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Biomedical subjects

M J Willemse

Publications and source records attributed to M J Willemse.

13 recordsLinked to original sources
PubMed2000

T cell receptor gamma (TCRG) gene rearrangements in T cell acute lymphoblastic leukemia refelct 'end-stage' recombinations: implications for minimal residual disease monitoring.

The T cell receptor gamma (TCRG) gene configuration was established in a large series of 126 T cell acute lymphoblastic leukemia (T-ALL) patients using combined Southern blotting (SB) and heteroduplex PCR analyses. The vast majority of TALL (96%) displayed clonal TCRG gene rearrangements, with biallelic recombination in 91% of patients. A small immature subgroup of CD3- T-ALL (n = 5) had both TCRG genes in germline configuration, three of them having also germline TCRD genes. In five patients (4%) combined SB and PCR results indicated oligoclonality. In five rearrangements detected by SB, the Vgamma gene segment could not be identified suggesting illegitimate recombination. Altogether, 83% of TCRG gene rearrangements involved either the most upstream Vgamma2 gene (including four cases with interstitial deletion of 170 bp in Vgamma2) and/or the most downstream Jgamma2.3 segment, which can be perceived as 'end-stage' recombinations. Comparative analysis of the TCRG gene configuration in the major immunophenotypic subgroups indicated that TCRgammadelta+ T-ALL display a less mature immunogenotype as compared to TCRalphabeta+ and most CD3- cases. This was reflected by a significantly increased usage of the more downstream Vgamma genes and the upstream Jgamma1 segments. Comparison between adult and pediatric T-ALL patients did not show any obvious differences in TCRG gene configuration. The high frequency, easy detectability, rare oligoclonality, and frequent 'end-stage' recombinations make TCRG gene rearrangements principal targets for PCR-based detection of minimal residual disease (MRD) in T-ALL. We propose a simple heteroduplex PCR strategy, applying five primer combinations, which results in the detection of approximately 95% of all clonal TCRG gene rearrangements in T-ALL. This approach enables identification of at least one TCRG target for MRD monitoring in 95% of patients, and even two targets in 84% of T-ALL.

Adult↗
PubMed2000

Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia.

Large-scale clinical studies on detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) have shown that quantification of MRD levels is needed for reliable MRD-based risk group classification. Recently, we have shown that 'real-time' quantitative PCR (RQ-PCR) can be applied for this purpose using patient-specific immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets with TaqMan probes at the position of the junctional region and two germline primers. Now, we tested an alternative approach on 35 immunoglobulin heavy chain (IGH) gene rearrangements, by designing three germline JH TaqMan probes to be used in combination with one of six corresponding germline JH primers and one allele specific oligonucleotide (ASO) primer complementary to the junctional region. In nine cases in which both approaches were compared, at least similar (n = 4) or slightly higher (n= 5) maximal sensitivities were obtained using an ASO primer. The ASO primer approach reached maximal sensitivities of at least 10(-4) in 33 out of 35 IGH rearrangements. The reproducible range for accurate quantification spanned four to five orders of magnitude in 31 out of 35 cases. In 13 out of 35 rearrangements the stringency of PCR conditions had to be increased to remove or diminish background signals; this only concerned the frequently occurring JH4, JH5 and JH6 gene rearrangements. After optimization of the conditions (mainly by increasing the annealing temperature), only occasional aspecific amplification signals were observed at high threshold cycle (CT) values above 42 cycles and at least six cycles above the CT value of the detection limit. Hence, these rare aspecific signals could be easily discriminated from specific signals. We conclude that the here presented set of three germline JH Taq-Man probes and six corresponding germline JH primers can be used to develop patient-specific RQ-PCR assays, which allow accurate and sensitive MRD analysis in almost all IGH gene rearrangements. These results will facilitate standardized RQ-PCR analysis for MRD detection in large clinical studies.

DNA Probes↗
PubMed2000

Regenerating normal B-cell precursors during and after treatment of acute lymphoblastic leukaemia: implications for monitoring of minimal residual disease.

