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J Goudsmit

Publications and source records attributed to J Goudsmit.

At least 199 records · Page 11Linked to original sources
PubMed1994

Development and evaluation of an HIV-1 transfection-neutralization assay.

We developed a transfection-neutralization assay for human immunodeficiency virus type 1 (HIV-1) infectious molecular clones. In this assay CD4 negative adherent cells, transfected in microtiter plates with fixed amounts of proviral DNA of molecular HIV-1 clones, are cocultivated with CD4 positive T cell lines or primary peripheral blood mononuclear cells (PBMC) in the presence of anti-HIV-1 sera or monoclonal antibodies (MAbs). Results obtained with this technique were reproducible and compared favorably with a conventional cell-free infection inhibition assay. The transfection-neutralization assay obviates the need for virus stock preparation and, therefore, is particularly suitable for the evaluation of HIV-1 clones with slow replication kinetics and of recombinant chimeric HIV-1 clones inclined to undergo additional mutations during stock preparation. The potential value of this assay for the analysis of the specificity of neutralizing sera and MAbs was demonstrated in experiments with V3 chimeric molecular clones.

Amino Acid Sequence↗
PubMed1993

Cytochemical analysis of human T cell leukaemia virus I LTR-regulated beta-galactosidase gene expression using a novel integrated cell system.

To develop a reporter system to study the response of an integrated retroviral LTR and cellular and viral events which influence transcription, the 5' LTR of HTLV-1 was coupled to the Escherichia coli beta-galactosidase gene (lacZ). This construct was assembled within a vector containing the neomycin resistance gene controlled by the SV40 promoter, and introduced into HeLa cells. Expression from the LTR in one clone was upregulated by positive regulators of HTLV-1 expression, including 12-O-tetradecanoylphorbol-13-acetate (TPA) and the HTLV-1 transregulatory protein (tax), as has been previously reported using transient transfection assays. This method proved to be a rapid and reproducible assay for the measurement of integrated viral LTR activation in a single cell system.

Escherichia coli↗
PubMed1993

Increasing antigenic and genetic diversity of the V3 variable domain of the human immunodeficiency virus envelope protein in the course of the AIDS epidemic.

Population-wide variation in genomic RNA of human immunodeficiency virus type 1 (HIV-1) encompassing the V3 loop of the envelope protein was studied in serum samples of 74 newly infected individuals from three Dutch cohorts: 30 homosexual men, 32 drug users, and 12 hemophiliacs. During acute infection, HIV-1 RNA sequences present in serum are relatively homogeneous, which makes direct sequencing feasible. This offered an opportunity to study the infecting virus variants before mutations had accumulated in the new host. The sampling dates ranged from 1980 to 1991, thus spanning the entire AIDS epidemic in The Netherlands. The diversity in the sequenced region increased over time in both the homosexual and the drug-user risk groups. Furthermore, this increase was associated with an increase in antigenic variation, as witnessed by serum reactivity to a V3 peptide panel. Despite this diversification, some 1990 sequences still closely resembled the earliest 1980 sequence, making ancestral inferences problematic. No evidence was found of a change in the master sequence of the virus quasi-species over time. At the amino acid level, no risk-group-associated variation was found, but at the nucleotide level, the drug-user and homosexual/hemophiliac sequences could be distinguished on the basis of a single silent nucleotide change in the sequence encoding the tip of the V3 loop. Hemophiliac sequences could not be distinguished from those of homosexuals. In spite of the large and increasing genetic variability, all sequences were more similar to the European/American HIV consensus sequence than to that of non-Western strains.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Binding of human immunodeficiency virus type-1 to follicular dendritic cells in vitro is complement dependent.

The authors studied the binding in vitro of HIV-1 virus particles, conjugated to fluorescein isothiocyanate, to follicular dendritic cells (FDC) isolated from human tonsils. Analysis was done using flow cytometry, fluorescence microscopy, and immunogold electron microscopy. The focus of study was on the effect of serum from various origins, including pooled fresh serum and heated serum from control donors and pooled heated serum from HIV-1-infected patients (containing anti-HIV-1 antibodies). In the presence of heated serum, either from controls or from HIV-1-infected patients, the fluorescence signal in flow cytometry was similar to the background value. In the presence of fresh serum, the signal was substantially increased, and an even higher signal was observed in the presence of fresh serum and serum from HIV-1-infected patients. This high fluorescence signal was also found in the presence of serum depleted of complement factor C5, but not with serum deficient in complement factor C3. The binding of HIV-1 virions to FDC in the presence of fresh serum was confirmed by fluorescence microscopy on cytospot preparations. After quenching of the extracellular fluorescence with trypan blue, the fluorescence was reduced to about 30% of the initial value, indicating that most of bound fluorescent virions were present extracellularly. Similar experiments using blood mononuclear cells showed that fluorescent HIV-1 particles after binding to these cells were present intracellularly. This flow cytometry data was confirmed in immunogold electron microscopy demonstrating that most HIV-1 gag p24 or FITC label was present at the outside of FDC and on adherent virus particles. We conclude that HIV-1 virions adhere to FDC in vitro in a complement component C3-dependent way. Anti-HIV-1 antibodies in serum from HIV-1 infected patients enhance binding but, by itself, are unable to mediate binding.

