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Biomedical subjects

J Wagstaff

Publications and source records attributed to J Wagstaff.

At least 19 recordsLinked to original sources

HPV16 E6 confers p53-dependent and p53-independent phenotypes in the epidermis of mice deficient for E6AP.

High-risk human papillomaviruses are the causative agents of cervical and other anogenital cancers. In these cancers, two viral oncogenes, E6 and E7, are expressed. E6 is best known for its ability to inactivate the tumor suppressor p53, which is thought to arise through ubiquitin-mediated degradation of p53 and involve a ternary complex between E6, p53 and the E3 ligase, E6AP. In mice transgenic for wild-type HPV16 E6, its expression leads to epithelial hyperplasia and an abrogation of normal cellular responses to DNA damage. Whereas only the latter phenotype is dependent upon E6's inactivation of p53, both are reduced in transgenic mice expressing an E6 mutant severely reduced in its binding to E6AP and other cellular proteins that bind E6 through a shared alpha-helix motif. Here, we investigated whether E6AP is required for the induction of the above phenotypes through the use of both E6AP-mutant and E6AP-null mice. E6, in the absence of E6AP retains an ability to induce epithelial hyperplasia, abrogate DNA damage responses and inhibit the induction of p53 protein following exposure to ionizing radiation. We conclude that E6 is able to induce both p53-dependent and p53-independent phenotypes through E6AP-independent pathways in the mouse.

Animals↗

Fast cerebellar oscillation associated with ataxia in a mouse model of Angelman syndrome.

Ataxia may result from various cerebellar cortex dysfunctions. It is included in the diagnostic criteria of Angelman syndrome, a human neurogenetic condition. In order to better understand the cerebellar dysfunction in this condition, we recorded in vivo cerebellar activity in a mouse model of Angelman syndrome produced by null mutation of the maternal Ube3a gene. We found fast oscillation (approximately 160 Hz) in the cerebellar cortex sustained by abnormally increased Purkinje cell firing rate and rhythmicity. This oscillation is inhibited by sensory stimulation and gap junction or GABA(A) receptor blockers. A physiologically similar oscillation was previously found in mice lacking calcium-binding proteins that also present ataxia, but never in wild-type mice. We propose that fast oscillation in the cerebellar cortex is implicated in the cerebellar symptomatology of Angelman syndrome.

Angelman Syndrome↗

Discordant phenotypes in first cousins with UBE3A frameshift mutation.

Mutations have been found in the UBE3A gene (E6-AP ubiquitin protein ligase gene) in many Angelman syndrome (AS) patients with no deletion, no uniparental disomy, and no imprinting defect. UBE3A mutations are more frequent in familial than in sporadic patients and the mutations described so far seem to cause similar phenotypes in the familial affected cases. Here we describe two first cousins who have inherited the same UBE3A frameshift mutation (duplication of GAGG in exon 10) from their asymptomatic mothers but present discordant phenotypes. The proband shows typical AS features. Her affected cousin shows a more severe phenotype, with asymmetric spasticity that led originally to a diagnosis of cerebral palsy. Proband's brain MRI shows mild cerebral atrophy while her cousin's brain MRI shows severe brain malformation. This family demonstrates that, although brain malformation is unusual in AS, presence of a brain malformation does not exclude the diagnosis of AS. Also, this UBE3A mutation was transmitted from the cousin's grandfather to only two sisters among eight full siblings, raising the hypothesis of mosaicism for this mutation.

Female↗

A systematic review of the relation between interleukin-2 schedule and outcome in patients with metastatic renal cell cancer.