We studied 57 childhood acute lymphoblastic leukaemia (ALL) patients who remained in continuous complete remission after treatment according to the Dutch Childhood Leukaemia Study Group ALL-8 protocols. The patients were monitored at 18 time points during and after treatment [640 bone marrow (BM) and 600 blood samples] by use of cytomorphology and immunophenotyping for the expression of TdT, CD34, CD10 and CD19. Additionally, 60 BM follow-up samples from six patients were subjected to clonality assessment via heteroduplex polymerase chain reaction (PCR) analysis of immunoglobulin VH-JH gene rearrangements. We observed substantial expansions of normal precursor B cells in regenerating BM not only after maintenance therapy but also during treatment. At the end of the 2-week intervals after consolidation and reinduction treatment, B-cell-lineage regeneration was observed in BM with a large fraction of immature CD34+/TdT+ B cells. In contrast, in regenerating BM after cessation of maintenance treatment, the more mature CD19+/CD10+ B cells were significantly increased, but the fraction of immature CD34+/TdT+ B cells was essentially smaller. Blood samples showed a profound B-cell lymphopenia during treatment followed by a rapid normalization of blood B cells after treatment, with a substantial CD10+ fraction (10-30%). Heteroduplex PCR analysis confirmed the polyclonal origin of the expanded precursor B cells in regenerating BM. This information regarding the regeneration of BM is essential for the correct interpretation of minimal residual disease studies.

Adolescent↗
PubMed1996

In vivo properties of a feline herpesvirus type 1 mutant carrying a lacZ insertion at the gI locus of the unique short segment.

The major cause of upper respiratory tract disease in cats in the feline herpesvirus type 1 (FHV-1). FHV-1 replicates predominantly in the mucosal epithelium of the oral and nasal cavities, local immunity should therefore be the key target for vaccine development. Recombinant DNA technology enables accurate manipulation of the genetic content of the FHV-1 genome, hopefully resulting in a next generation of safe vaccine strains that can be used intranasally in cats. Integration of a reporter gene into the glycoprotein I (gI) homologous gene of FHV-1, resulted in strain C4-1-4-1 which displayed reduced replication not only in cell culture but also in the respiratory tract of infected cats. Oronasal application of strain C4-1-4-1 caused less severe clinical signs than local administration of the parent virus. In addition, oronasally vaccinated cats were better protected against the clinical signs of a challenge infection than cats vaccinated subcutaneously.

Alphaherpesvirinae↗
PubMed1996

Evaluation of subunit vaccines against feline immunodeficiency virus infection.

Subunit vaccines prepared against feline immunodeficiency virus (FIV) infection were evaluated in two trials. First, cats were immunized with bacterial expression products of an envelope fragment that contained the V3 neutralization domain of the FIV surface protein fused to either galactokinase (K-SU3) or glutathione-S-transferase (G-SU3). Quantitative and qualitative differences in the humoral immune response were observed with three adjuvants of which Quil A was the best in terms of total and virus neutralizing antibody. Notwithstanding the responses induced, 19 of 20 immunized cats did not resist challenge and became infected. To determine whether priming with a live viral vector would confer protection, cats were inoculated oronasally and subcutaneously with a feline herpesvirus (FHV) mutant expressing the FIV env gene; two booster immunizations followed using the K-SU3 product in either Quil A or a mineral oill Al(OH)3 adjuvant. FIV-specific antibody responses were only weak, and the vaccinates did not withstand challenge with a low dose of homologous virus.

Animals↗
PubMed1996

Vaccination against feline leukaemia using a new feline herpesvirus type 1 vector.

A recombinant feline herpesvirus type 1 (FHV-1) was constructed expressing the envelope glycoprotein gene from feline leukaemia virus (FeLV). The expression cassette containing the long terminal repeat promoter from Rous sarcoma virus was stably integrated at the locus downstream of the gC homologue in FHV-1. Oronasal vaccination with recombinant FHV-1 engendered significant protection against challenge with the homologous FelV-A/Glasgow-1 isolate. Three of four vaccinated cats did not become viraemic for FeLV and developed serum neutralizing antibodies while five of six controls became persistently infected after challenge. However, latent FeLV was detected at 12 weeks after challenge in bone marrow cultures of all animals except one. The potential of this new vector to protect against FeLV was compared with previous reports using live recombinant vaccines.