Adhesiveness↗
PubMed1993

Predictors of rapid progression to AIDS in HIV-1 seroconverters.

OBJECTIVE: To determine whether at the time of HIV-1 seroconversion rapid progressors to AIDS and a low CD4+ count can be distinguished by the clinical presentation of primary HIV-1 infection and serological and immunological characteristics. DESIGN: Prospective cohort study on HIV-1 infection in homosexual men. SETTING: The Municipal Health Service, Amsterdam, The Netherlands. SUBJECTS: One hundred and eight men who seroconverted for HIV-1 during follow-up. MAIN OUTCOME MEASURES: Progression to AIDS and progression to a CD4+ lymphocyte count < 200 x 10(6)/l. RESULTS: Symptomatic primary HIV infection with fever and skin rash, absence of anti-HIV core and transient HIV p24 antigenemia were independent predictors of progression to AIDS at the time of HIV-1 seroconversion. A low CD4+ count immediately after seroconversion and the calendar year were independent predictors of progression to a low CD4+ count at the time of HIV-1 seroconversion. CONCLUSIONS: Even in the earliest stage of HIV-1 infection a small group of individuals at high risk for rapid progression to AIDS can be recognized by the clinical presentation of primary HIV infection, the presence of HIV p24 antigenaemia and the absence of a serological response to HIV core protein.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Antigenic variation of the dominant gp41 epitope in Africa.

OBJECTIVE: To determine the value of (combinations of) synthetic peptides representing immunodominant sites on HIV-1/HIV-2 transmembrane proteins for the detection and discrimination between HIV-1 and HIV-2 infection in various populations. DESIGN AND METHODS: Two 24-mer synthetic peptides derived from immunodominant sites on the HIV-1 and HIV-2 transmembrane proteins were used separately, in combination (env 1/2), and in combination with recombinant p24 (p24/env) in enzyme-linked immunosorbent assays. RESULTS: Positive reactions with env-1 were found in 150 out of 150 (100%) samples from Dutch AIDS patients, 60 out of 60 (100%) samples from Dutch homosexual men obtained 1 year after HIV-1-antibody seroconversion, 29 out of 30 (96.7%) samples from these men obtained at the time of HIV-1-antibody seroconversion, 40 out of 41 (97.6%) samples from East Africans with AIDS-related symptoms, and three out of 29 (10.3%) samples from West Africans with HIV-2 infection (including a sample from an individual infected with both HIV-1 and HIV-2). Positive reactions with env-2 in these study populations were 11 out of 150 (7.3%), nine out of 60 (15%), none out of 30 (0%), 25 out of 41 (60.9%) and 29 out of 29 (100%), respectively. In the samples with dual reactivity, true versus cross-reactivity could generally be differentiated on the basis of large differences in optical density values in the respective assays. All samples reacted positively with p24/env; 308 out of 310 (99.3%) were positive in the env 1/2 assay. Four East African samples that had negative or only weakly positive reactions with env-1 showed a noticeably stronger reaction with variant peptides derived from Central African isolate sequences. In all samples from HIV-1-infected Dutch homosexual men, the strongest signal was detected using the env-1 peptide sequence, which is derived from European and American isolates. CONCLUSIONS: Small peptide antigens may permit the detection of strain-specific antibodies, allowing serological characterization of HIV isolates.

Africa↗
PubMed1993

Greater diversity of the HIV-1 V3 neutralization domain in Tanzania compared with The Netherlands: serological and genetic analysis.

