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Biomedical subjects

J Goudsmit

Publications and source records attributed to J Goudsmit.

At least 37 records · Page 2Linked to original sources
PubMed2001

[Ethiopia-Netherlands AIDS research project].

The 'Ethiopia-Netherlands AIDS Research Project' (ENARP), started in 1994, is a long-term collaboration between AIDS researchers in Amsterdam and the Ethiopian Health and Nutrition Research Institute in Addis Ababa. The ENARP's primary objectives include conducting studies on HIV and AIDS in Ethiopia, especially by means of some large-scale prospective cohort studies, training Ethiopian scientists in PhD programmes in epidemiology, immunology and virology and establishing a reference laboratory for HIV and AIDS in Ethiopia and neighbouring countries. External funding for ENARP amounts to 32 million Dutch guilders for two periods of four years and is being provided by the Dutch Government. ENARP is the largest third world biomedical project supported by the Dutch Government. In 2000 two Ethiopian students obtained their doctorates from the University of Amsterdam. Five new PhD students commenced their training in 1999. ENARP hopes to set up HIV-1 vaccine phase I and phase II trials in the near future.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Selection by AZT and rapid replacement in the absence of drugs of HIV type 1 resistant to multiple nucleoside analogs.

We studied the intrahost evolution and dynamics of a multidrug-resistant HIV-1, which contains an insertion of two amino acids (aa) and several aa changes within the reverse transcriptase (RT) gene. From an individual receiving intermittent therapy, sequences of 231 full-length molecular clones of HIV-1 RT were obtained from serum-derived viruses at 12 consecutive time points over a period of 6 years, 17 to 20 clones per time point. In the 3.5-year period prior to the first course of therapy, only wild-type (wt) viruses were found. As soon as 6 months after the start of zidovudine (AZT) monotherapy, all viruses contained an insertion of two aa between positions 68 and 69 of the RT and aa changes at positions 67 and 215, a combination conferring resistance to multiple nucleoside analogs. After termination of therapy, the insertion mutants were rapidly and completely replaced by the wt viruses. In turn, the insertion mutants replaced the wt viruses after initiation of therapy with 3TC, d4T, and saquinavir. After termination of triple therapy, the wt viruses completely replaced the mutants within 1 month, which is markedly faster than has been observed earlier for the replacement of AZT-resistant viruses. Fast replacements of the mutant virus populations after termination of therapy indicate gross competitive disadvantage of the insertion mutant in the absence of therapy, which we estimated by using several models. The insertion mutants attained high virus loads, demonstrating that virus load cannot be used as a direct measure of virus fitness.

Amino Acid Sequence↗
PubMed2001

Timing of the introduction into Ethiopia of subcluster C' of HIV type 1 subtype C.

Viruses circulating in Ethiopia during the 1990s cluster with main subtype C, but a significant subcluster, C', was noted in multiple analyses. This subcluster of subtype C(C') was in a fifty-fifty equilibrium with the main subtype C (Abebe et al., AIDS Res Hum Retroviruses 2000;16:1909-1914). To analyze genetic diversification within the subcluster of HIV-1 subtype C designated C' in the course of the epidemic in Ethiopia, we analyzed 165 env gp120 V3 sequences obtained between 1988 and 1999. We observed a highly significant positive correlation between sampling years of individual sequences and their synonymous distances to the reconstructed common ancestor of the HIV-1 subtype C' subcluster. The extrapolation of the regression line of synonymous distances back to the date when no synonymous heterogeneity was present among the Ethiopian HIV-1 C' population allowed us to estimate 1982 (95% CI, 1980-1983) as the year of the onset of HIV-1 C' genetic diversification and expansion in Ethiopia. These results are in agreement with retrospective epidemiological and serological data, which demonstrated the absence of an HIV-1 epidemic in the Ethiopian population before the 1980s.

Amino Acid Sequence↗
PubMed2001

Sexual risk behaviour relates to the virological and immunological improvements during highly active antiretroviral therapy in HIV-1 infection.