In Europe, interleukin 2 (IL-2) is one of the two treatment modalities officially approved for patients with metastatic renal cell cancer. Traditionally, IL-2 has been administered by three different routes: intermittent bolus injection (BIV), continuous intravenous infusion (CIV) and subcutaneous injection (SC). There have been few randomized trials designed to compare these routes of administration. This paper describes a systematic review of the literature in which an attempt has been made to determine which schedule of administration is superior. Heterogeneity of the data makes firm conclusions difficult. It appears that the number of complete remissions (CR) is similar between BIV and SC routes and that these are higher than for CIV schedules. The durability of the CRs induced by BIV appeared superior to those induced by SC IL-2 and definitely higher than with CIV protocols. This analysis highlights some of the difficulties of using evidence-based medicine to determine standard of care when the clinical-trial data are heterogeneous. These data emphasize the importance of randomized clinical trials in determining what should be regarded as optimum therapy.

Antineoplastic Agents↗

PIP2 binding residues of Kir2.1 are common targets of mutations causing Andersen syndrome.

BACKGROUND: Mutations in KCNJ2, the gene encoding the inward-rectifying K+ channel Kir2.1, cause the cardiac, skeletal muscle, and developmental phenotypes of Andersen-Tawil syndrome (ATS; also known as Andersen syndrome). Although pathogenic mechanisms have been proposed for select mutations, a common mechanism has not been identified. METHODS: Seventeen probands presenting with symptoms characteristic of ATS were evaluated clinically and screened for mutations in KCNJ2. The results of mutation analysis were combined with those from previously studied subjects to assess the frequency with which KCNJ2 mutations cause ATS. RESULTS: Mutations in KCNJ2 were discovered in nine probands. These included six novel mutations (D71N, T75R, G146D, R189I, G300D, and R312C) as well as previously reported mutations R67W and R218W. Six probands possessed mutations of residues implicated in binding membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2). In total, mutations in PIP(2)-related residues accounted for disease in 18 of 29 (62%) reported KCNJ2 -based probands with ATS. Also reported is that mutation R67W causes the full clinical triad in two unrelated males. CONCLUSIONS: The novel mutations corresponding to residues involved in Kir2.1 channel-PIP2 interactions presented here as well as the overall frequency of mutations occurring in these residues indicate that defects in PIP2 binding constitute a major pathogenic mechanism of ATS. Furthermore, screening KCNJ2 in patients with the complex phenotypes of ATS was found to be invaluable in establishing or confirming a disease diagnosis as mutations in this gene can be identified in the majority of patients.

Abnormalities, Multiple↗

Neurons but not glial cells show reciprocal imprinting of sense and antisense transcripts of Ube3a.

The human UBE3A gene shows brain-specific partial imprinting, and lack of a maternally inherited allele causes Angelman syndrome (AS), which is characterized by neurobehavioral anomalies. In several AS model mice, imprinted Ube3a expression is detected predominantly in the hippocampus, cerebellar Purkinje cells and the olfactory bulb. Therefore, imprinting of mouse Ube3a is thought to be region-specific with different levels of silencing of the paternal Ube3a allele in different brain regions. To determine cell types of imprinted Ube3a expression, we analyzed its imprinting status in embryonic brain cells by using primary cortical cell cultures. RT-PCR and immunofluorescence were performed to determine the allelic expression of the gene. The Ube3a gene encodes two RNA transcripts in the brain, sense and antisense. The sense transcript was expressed maternally in neurons but biallelically in glial cells in the embryonic brain, whereas the antisense transcript was expressed only in neurons and only from the paternal allele. Our data present evidence of brain cell type-specific imprinting, i.e. neuron-specific imprinting of Ube3a in primary brain cell cultures. Reciprocal imprinting of sense and antisense transcripts present only in neurons suggests that the neuron-specific imprinting mechanism is related to the lineage determination of neural stem cells.

Angelman Syndrome↗

Flow cytometric quantification of tumour endothelial cells; an objective alternative for microvessel density assessment.