OBJECTIVE: To compare variation in the HIV-1 V3 neutralization domain in Tanzania and The Netherlands. METHODS: For serologic analysis, the specificity of anti-V3 antibodies (immunoglobulin G) for a panel of V3 peptides was determined in sera from 55 symptomatic HIV-1-infected Tanzanians and 51 Dutch AIDS patients. For genetic analysis, viral RNA was isolated from 15 of the Tanzanian sera and six of the Dutch sera. The V3 encoding region was reverse-transcribed, polymerase chain reaction-amplified and bacterially cloned, and sequences were determined over a stretch of at least 207 nucleotides. RESULTS: Thirty-five per cent of the Tanzanian sera, versus 2% of the Dutch sera, showed the highest reactivity to a V3 sequence of Zairian origin (RKSIHVGPGQAFYATG). Twenty-nine per cent of the Tanzanian sera, versus 82% of the Dutch sera, showed the highest reactivity to V3 sequences of US/European origin (RKSIXIGPGRAFYTTG; X = H, P or N). The Tanzanian RNA sequences showed greater diversity (mean distance, 19%) than the Dutch RNA sequences (10%). The measured anti-V3 specificities of the Tanzanian sera did not match accurately with the V3 sequences recovered from these sera. However, reactivity to the Zairian V3 peptide was associated with the sequence GPGQ, found in the centre of the V3 in 50% of the Tanzanian sequences. Sera from both countries that showed similar reactivities to the peptide panel contained RNA sequences that were relatively distant. CONCLUSIONS: The diversity of the HIV-1 population in Tanzania is much greater than that in The Netherlands. An indication of the HIV-1 V3 variation in a particular geographic region can be obtained by serological methods, but sequence analysis should not be omitted.

Adolescent↗
PubMed1993

Further characterization of an antigenic site of HIV-1 gp120 recognized by virus neutralizing human monoclonal antibodies.

OBJECTIVE: The aim of this study is to characterize antigenic sites on HIV-1 gp120 which may be important for the development of active and passive immunization strategies against HIV-1 infection. DESIGN: Two HIV-1-seropositive individuals were selected from the Amsterdam cohort and Epstein-Barr virus (EBV)-transformed B cells were generated from their peripheral blood mononuclear cells, which produce HIV-1-specific human monoclonal antibodies (HuMAb). METHODS: HuMAb were generated and selected based on their reactivities with native gp120. Reactivity with HIV-1 strains from phylogenetically different subfamilies was determined by immunostaining and virus neutralization assays. Specificity for the CD4-binding site was tested by an inhibition enzyme-linked immunosorbent assay and amino acids (aa) involved in the binding of the HuMAb were identified with a set of gp120 molecules with single aa substitutions. RESULTS: Three HuMAb (GP13, GP44, GP68) were generated, all recognizing a conserved conformation dependent epitope within, or topographically near, the CD4-binding site of gp120. HuMAb GP13 and GP68 neutralized a broad range of HIV-1 strains from phylogenetically different subfamilies, whereas HuMAb GP44 exhibited a more restricted pattern of neutralizing activity. The patterns of gp120 aa involved in their binding were unique for each of these HuMAb. CONCLUSIONS: The pattern of reactivities of these three HIV-1-neutralizing HuMAb developed in these studies is similar to, but distinct from other human and rodent MAb that recognize this antigenic site of HIV-1 gp120.

Adult↗
PubMed1993

Qualitative and quantitative detection of HIV-1 RNA by nucleic acid sequence-based amplification.

AIM: To develop a method to detect HIV-1 viral RNA by amplifying a specific nucleic acid sequence. METHOD: The nucleic acid sequence-based amplification (NASBA) method uses the simultaneous activity of avian myeloblastosis virus reverse transcriptase, T7 RNA polymerase and RNase H to amplify a specific nucleic acid target sequence. VALIDATION: An in vitro cultured HIV-1 stock solution was used to validate the NASBA method and determine the variation in RNA measurement. CONCLUSION: Although NASBA is theoretically capable of specific amplification of RNA or DNA, it is most suitable for amplification of RNA, and therefore for detection of HIV-1 viral RNA.

Gene Amplification↗
PubMed1993

Host cell membrane proteins on human immunodeficiency virus type 1 after in vitro infection of H9 cells and blood mononuclear cells. An immuno-electron microscopic study.

