treatment. During six 3-weekly courses of paclitaxel at a dose level of 157.5 mg/m(2) (viz. a 10% dose reduction), the renal function remained stable. Pharmacokinetic evaluation revealed a reproducible and surprisingly high paclitaxel area under the plasma concentration-time curve of 26.0 +/- 1.11 microM.h (mean +/- S.D.; n = 6; c.v. = 4.29%), and a terminal disposition half-life of approximately 29 h. Both parameters are substantially increased ( approximately 1.5-fold) when compared with kinetic data obtained from patients with normal renal function. The cumulative urinary excretion of the parent drug was consistently low and averaged 1.58 +/- 0.417% (+/- S.D.) of the dose. Total fecal excretion (measured in one course) was 52.9% of the delivered radioactivity, and mainly comprised known mono- and dihydroxylated metabolites, with unchanged paclitaxel accounting for only 6.18%. The plasma area under the plasma concentration-time curve of the paclitaxel vehicle Cremophor EL, which can profoundly alter the kinetics of paclitaxel, was 114.9 +/- 5.39 microl.h/ml, and not different from historic data in patients with normal or mild renal dysfunction. Urinary excretion of Cremophor EL was less than 0.1% of the total amount administered. These data indicate that the substantial increase in systemic exposure of the patient to paclitaxel relates to decreased renal metabolism and/or urinary elimination of polar radioactive species, most likely lacking an intact taxane ring fragment.

Aged↗

PubMed1995

Human herpesvirus type 7 in blood donors: detection by the polymerase chain reaction.

In order to evaluate the prevalence of human herpesvirus type 7 (HHV-7) in adult blood donors oral lavage fluid, buffy coat, and urine samples from 112 persons were examined by the polymerase chain reaction (PCR) at one time point. In addition, 11 donors were studied longitudinally over 11 weeks. When the results of the initial and the longitudinal study were combined HHV-7 DNA was found in samples from 109 of 112 (97.3%) adult blood donors. On the basis of different sensitivity levels of the first and the nested PCR differences were detected in the viral DNA load in the samples. It was found that lavage fluid regularly carried significantly higher DNA concentrations than buffy coat. Out of 112 donors, 102 (91.1%) and 8 (7.1%) were positive in the first, less sensitive PCR in lavage fluid and buffy coat, respectively (P < .0001). After nested PCR, 107 (95.5%) and 74 (66.1%) were positive in lavage fluid and buffy coat, respectively (P < .0001). Urine samples were found positive only sporadically. The longitudinal study showed that the oral lavage fluid of most of the donors consistently carried HHV-7 over up to 53 weeks, whereas buffy coat samples were positive less often. In conclusion, HHV-7 is found frequently in adult blood donors in the oral lavage fluid and buffy coat, which are, therefore, potential sources of HHV-7 transmission.

Adult↗

PubMed1995

Oral manifestations of alpha-mannosidosis: report of a case with ultrastructural findings.

Alpha-mannosidosis is a rare storage disease with distinct biochemical, clinical, histological and ultrastructural features. The oro-facial findings in a 26-year-old man are described. Gingival and oral mucosal hyperplasias were studied using histology and transmission electron microscopy (TEM). The TEM findings were comparable to those of other tissues examined in previous reports, consisting of histiocytic cells containing storage vacuoles with fine reticulo-granular material.

Adult↗

PubMed1994

Herpesvirus type 6 in patients undergoing bone marrow transplantation: serologic features and detection by polymerase chain reaction.