Assessment of microvessel density by immunohistochemical staining is subject to a considerable inter-observer variation, and this has led to variability in correlation between microvessel density and clinical outcome in different studies. In order to improve the method of microvessel density measurement in tumour biopsies, we have developed a rapid, objective and quantitative method using flow cytometry on frozen tissues. Frozen tissue sections of archival tumour material were enzymatically digested. The single-cell suspension was stained for CD31 and CD34 for flow cytometry. The number of endothelial cells was quantified using light scatter- and fluorescence-characteristics. Tumour endothelial cells were detectable in a single cell suspension, and the percentage of endothelial cells detected in 32 colon carcinomas correlated highly (r=0.84, P<0.001) with the immunohistochemical assessment of microvessel density. Flow cytometric endothelial cells quantification was found to be more sensitive especially at lower levels of immunohistochemical microvessel density measurement. The current method was found to be applicable for various tumour types and has the major advantage that it provides a retrospective and quantitative approach to the angiogenic potential of tumours.

Cell Count↗

A phase II study of active specific immunotherapy and 5-FU/Leucovorin as adjuvant therapy for stage III colon carcinoma.

Active specific immunotherapy, using vaccines with autologous tumour cells and BCG, significantly reduces the rate of tumour recurrence in stage II colon cancer patients, while no clinical benefit has yet been observed in stage III patients. Adjuvant treatment with 5-Fluorouracil/Leucovorin is now considered standard therapy for stage III colon carcinoma and results in an absolute survival benefit of approximately 10%. Yet, the 5-year overall survival rate of stage III colon cancer patients is only 40-50%. Combining chemotherapy and immunotherapy might improve prognosis for stage III patients, especially when considering that active specific immunotherapy and chemotherapy have shown synergistic effects in pre-clinical tumour models. We performed a phase II study with 56 patients, using the combination of active specific immunotherapy and chemotherapy as an adjuvant therapy in stage III colon cancer patients to assess the influence of 5-Fluorouracil/Leucovorin on anti-tumour immunity induced by autologous tumour cell vaccinations. Anti-tumour immunity was measured before and after chemotherapy by means of delayed type hypersensitivity reactions, taken 48 h after the third and the fourth vaccination. We also investigated the toxicity of this combined immuno-chemotherapy treatment. Delayed type hypersensitivity reactions before chemotherapy had a median size of 20.3 mm, while after chemotherapy delayed type hypersensitivity size was 18.4 mm (P=0.01), indicating that chemotherapy hardly affected anti-tumour immunity. The severity of ulcers at the BCG vaccination sites was comparable to previous studies. In 30% of the patients grade III or grade IV chemotherapy related toxicity was seen; this is comparable to what is normally observed after adjuvant chemotherapy alone. This study shows that the active specific immunotherapy-induced anti-tumour immune response is only minimally impaired by consecutive chemotherapy and that the combined treatment of stage III colon cancer patients with active specific immunotherapy and 5-Fluorouracil/Leucovorin does not cause unexpected toxicity.

Adult↗

Non-myeloablative allogeneic stem cell transplantation in patients with solid tumours and patients with a haematological malignancy.

Allogeneic stem cell transplantation (allo-SCT) is a treatment option for several haematological tumours, not only to regain haematopoiesis after myeloablative chemotherapy and/or radiotherapy, but also for its apparent antitumour effect. This so-called graft-versus-tumour effect might not only be effective against haematological tumours, where it has best been proven, but also against solid tumours. To reduce morbidity and treatment-related mortality of allo-SCT, efforts are being made to establish engraftment of allogeneic stem cells after a non-myeloablative conditioning regimen and create a 'mini transplantation'. Such a therapy relies more heavily on the graft-versus-malignancy effect than on the antitumour effect exerted by the chemotherapy/radiotherapy. Here, we report the outcomes of 15 patients with haematological disease or solid tumours who underwent an SCT in the University Hospital Maastricht after a non-myeloablative fludarabine/cyclophosphamide conditioning regimen. Although results are promising, adjustments will be needed to ensure long-term stable engraftment and optimise the antitumour effect.

Adult↗

Parent-specific complementary patterns of histone H3 lysine 9 and H3 lysine 4 methylation at the Prader-Willi syndrome imprinting center.