OBJECTIVES: To evaluate the effect of highly active antiretroviral therapy (HAART) on the sexual behaviour of homosexual men, we conducted (i) an ecological study of time trends in sexual behaviour and sexually transmitted diseases; (ii) a HAART-effect study focused on the practice of unprotected anogenital sex. DESIGN: Subjects were participants in the ongoing Amsterdam Cohort Studies (ACS) among homosexual men, initiated in 1984. Data for (i) represented all ACS visits by HIV-1-positive and -negative participants who entered ACS at or below 30 years of age and were followed until 35 years (n = 1062). Data for (ii) represented all ACS visits of HIV-1-positive men from 1992 to 2000 (n = 365), of whom 84 were HAART recipients with at least 2 months of behavioural follow-up. RESULTS: (i) After HAART became generally available in July 1996, unprotected sex was practised more frequently and the incidence of gonorrhoea was higher compared to March 1992-June 1996 among HIV-1-negative and -positive men, respectively. (ii) Among HIV-1-positive men, a higher level of unprotected sex with casual partners was observed after HIV-1 RNA became undetectable and CD4 cell counts increased with the use of HAART. Notably, in individuals who did not receive HAART, high HIV-1-RNA levels (above 10(5) copies/ml) were likewise related to unprotected sex with casual partners. CONCLUSION: Data support the need for the reinforcement of safe sex prevention messages among HIV-1-negative men, and our data also provide a lead for redirecting and tailoring current prevention strategies to the needs of HIV-1-positive men.

Adult↗
PubMed2001

Using phylogenetic analysis to trace HIV-1 migration among western European injecting drug users seroconverting from 1984 to 1997.

OBJECTIVE: To reconstruct the epidemiological relationships of the HIV epidemics among injecting drug users (IDU) in western Europe. METHODS: HIV env V3 sequences of and epidemiological data were obtained from 145 IDU who seroconverted in three sequential periods: 1984-1988, 1989-1992 and 1993-1997. The sequences were phylogenetically analysed and examined for signature patterns characteristic of northern European IDU, including the conserved GGC codon in the V3 loop. RESULTS: Subpopulations of genetically related HIV strains were observed in Italy, France, Scotland and Spain, in contrast to the Netherlands, Austria and Switzerland. This difference between the two groups of countries suggests that the HIV epidemics amongst IDU in the latter group was caused by multiple virus introductions. In Edinburgh and the surrounding area, most IDU were infected with the same GGC strain over the 12-year study period. The epidemic among IDU in north-western Europe started with GGC viruses, whereas in south-western Europe non-GGC viruses predominated. This geographical separation has faded during the course of the epidemic, most likely because of virus exchange among IDU populations.

Base Sequence↗
PubMed2001

N-linked glycosylation of the HIV type-1 gp120 envelope glycoprotein as a major determinant of CCR5 and CXCR4 coreceptor utilization.

The variable V1V2 and V3 regions of the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein (gp120) can influence viral coreceptor usage. To substantiate this we generated isogenic HIV-1 molecularly cloned viruses that were composed of the HxB2 envelope backbone containing the V1V2 and V3 regions from viruses isolated from a patient progressing to disease. We show that the V3 amino acid charge per se had little influence on altering the virus coreceptor phenotype. The V1V2 region and its N-linked glycosylation degree were shown to confer CXCR4 usage and provide the virus with rapid replication kinetics. Loss of an N-linked glycosylation site within the V3 region had a major influence on the virus switching from the R5 to X4 phenotype in a V3 charge-dependent manner. The loss of this V3 N-linked glycosylation site was also linked with the broadening of the coreceptor repertoire to incorporate CCR3. By comparing the amino acid sequences of primary HIV-1 isolates, we identified a strong association between high V3 charge and the loss of this V3 N-linked glycosylation site. These results demonstrate that the N-linked glycosylation pattern of the HIV-1 envelope can strongly influence viral coreceptor utilization and the R5 to X4 switch.

Amino Acid Sequence↗
PubMed2001

OKT3 and IL-2 treatment for purging of the latent HIV-1 reservoir in vivo results in selective long-lasting CD4+ T cell depletion.

Activation of resting T cells has been proposed to purge the reservoir of HIV-1-infected resting CD4+ T cells. We therefore treated three HIV-1-infected patients on antiretroviral therapy with OKT3, a CD3 monoclonal antibody, and recombinant human IL-2. Here we report the profound and partially long-lasting host responses induced by the OKT3 and IL-2 treatment. OKT3/IL-2 induced a strong but transient release of plasma cytokines and chemokines. The percentage CD4+ and CD8+ cells in the blood expressing the activation marker CD38 transiently increased to almost 100%, and in lymph nodes we "observed" a 10-fold increase in the number of dividing Ki67+ cells and increased numbers of apoptotic cells. Following OKT3/IL-2 treatment, a long-lasting depletion of CD4+ cells in the peripheral blood and lymph nodes occurred, suggesting the physical deletion of these cells. Increases in CD4+T cell numbers during the two year followup period were due mainly to increased memory cell numbers. CD8+ cells were also depleted in the blood, but less severely in lymph nodes, and returned to baseline levels within several weeks.