To evaluate the potential role of human herpesvirus type 6 (HHV-6) infection in patients after bone marrow transplantation (BMT) we sequentially analyzed buffy coat leukocytes, oral lavage fluid, and urine from 57 patients for the presence of HHV-6 DNA by polymerase chain reaction (PCR) before and after 60 BMTs. Twenty-four patients undergoing autologous BMT and 36 with allogeneic BMT were studied. Thirty-six patients (60%) were PCR positive in one or more tests. The majority of PCR-positive patients had positive results only sporadically, in 1 (n = 23) or 2 weeks (n = 5). Six patients were positive in 3 to 5 weeks. In 2 patients, we found a high frequency of positive tests, in 7 of 7 and 10 of 10 weeks analyzed. Twenty-four patients (40%) remained PCR negative throughout the post-BMT period. There was a significant correlation between the results of HHV-6 PCR and the occurrence of acute graft-versus-host disease (aGVHD). In grade II-IV, 6 of 8 (75%) patients had 2 or more positive PCR tests, compared with 5 of 25 (20%) patients without or with grade I aGVHD (P = .01). There was no difference in the outcome of PCR tests with respect to the type of BMT or pre-BMT HHV-6 enzyme-linked immunosorbent assay titers. Restriction enzyme analysis of PCR amplificates from 18 patients showed HHV-6 variant B in 16 (88.9%) and variant A in 2 cases (11.1%). We conclude that HHV-6 DNA can be detected in 60% of the patients after BMT. HHV-6 DNA can be detected more frequently in patients with moderate and severe aGVHD than in patients without aGVHD or with mild aGVHD.

Adolescent↗

PubMed1994

Human adenovirus type 41 contains two fibers.

DNA sequencing of the subgroup F human adenovirus serotype 41 (TAK, Ad41) fiber gene revealed the presence of two adjacent open reading frames encoding information for proteins with molecular weights of 60.6 kDa and 41.4 kDa (Pieniazek, et al; Nucleic Acids Res. 18: p. 1901, 1990). In this paper, various approaches were used to characterize the two proteins and determine whether both fibers were expressed in infected cells as well as on viral particles. We initially used a reverse transcriptase-polymerase chain reaction with primers for the short and long fiber genes to amplify mRNA from Ad41 infected HEp-2 cells at 48 h post-infection. Two distinct DNA bands; one slightly larger than 1.1 kbp and the other at about 1.7 kbp were identified. Second, we used polyclonal anti-Ad41 virion and monoclonal anti-Ad5 fiber antibodies to demonstrate that at both 24 and 36 h post-infection, Ad41 expressed two fiber proteins of the expected size. Specifically, by SDS-PAGE, one fiber (short) had a molecular weight of 40 kDa, while the other (long) had a molecular weight of 60 kDa. Third, by electron microscopy, two sizes of fibers were released from CsCl purified virions, both having a characteristic adenovirus morphology, with a knob at one end. The long fiber measured 315A in length and the short fiber was 250A long. These measurements are consistent with the two Ad41 fibers being encoded by the above open reading frames. We also performed a computer search to compare fiber sequences from other human adenovirus serotypes with that of the Ad41 short and long fiber proteins. The primary structure of both Ad41 fibers were found to be similar in that they contained tail, shaft and knob regions. Further, the tail region of both fibers (amino acids 1-42) showed a 74% overall homology to each other and contained the Ad conserved sequence NH2-F-N-P-V-Y-P-Y-COOH. An interesting difference, however, was observed in the shaft region where the long fiber (amino acids 43-389) had twenty-two 16-amino acid repeat motifs, while the short fiber (amino acids 43-233) had only twelve. Finally, we noted that the long fiber knob region was about 15% longer than that of the short fiber, and showed little overall homology. In conclusion, human adenovirus subgroup F (type 41) virions appear to differ from those of all other human adenoviruses (subgenera A-E) in that they contain two fiber genes and correspondingly, two different sized fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoviruses, Human↗

PubMed1994

Enterocytozoon bieneusi infection in an immunocompetent patient who had acute diarrhea and who was not infected with the human immunodeficiency virus.

Enterocytozoon bieneusi is a microsporidian species that has been detected exclusively in patients infected with human immunodeficiency virus (HIV) who are severely immunocompromised. We report what we believe is the first case of E. bieneusi infection in an HIV-negative patient with acute, self-limited diarrhea whose CD4+ cell count was not depressed.

Acute Disease↗

PubMed1993

Antiviral effects of different CD4-immunoglobulin constructs against HIV-1 and SIV: immunological characterization, pharmacokinetic data and in vivo experiments.