Human immunodeficiency virus type 1 (HIV-1)-infected H9 and blood mononuclear cells (MNCs) were studied by immunogold electron microscopy for the presence of HIV-1 gag p24 protein, env gp41 and gp120 proteins, and host cell molecules CD4, CD11a, CD25, CD54, CD63, HLA class I and HLA-DR. Uninfected H9 cells and MNC membranes labelled for CD4, HLA class I and class II, and, at low density, CD11a and CD54; lysosomal structures in the cytoplasm labelled for CD63. The infected cell surface showed immunolabelling for HIV-1 proteins, as did budding particle-like structures. Immunogold labelling of the cell membrane for CD4 was almost non-existent. The level of immunolabelling for CD11a and CD54 on infected cells was greater than that on uninfected cells; this is presumably related to a state of activation during virus synthesis. Budding particle-like structures and free virions in the intercellular space were immunogold-labelled for all host cell markers investigated. This was confirmed by double immunogold labelling using combinations of HIV-1 gag p24 labelling and labelling for the respective host cell molecule. We conclude that virions generated in HIV-1-infected cells concentrate host-derived molecules on their envelope. Also molecules with a prime function in cellular adhesion concentrate on the virion.

Antigens, CD↗
PubMed1993

Genomic human immunodeficiency virus type 1 RNA variation in mother and child following intra-uterine virus transmission.

In order to study the relationship between virus populations in a human immunodeficiency virus type 1 (HIV-1)-infected mother and her infant, we analysed a 276 bp fragment, including the V3 region, of genomic HIV-1 RNA purified from serum. Samples were collected from the mother 6, 4 and 2 months prior to delivery, during delivery and 10 months after childbirth (samples MA to ME, respectively) and from the infant at birth (cord blood) and the ages of 6 weeks and 9 months. A heterogeneous sequence population was observed in the maternal samples (mean nucleotide variation of 2.4 to 4.2%, range 0 to 8.3%). Until the age of 6 weeks the sequence population in the infant was highly homogeneous (mean nucleotide variation < or = 0.7%, range 0 to 2.5%). At 9 months of age, the infant's virus population showed more heterogeneity (mean nucleotide variation of 1.8%, range 0.4 to 3.6%) and a drift in the consensus sequence was observed. The evolution of the V3 region in the mother was characterized by accumulation of amino acid substitutions diverging from the virus population observed in the infant. The mean nucleotide distance between the maternal sequence populations and the sequence population of the child at birth was 2.8, 2.6, 3.7, 5.2 and 5.3% for the samples MA, MB, MC, MD and ME, respectively. Nearly complete replacement at position 308, previously described as antigenically important, from a proline to a histidine was observed during pregnancy, whereas all clones of the child's virus at birth and at 6 weeks contained a proline at that position. In conclusion, intra-uterine transmission is associated with a homogeneous sequence population in the child at birth, which is more closely related to the sequence population present in the mother during the first and second trimester of pregnancy than to the sequence population at delivery.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Mutations in human immunodeficiency virus type 1 gp41 affect sensitivity to neutralization by gp120 antibodies.

Three closely related molecular human immunodeficiency virus type 1 (HIV-1) clones, with differential neutralization phenotypes, were generated by cloning of an NcoI-BamHI envelope (env) gene fragment (HXB2R nucleotide positions 5221 to 8021) into the full-length HXB2 molecular clone of HIV-1 IIIB. These env gene fragments, containing the complete gp120 coding region and a major part of gp41, were obtained from three different biological clones derived from a chimpanzee-passaged HIV-1 IIIB isolate. Two of the viruses thus obtained (4.4 and 5.1) were strongly resistant to neutralization by infection-induced chimpanzee and human polyclonal antibodies and by HIV-1 IIIB V3-specific monoclonal antibodies and weakly resistant to soluble CD4 and a CD4-binding-site-specific monoclonal antibody. The third virus (6.8) was sensitive to neutralization by the same reagents. The V3 coding sequence and the gp120 amino acid residues important for the discontinuous neutralization epitope overlapping the CD4-binding site were completely conserved among the clones. However, the neutralization-resistant clones 4.4 and 5.1 differed from neutralization-sensitive clone 6.8 by two mutations in gp41. Exchange experiments confirmed that the 3' end of clone 6.8 (nucleotides 6806 to 8021; amino acids 346 to 752) conferred a neutralization-sensitive phenotype to both of the neutralization-resistant clones 4.4 and 5.1. From our study, we conclude that mutations in the extracellular portion of gp41 may affect neutralization sensitivity to gp120 antibodies.

Amino Acid Sequence↗
PubMed1993

Progression of HIV infection among injecting drug users: indications for a lower rate of progression among those who have frequently borrowed injecting equipment.