Administration, Intranasal↗
PubMed1995

Transcriptional analysis of the short segment of the feline herpesvirus type 1 genome and insertional mutagenesis of a unique reading frame.

Transcription mapping was performed in the short region of the feline herpesvirus type 1 (FHV-1) genome as a first approach to the functional analysis of open reading frames encoding the homologs of the herpes simplex virus type 1 (HSV-1) gD, gl, gE, US9, and probably also US8.5. All reading frames appeared to be transcribed. Transcripts were grouped into two nested RNA sets; namely, the coterminal transcripts of gD and gl and the coterminal transcripts of gE, US8.5, and US9. This situation was similar to that found in the equivalent region of HSV-1. The FHV-1 ORFs US8.5 and US9 have not been described previously. Sequence analysis and comparison of the putative polypeptide encoded by US8.5 revealed that this ORF was unique to FHV-1. However, US8.5 of FHV-1 might be functionally related to its positional homologs in HSV-1 and equine herpesvirus type 1. In all three viruses, US8.5 does not seem to be critical for virus propagation in cell culture. This was shown for FHV-1 by isolating a mutant containing an insertion in US8.5 and comparing its growth properties in cell culture to those of the parent virus G2620. With regard to US9, conscientious amino acid sequence alignment of FHV-1 US9 and homologs in related herpesviruses suggests that this particular protein could contribute to the virus infectivity in vivo. This speculation was based on the highly conserved C-terminus of US9, starting with a characteristic YYSES motif and followed by a nuclear target sequence and a transmembrane region.

Alphaherpesvirinae↗
PubMed1994

The gene downstream of the gC homologue in feline herpes virus type 1 is involved in the expression of virulence.

Feline herpesvirus type 1 (FHV-1) mutants were constructed, carrying a beta-galactosidase marker gene integrated into the region downstream of the gene encoding the homologue of glycoprotein C (gC) of herpes simplex virus type 1. In cell culture, no differences in replication were observed between mutants and the parent FHV-1 strain. However, in experimentally infected cats, mutants caused fewer clinical signs after oronasal administration although they replicated to the same extent as the parental strain. Sequence analysis in the region of the UL segment surrounding the insertion site revealed an open reading frame (ORF 2) encoding a putative polypeptide of 21K. RNA analysis indicated a corresponding transcript of 0.8 kb that was detected late after infection of cells in culture. This particular UL locus downstream of the gC gene has not been thoroughly investigated in any of the herpesviruses. The putative gene product showed only limited evolutionary conservation since similarity could be found only with the assumed homologue of equine herpesvirus type 1. Further characterization of this newly identified FHV-1 gene involved in virulence may provide insight into the development of disease owing to herpesvirus infection.

Algorithms↗
PubMed1993

Avian herpesvirus as a live viral vector for the expression of heterologous antigens.

Control of Marek's disease in the poultry industry has been successfully achieved for several decades by large-scale vaccination of day-old chickens with live herpesvirus of turkeys (HVT) strains. Several features of this virus including lack of pathogenicity and long-term immune protection due to a persistent viraemic infection made us decide to use HVT as a live viral vector for the expression of foreign antigens. Potential sites for the integration of foreign DNA in the unique short region of the HVT genome were identified by the insertion of a beta-galactosidase expression cassette. Vaccination trials with recombinant virus strains indicated that the marker gene was expressed and stably maintained during animal passage. Based on an insertion site mapping in one of the open reading frames of the unique short region, a general recombination vector was designed for the integration of foreign genes into HVT. Recombinant virus-directed expression of individual antigens from Newcastle disease virus was driven by a strong promoter element derived from the lung terminal repeat sequence of Rous sarcoma virus.

Amino Acid Sequence↗