The CD4 cell surface antigen belongs to the immunoglobulin superfamily and is the primary receptor for the human immunodeficiency virus 1 (HIV-1). The high affinity interaction between HIV-1 and CD4 is mediated by the viral envelope glycoprotein gp120. Recombinant soluble CD4 (rsCD4) has been shown in vitro to be an effective inhibitor of HIV-1 and HIV-2 propagation in lymphoid cells. A variety of antibody-like molecules were constructed, consisting of different parts of the extracellular domain of CD4 fused to immunoglobulin constant regions. The fusion proteins were expressed in mammalian cell lines and purified via affinity chromatography. The specificity and anti-viral effects of the different CD4-immunoglobulin constructs against HIV were analysed by different immunological tests, i.e., immunofluorescence, neutralisation and in vitro assays. In pharmacokinetic studies, differences were found in serum half-life between the four- and two-domain CD4 constructs in cynomolgus monkeys and between glycosylated and deglycosylated CD4-Fc constructs in rabbits. In two in vivo experiments using the four-domain CD4-Fc in SIV-infected macaques, no beneficial effects were observed.

Animals↗

PubMed1993

Characterization of murine monoclonal antibodies directed against the submembrane protein p17 of HIV-1.

Monoclonal antibodies (MAbs) were raised against the gag protein of human immunodeficiency virus type 1 (HIV-1). The reactivity of the selected Mabs with the matrix protein p17 of HIV-1 were investigated in several tests, i.e. ELISA, immunostaining of Western blots, and alkaline phosphatase anti-alkaline phosphatase immunocytochemistry (APAAP). All three Mabs reacted exclusively with HIV-1 and showed specific binding to the virus surface in pre-embedding immuno-electron-microscopy and useful as diagnostic agents. In an "in vitro" cultivation experiment the MAbs showed antiviral activity in concentrations in the range of 25-100 micrograms/ml. No binding region could be defined using overlapping peptides consisting of the p17 protein sequence of HIV-1 in an epitope mapping system and therefore we concluded, that the MAbs recognize a conformational epitope.

Animals↗

PubMed1992

Interferon-gamma-induced assembly block in the replication cycle of adenovirus 2: augmentation by tumour necrosis factor-alpha.

Replication of adenovirus 2 (Ad-2) is inhibited in A 549 cells pretreated with interferon-gamma (IFN-gamma). The antiviral effect is synergistically enhanced by the simultaneous presence of tumor necrosis factor-alpha (TNF-alpha) before infection. Under conditions of strong inhibition of virus progeny formation, viral DNA synthesis and [35S]methionine incorporation into most late viral proteins are only marginally impaired. Pulse chase experiments indicate a partial inhibition of processing of viral proteins. Viral proteins are not degraded and capsomeres accumulate in the inhibitor-treated cells. Capsid formation, on the other hand, is strongly inhibited in the cytokine-treated cells. The inhibition of Ad-2 replication in A 549 cells by IFN-gamma and TNF-alpha is caused, therefore, by a block in the maturation of Ad-2.

Adenoviruses, Human↗

PubMed1992

Development of a Langerhans cell phenotype from peripheral blood monocytes.

Epidermal Langerhans cells (ELC) are definitively primed to differentiate into dendritic cells (DC). It is unknown at what stage of monocyte development this priming occurs. In a culture system characterized by low paracrine stimulation, i.e. Iscove's modified Dulbecco medium (IMDM) with 2% FCS, we tested the ability of peripheral blood monocytes to turn to the route of the LC-DC lineage. In this system monocytes did not develop significant yeast cell phagocytosis, although mannose receptors were available. However, they became strong stimulators of mannan specific T cell proliferation. Phenotype development was analysed by flow cytometry using the monoclonal antibodies OKT6 (CD1a), IOT2 (HLA-DR), IOM2 (CD14) and the ligand Man-BSA-FITC. CD1a was the first marker which distinguished cultured monocytes from developing macrophages, obtained by addition of 8% human serum. Like cord blood Langerhans cells (CBLC) they internalized OKT6 in deep coated pits. They maintained a phenotype of monocyte derived Langerhans cells (MoLC) during eight days of in vitro culture, expressing CD1a, mannose receptors and HLA-DR and decreasing CD14, if left in their own conditioned medium. MoLC could be converted into macrophages by addition of human serum only within the first four days in vitro. Our data suggest that monocytes acquire an LC phenotype by autocrine stimulation.