CD4 Lymphocyte Count↗
PubMed2001

Quantitative detection of hepatitis B virus DNA by real-time nucleic acid sequence-based amplification with molecular beacon detection.

We have developed a hepatitis B virus (HBV) DNA detection and quantification system based on amplification with nucleic acid sequence-based amplification (NASBA) technology and real-time detection with molecular beacon technology. NASBA is normally applied to amplify single-stranded target RNA, producing RNA amplicons. In this work we show that with modifications like primer design, sample extraction method, and template denaturation, the NASBA technique can be made suitable for DNA target amplification resulting in RNA amplicons. A major advantage of our assay is the one-tube, isothermal nature of the method, which allows high-throughput applications for nucleic acid detection. The homogeneous real-time detection allows a closed-tube format of the assay, avoiding any postamplification handling of amplified material and therefore minimizing the risk of contamination of subsequent reactions. The assay has a detection range of 10(3) to 10(9) HBV DNA copies/ml of plasma or serum (6 logs), with good reproducibility and precision. Compared with other HBV DNA assays, our assay provides good sensitivity, a wide dynamic range, and high-throughput applicability, making it a viable alternative to those based on other amplification or detection methods.

Centrifugation, Density Gradient↗
PubMed2001

Single rapid real-time monitored isothermal RNA amplification assay for quantification of human immunodeficiency virus type 1 isolates from groups M, N, and O.

Because human immunodeficiency virus type 1 (HIV-1) subtypes and circulating recombinant forms (CRFs) are spreading rapidly worldwide and are becoming less confined to a geographical area, RNA assays that can detect and quantify all HIV-1 isolates reliably are in demand. We have developed a fast, real-time monitored RNA assay based on an isothermal nucleic acid sequence-based amplification technology that amplifies a part of the long terminal repeat region of the HIV-1 genome. Real-time detection was possible due to the addition of molecular beacons to the amplification reaction that was monitored in a fluorimeter with a thermostat. The lower level of detection of the assay was 10 HIV-1 RNA molecules per reaction, and the lower level of quantification was 100 copies of HIV-1 RNA with a dynamic range of linear quantification between 10(2) and 10(7) RNA molecules. All HIV-1 groups, subtypes, and CRFs could be detected and quantified with equal efficiency, including the group N isolate YBF30 and the group O isolate ANT70. To test the clinical utility of the assay, a series of 62 serum samples containing viruses that encompassed subtypes A through G and CRFs AE and AG of HIV-1 group M were analyzed, and these results were compared to the results of a commercially available assay. This comparison showed that the quantification results correlated highly (R(2) = 0.735) for those subtypes that could be well quantified by both assays (subtypes B, C, D, and F), whereas improved quantification was obtained for subtypes A and G and CRFs AE and AG. A retrospective study with six individuals infected with either a subtype A, B, C, or D or an AG isolate of HIV-1 group M, who were treated with highly active antiretroviral therapy, revealed that the assay was well suited to the monitoring of therapy effects. In conclusion, the newly developed real-time monitored HIV-1 assay is a fast and sensitive assay with a large dynamic range of quantification and is suitable for quantification of most if not all subtypes and groups of HIV-1.

Anti-HIV Agents↗
PubMed2001

One-tube real-time isothermal amplification assay to identify and distinguish human immunodeficiency virus type 1 subtypes A, B, and C and circulating recombinant forms AE and AG.

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires interventions that can prevent transmission of numerous HIV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay that identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the gag gene sequence of the genome of all currently known HIV-1 subtypes and can identify and distinguish among the targeted subtypes as the reaction proceeds, because of the addition of subtype-specific molecular beacons with multiple fluorophores. The combination of isothermal nucleic acid sequence-based amplification and molecular beacons is a new approach in the design of real-time assays. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay on a panel of the culture supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a group N isolate. Assay sensitivity on this panel was 92%, with 89% correct subtype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10(3) copies of HIV-1 RNA per reaction. In 38% of 50 serum samples from HIV-1-infected individuals with a detectable amount of virus, we could identify subtype sequences with a specificity of 94% by using sequencing and phylogenetic analysis as the "gold standard." In conclusion, we showed the feasibility of the approach of using multiple molecular beacons labeled with different fluorophores in combination with isothermal amplification to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would not be suited for clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies.