OBJECTIVE: To study markers of progression in a cohort of HIV-infected intravenous drug users (IDU). DESIGN: A prospective epidemiologic study. SETTING AND PATIENTS: We studied progression of HIV infection among 126 IDU attending the Municipal Health Service in Amsterdam. MAIN OUTCOME MEASURES: Progression was defined as a decline of the CD4 cell count to < 200 x 10(6)/l on two consecutive follow-up visits or AIDS. RESULTS: Using Cox modelling, the following baseline variables were predictive of progression. Enhanced progression was associated with: age > 30 years [relative hazard (RH), 7.7 [95% confidence intervals (CI), 1.7-36.0]], core antibody negativity [RH, 5.3 (95% CI, 1.6-17.6)], CD4 cell count [for CD4 cells 350-500 x 10(6)/l, RH, 1.38 (95% CI, 0.37-5.16); for CD4 cells 200-350 x 10(6)/l, RH, 9.20 (95% CI, 2.73-31.05) compared with a CD4 count > 500 x 10(6)/l]. A lower rate of progression was associated with borrowing used injecting equipment. IDU who reported borrowing injecting equipment between 1980 and baseline 10-99 times or > 99 times had a RH of 0.44 (95% CI, 0.22-0.88) and 0.19 (95% CI, 0.03-0.37), respectively, compared with IDU who had borrowed < 10 times. p24 antigen positivity was more predictive than core antibody negativity in a model with time-dependent variables, the relative risk for p24 antigen-positive participants was 3.5 (95% CI, 1.3-9.3). Additional analysis of progression to AIDS in a larger group of IDU showed comparable results with regard to the effect of borrowing on progression. CONCLUSIONS: Our observation that those IDU who reported borrowing injecting equipment most frequently appeared to have the lowest rate of progression, corrected for some sources of potential confounding, requires further epidemiologic confirmation and extended laboratory studies since other sources of bias might have been present. Baseline CD4 count, age and core antibody or p24 antigen were predictive of progression in IDU. We wish to emphasize that our results do not imply that borrowing should be encouraged, but may have implications for our understanding of HIV pathogenesis.

Adult↗
PubMed1993

Presentation and course of AIDS dementia complex: 10 years of follow-up in Amsterdam, The Netherlands.

OBJECTIVE: To assess the clinical presentation and course of the AIDS dementia complex (ADC). DESIGN: Retrospective study of a consecutive series of symptomatic HIV-1-infected patients [Centers for Disease Control and Prevention (CDC) stages IVA, B, C and D] evaluated for neurological symptoms between 1982 and 1992. SETTING: An academic referral centre for AIDS. PATIENTS: A total of 536 symptomatic HIV-1-infected patients evaluated for neurological symptoms between 1982 and 1992. INTERVENTIONS: Zidovudine treatment, which was introduced in The Netherlands on 1 May 1987 for patients with severe symptoms of HIV infection (CDC stages IVA, B, C and D). MAIN OUTCOME MEASURES: Diagnosis of ADC and CD4 cell count, clinical features, neuropsychological abnormalities, computed tomography (CT) and magnetic resonance imaging (MRI) abnormalities, cerebrospinal fluid (CSF) findings and course in patients with ADC. RESULTS: ADC was diagnosed in 40 out of 536 (7.5%) immunosuppressed, neurologically symptomatic HIV-1-infected patients in CDC stage IV, and was the AIDS-defining illness in six. The mean CD4 cell count of the 40 patients with ADC was 109 x 10(6)/l. Neuropsychological abnormalities in 15 out of 17 patients tested were in accordance with subcortical dementia. On CT scan of the brain, 70% showed no or only mild cortical atrophy. MRI was more sensitive than CT scan for detecting white matter abnormalities (73 versus 35%; P = 0.02). CSF examination showed mononuclear pleocytosis in 25%, protein level increase in 55%, and HIV-1 p24 core protein in 38% (13 out of 34). The mean survival was 6.7 months in the 40 ADC patients, but 4 months in 20 patients who had never used zidovudine, compared with 14.8 months in 10 patients who started zidovudine after they were classified as having ADC (P < 0.001). Three of these 10 patients improved remarkably, and two slightly, after starting zidovudine. ADC developed after discontinuation of zidovudine in nine patients. Only one patient developed ADC while receiving 600 mg zidovudine. CONCLUSIONS: MRI is more sensitive than CT for detecting white matter abnormalities. To date, there is no specific or sensitive CSF marker for ADC. Zidovudine may improve symptoms and prolong survival in patients with ADC, which rarely developed with continued zidovudine use in our study.

AIDS Dementia Complex↗