Antigens, CD↗

PubMed1992

Morphogenesis of recombinant HIV-2 gag core particles.

The gag-pol coding region of the HIV-2BEN genome was expressed in CV-1 cells infected with four recombinant vaccinia viruses (VV). These recombinant VV encoded either the whole gag-pol region or the gag gene including the protease-coding region of the pol gene or the gag gene truncated at its 3'-end or only the pol gene. The HIV-2BEN gag precursor p55, its mature cleavage products p24 and p17 as well as the pol reverse transcriptase (RT) p66 were detected in VV-infected CV-1 cells. The p55 and two intermediate cleavage products p40 and p35 were myristilated. Comparison to lysates of permanently HIV-2BEN-infected Molt 4 clone 8 cells revealed that several additional gag and pol proteins were present in the VV-infected CV-1 cells. Deletion of the gag and pol overlapping region coding for the viral protease prevented cleavage of the recombinant gag precursor. Electron microscopy of VV-infected CV-1 cells revealed budding structures and immature as well as mature retroviral particles formed by the recombinant gag proteins. Striking differences in the ability to form complete particles were observed between the different recombinant VV. Expression of the truncated gag gene led to the formation of budding structures, but completely budded circular particles were not detectable. Such particles were produced by expression of the whole gag gene and the protease. Mature virions with an internal core structure were only detected in VVgagpol-infected cells. From these findings we conclude that the 3'-end of the gag gene coding for the p16 protein is essential for the formation of complete HIV-2 particles and that the pol proteins support the assembly of the viral core.

Base Sequence↗

PubMed1992

Spatial visualization of the maturing HIV-1 core and its linkage to the envelope.

The core of late states of maturing human immunodeficiency virus type 1 (HIV-1) has been visualized in three dimensions at approximately 7 nm resolution by electron microscopic tomography. After budding, approximately 25 nm thick precursor material is observed densely assembled inside the viral envelope. Upon proteolysis the core material is transported and condensed in the center of the virion. The core, 100 nm in length, spans the entire diameter of the virion showing a 40-60 nm wide free end and a narrow end approximately 20 nm. A model of the core is derived consisting of two fibers packed into a bilateral, elongated structure. Two ends of the fibers are compacted together, forming one narrow end of the core, while the two other fiber ends are situated more loosely together allowing for flexibility. Structural maturation of the virus could be reflected by the degree of compactness of the core. The narrow end of the core is observed attached to the envelope with a conspicuous core-envelope link (CEL).

HIV-1↗

PubMed1991

Expression of proteins encoded by Epstein-Barr virus trans-activator genes depends on the differentiation of epithelial cells in oral hairy leukoplakia.

The Epstein-Barr virus (EBV) immediate early gene product BZLF1 was localized by indirect immunofluorescence to the cytoplasm of the basal epithelial layer at the lateral border and dorsum of tongue in human immunodeficiency virus-infected and -seronegative patients. Two biopsies of oral hairy leukoplakia revealed a sporadic cytoplasmic staining of the BHRF1 and BRLF1 gene products in the basal epithelial layer. The widespread presence of BZLF1 in the basal epithelial layer indicated that this cell layer contained EBV DNA and was probably directly infected by EBV. Nuclear localization of the immediate early and early gene products BZLF1, BHRF1, BRLF1, and BMLF1 was limited to oral hairy leukoplakia in human immunodeficiency virus-seropositive patients and revealed a codistribution with the virus capsid antigen. Our results indicate that the epithelium of the tongue is a potential reservoir for EBV and that in heavily immunocompromised patients EBV may move from the cytoplasm to the nucleus with increasing differentiation and be coactivated there during the terminal differentiation of epithelial cells at the lateral border and dorsum of tongue.

Cell Differentiation↗

PubMed1990

A CD 4: immunoglobulin fusion protein with antiviral effects against HIV.