Base Sequence↗
PubMed2001

Establishment of new transmissible and drug-sensitive human immunodeficiency virus type 1 wild types due to transmission of nucleoside analogue-resistant virus.

Sequence analysis of human immunodeficiency virus type 1 (HIV-1) from 74 persons with acute infections identified eight strains with mutations in the reverse transcriptase (RT) gene at positions 41, 67, 68, 70, 215, and 219 associated with resistance to the nucleoside analogue zidovudine (AZT). Follow-up of the fate of these resistant HIV-1 strains in four newly infected individuals revealed that they were readily replaced by sensitive strains. The RT of the resistant viruses changed at amino acid 215 from tyrosine (Y) to aspartic acid (D) or serine (S), with asparagine (N) as a transient intermediate, indicating the establishment of new wild types. When we introduced these mutations and the original threonine (T)-containing wild type into infectious molecular clones and assessed their competitive advantage in vitro, the order of fitness was in accord with the in vivo observations: 215Y < 215D = 215S = 215T. As detected by real-time nucleic acid sequence-based amplification with two molecular beacons, the addition of AZT or stavudine (d4T) to the viral cultures favored the 215Y mutant in a dose-dependent manner. Our results illustrate that infection with nucleoside analogue-resistant HIV leads in newly infected individuals to mutants that are sensitive to nucleoside analogues, but only a single mutation removed from drug-resistant HIV. Such mutants were shown to be transmissible, stable, and prone to rapid selection for resistance to AZT or d4T as soon as antiretroviral therapy was administered. Monitoring of patients for the presence of new HIV-1 wild types with D, S, or N residues at position 215 may be warranted in order to estimate the threat to long-term efficacy of regimens including nucleoside analogues.

Drug Resistance, Microbial↗
PubMed2001

Evolutionary relationships among parvoviruses: virus-host coevolution among autonomous primate parvoviruses and links between adeno-associated and avian parvoviruses.

The current classification of parvoviruses is based on virus host range and helper virus dependence, while little data on evolutionary relationships among viruses are available. We identified and analyzed 472 sequences of parvoviruses, among which there were (virtually) full-length genomes of all 41 viruses currently recognized as individual species within the family Parvoviridae. Our phylogenetic analysis of full-length genomes as well as open reading frames distinguished three evolutionary groups of parvoviruses from vertebrates: (i) the human helper-dependent adeno-associated virus (AAV) serotypes 1 to 6 and the autonomous avian parvoviruses; (ii) the bovine, chipmunk, and autonomous primate parvoviruses, including human viruses B19 and V9; and (iii) the parvoviruses from rodents (except for chipmunks), carnivores, and pigs. Each of these three evolutionary groups could be further subdivided, reflecting both virus-host coevolution and multiple cross-species transmissions in the evolutionary history of parvoviruses. No parvoviruses from invertebrates clustered with vertebrate parvoviruses. Our analysis provided evidence for negative selection among parvoviruses, the independent evolution of their genes, and recombination among parvoviruses from rodents. The topology of the phylogenetic tree of autonomous human and simian parvoviruses matched exactly the topology of the primate family tree, as based on the analysis of primate mitochondrial DNA. Viruses belonging to the AAV group were not evolutionarily linked to other primate parvoviruses but were linked to the parvoviruses of birds. The two lineages of human parvoviruses may have resulted from independent ancient zoonotic infections. Our results provide an argument for reclassification of Parvovirinae based on evolutionary relationships among viruses.

Animals↗
PubMed2001

In vitro assay for site-specific proteases using bead-attached GFP substrate.

Site-specific proteases, which catalyze cleavage of peptide bonds in specific amino acid sequences of target proteins, play important roles in various biological events of many living organisms. In humans, disruption in regulation of these site-specific proteases can lead to pathological consequences. Here, we report a simple in vitro assay for enzymatic activities of site-specific proteases. This assay system employs a protein substrate molecule that is comprised of (i) His-tag binding module, (ii) cleavage sites, and (iii) green fluorescent protein (GFP) detection module. In this study, prostate-specific antigen (PSA) and Thrombin-specific cleavage sites were introduced into the substrate molecules. The overexpressed GFP substrate protein was purified with the aid of Ni++-charged magnetic beads. On cleavage by either PSA or Thrombin, GFP was released from the bound magnetic beads, enabling a direct measurement of the cleaved product by fluorescence. Detection sensitivity, as well as the kinetics of reaction of PSA cleavage with the GFP substrate, was similar or better than commercially available PSA fluorogenic peptide substrate. This bead-attached GFP substrate was also used for an inhibition assay using a competitive inhibitor of Thrombin. In conclusion, this assay offers a simple fluorescent method for monitoring the activity of the site-specific proteases. Furthermore, this system provides flexible means of incorporating varying sizes of flanking sequences adjacent to the cleavage site, which can be essential for studying the regulatory macromolecular interactions between proteases and their substrates.