Antibody-like molecules consisting of the human CD 4 extracellular domain fused to human IgG1 heavy chain constant regions were genetically constructed and expressed in a BHK cell stable transfectant. Purified chimeric antibodies bound to HIV particles as it was shown by immuno electron microscopy, inhibited fusions of HIV-1-infected cells with uninfected cells, neutralized HIV-1, and were able to inhibit the spread of a cellular HIV-1 infection in CD 4+ cells. Plaque reduction assays with CD 4(+)-transfected Hela-cells showed a comparable inhibition of HIV-1 and HIV-2. Inhibitory functions were enhanced in the presence of complement. HIV-1- and HIV-2-infected CD 4+ cells were efficiently lysed by a slow, complement-dependent mechanism, whereas uninfected CD 4+ cells and HLA-DR+ cells were not affected.

Animals↗

PubMed1990

Clonal diversity and virulence factors in strains of Escherichia coli of the classic enteropathogenic serogroup O114.

Eighty-eight Escherichia coli strains of the enteropathogenic (EPEC) group O114 that were isolated from humans and animals in geographically different places and over more than 30 years were examined for virulence markers, O:H serotypes, and for electrophoretic types by multilocus enzyme electrophoresis. Four major genetically tightly related clusters of strains showed close correlation between electrophoretic types and other phenotypic characters. Cluster I contained 35 EPEC class II strains of serotypes O114:H9 and O114:H- and 5 enterotoxigenic E. coli belonging to O114:H21 and O114:H49. Clusters II and III comprised 36 O114:H4, O114:H32, and O114:H- strains; most were of doubtful pathogenicity except one Verotoxin-positive O114:H4 strain isolated from a human with diarrhea. Cluster IV contained 9 classic EPEC strains of serogroup O114:H2 that were characterized by localized adherence to HEp-2 cells and by the EPEC adherence factor.

Alleles↗

PubMed1990

[Hyperpigmentation of the oral mucosa in HIV infection].

In ten HIV-infected patients oral hyperpigmentations (HP) were observed, which seemed to be unrelated to any of the known endogenous or exogenous etiological factors. Light microscopy revealed accumulation of Fe-negative pigment in keratinocytes of the stratum basale as well as extra- and intracellularly within the subepithelial connective tissue. The ultrastructure of HP was characterised by the occurrence of melanosomes and premelanosomes within keratinocytes, melanocytes, macrophages and occasionally in fibroblasts, while especially in keratinocytes of the superficial cell layers immature melanosomes were found. Immunohistochemical examination pointed to an increase of HLA-DR positive cells and an altered distribution pattern of immunocompetent cells within the hyperpigmented mucosa. The finding of oral HP in HIV-infected patients may occur due to an increased activity of melanocytes, an increased production of melanosomes and their increased transfer to keratinocytes.

Adult↗

PubMed1989

HBcAg expressed on the surface of circulating Dane particles in patients with hepatitis B virus infection without evidence of anti-HBc formation.

Circulating immune complexes composed of HBcAg and anti-HBc have been demonstrated recently in patients with hepatitis B virus replication. After dissociation of immune complexes by chaotropic ions, HBcAg was quantified radioimmunologically. In the present study, we describe 10 patients with hepatitis B virus replication, absent or delayed anti-HBc formation and exposed HBcAg in serum. Four of the 10 patients had acute hepatitis, and six patients had chronic persistent hepatitis. In seven of 10 patients, a secondary immune defect was apparent due to acquired immunodeficiency syndrome, leukemia, histiocytosis X, sarcoidosis or end-stage renal disease. Electron microscopy demonstrated that Dane particles from anti-HBc-negative patients were agglutinated after addition of monoclonal anti-HBc antibodies, whereas Dane particles from anti-HBc-positive sera did not show agglutination. Monoclonal HBsAg-specific antibodies aggregated Dane particles independent of the presence of anti-HBc. Circulating HBcAg was always associated with the Dane particle fraction after density gradient separation. Hepatitis B virus core proteins from patients with and without anti-HBc studied by immunoblotting after sodium dodecyl sulfate-gel electrophoresis showed identical patterns. Hepatocytes from anti-HBc-negative patients were positive for HBcAg but negative for immunoglobulin G by immunofluorescence technique. The data indicate that HBcAg may also be expressed on the surface of Dane particles, where it is commonly masked by anti-HBc.

Acute Disease↗