Animals↗
PubMed2000

Slower decline of plasma HIV-1 RNA following highly suppressive antiretroviral therapy in primary compared with chronic infection.

OBJECTIVES: To study the effect of highly suppressive antiretroviral therapy on the slopes of HIV-1 RNA decline in primary compared with chronic HIV-1 infection. METHODS: Slopes of HIV-1 RNA decline in plasma were compared before and after the start of highly suppressive antiretroviral therapy from five acutely infected patients who started treatment 2 to 5 weeks following the onset of clinical symptoms. Slopes of decline after the initiation of therapy were also compared with those found in 12 chronically infected individuals on the same therapy. Numbers and percentages of activated CD4 and CD8 T cells at baseline were compared as well. RESULTS: The pre-treatment slopes of HIV-1 RNA decline in the acutely infected individuals increased significantly (P = 0.0001) after the start of anti-retroviral therapy. However, these post-treatment slopes were lower than those found in the chronically infected individuals (P= 0.012). Slopes were inversely correlated (P= 0.012) with baseline HIV-1 RNA. Although the number of CD38+HLA-DR+ CD4 cells was higher in primary infection (P= 0.02), the percentage did not differ between primary and chronic infection. CONCLUSIONS: These findings indicate that antiretroviral therapy contributes significantly to the clearance of HIV-1 during primary infection. Based on the mathematical model the less steep RNA slope following the start of treatment in primary infection can be predicted to be the result of lower clearance of productively infected cells and higher burst size per cell per unit time. This may indicate a growing immune response to HIV-1 in this very early stage of infection.

Acute Disease↗
PubMed2000

Increasing cerebrospinal fluid chemokine concentrations despite undetectable cerebrospinal fluid HIV RNA in HIV-1-infected patients receiving antiretroviral therapy.

Only limited data on cerebrospinal fluid (CSF) HIV-1 RNA responses and markers of local inflammation in CSF during antiretroviral therapy are available. HIV-RNA, soluble tumor necrosis factor (TNF)-receptor (sTNFr)-II, monocyte chemoattractant protein (MCP)-1, and interferon-gamma-inducible protein (IP)-10 were measured in the peripheral blood and CSF of 26 antiretroviral-naive HIV-1-positive patients, who were treated with ritonavir (RTV)/saquinavir (SQV) (n = 5), RTV/SQV/stavudine (d4T; n = 8) or zidovudine (AZT)/lamivudine (3TC)/abacavir/nevirapine/indinavir (n = 13). After 8 to 12 weeks of treatment, CSF HIV-RNA dropped to <400 copies/ml in 1 of 5 patients in the RTV/SQV group, 8 of 8 patients in the RTV/SQV/d4T group, and 9 of 10 patients in the five-drug group. CSF sTNFr-II and IP-10 levels increased in patients with detectable CSF HIV-RNA. However, increases in CSF chemokine and sTNFr-II concentrations were also observed in some patients with good CSF HIV-RNA responses. Moreover, CSF MCP-1 concentrations increased in the whole population after 2 months of treatment. Ongoing residual HIV replication in the central nervous system, which cannot be detected with CSF HIV-RNA measurements, may account for this phenomenon.

Anti-HIV Agents↗
PubMed2000

Identification of a genetic subcluster of HIV type 1 subtype C (C') widespread in Ethiopia.

Others and we have previously shown that subtype C is the predominant HIV-1 subtype and the major cause of AIDS in Ethiopia. The present study shows that subtype C in Ethiopia has a genetic subcluster, designated C', has not increased in frequency, or spread geographically, over the period 1988 (%C' = 23/53) to 1996-1997 (%C' = 26/50). There is no association of the HIV-1 subtype C or subcluster C' with geographic location, time of sample collection, or risk group in Ethiopia. Of 105 randomly collected samples representing 7 different towns in Ethiopia, all but 2 (1 subtype A from Addis Ababa, 1997 and 1 subtype D from Dessie, 1996) belong to subtype C.

Amino Acid Sequence↗