The Prader-Willi syndrome (PWS)/Angelman syndrome (AS) region, on human chromosome 15q11-q13, exemplifies coordinate control of imprinted gene expression over a large chromosomal domain. Establishment of the paternal state of the region requires the PWS imprinting center (PWS-IC); establishment of the maternal state requires the AS-IC. Cytosine methylation of the PWS-IC, which occurs during oogenesis in mice, occurs only after fertilization in humans, so this modification cannot be the gametic imprint for the PWS/AS region in humans. Here, we demonstrate that the PWS-IC shows parent-specific complementary patterns of H3 lysine 9 (Lys9) and H3 lysine 4 (Lys4) methylation. H3 Lys9 is methylated on the maternal copy of the PWS-IC, and H3 Lys4 is methylated on the paternal copy. We suggest that H3 Lys9 methylation is a candidate maternal gametic imprint for this region, and we show how changes in chromatin packaging during the life cycle of mammals provide a means of erasing such an imprint in the male germline.

Alleles↗

Genetics beyond Mendel. Understanding nontraditional inheritance patterns.

Many medical conditions that clearly have a strong genetic component are not transmitted in a straightforward dominant, recessive, or X-linked pattern. Recent progress in understanding other modes of inheritance, such as imprinting, trinucleotide repeat expansion, mitochondrial inheritance, and mosaicism, has allowed us to solve many of these hereditary puzzles. Such advances have led to improvements in diagnosis and genetic counseling for patients affected with these disorders and should be valuable in development of effective therapies for some of these disorders in the future.

Angelman Syndrome↗

Skin tests predict survival after autologous tumor cell vaccination in metastatic melanoma: experience in 81 patients.

BACKGROUND: Currently there is no standard adjuvant treatment following surgical resection of metastatic melanoma. We investigated whether surgery followed by autologous tumor cell-BCG vaccination was beneficial for malignant melanoma patients. In this study we focus on the prognostic value of DTH response following vaccination therapy. PATIENTS AND METHODS: Eighty-one patients with AJCC stage III and IV melanoma were selected. Whenever feasible, radical metastasectomy was performed. ASI was initiated by the administration of three weekly intra-cutaneous vaccinations with 10(7) irradiated autologous tumor cells, starting four weeks after surgery. Depending on the size of DTH response to the first three injections, subsequent vaccinations were planned. The first two vaccines also contained 10(7) BCG organisms as an immune stimulatory adjuvant. RESULTS: Induration as well as erythema correlated strongly with survival (P < 0.0001 and P = 0.0004). After radical metastasectomy in stage III melanoma patients a five-year survival of 48% was observed. In stage IV disease, a five-year survival of 34% was seen, after radical surgery had been performed. When macroscopic disease was present at start of vaccination treatment, no clinical responses occurred. Apart from transient skin ulceration at the site of BCG-containing vaccinations, no serious side effects were observed. CONCLUSIONS: This study shows that large-scale preparation of autologous melanoma cell vaccines is feasible. while vaccination results in DTH responses that correlate significantly with survival. ASI seemed to be beneficial in stage III and stage IV melanoma when given in the adjuvant setting, while causing only very mild side effects.

Adjuvants, Immunologic↗

[Angiogenesis and the regulation of antitumor defense].

Formation of new blood vessels is a prerequisite for outgrowth of solid tumours and metastasis. Leukaemia and lymphoma are also dependent on angiogenesis. Inhibition of angiogenesis is therefore a promising therapy for all cancer types. Angiogenic factors reduce the expression of tumour endothelial adhesion molecules for leukocytes, which enables tumours to escape the inflammation response. Antiangiogenic factors induce not only starvation of the tumour owing to deprivation of vasculature, but also re-expression of adhesion molecules, resulting in increased leukocyte infiltration. On the basis of de novo design of chemokines with antiangiogenic properties, novel inhibitors of angiogenesis are developed and selected for their ability to induce a tumour inflammatory reaction.

Angiogenesis Inducing Agents↗

Prolonged neoadjuvant chemotherapy with GM-CSF in locally advanced breast cancer.

BACKGROUND: Neoadjuvant chemotherapy improves survival in patients with locally advanced breast cancer (LABC). Usually three to four cycles of conventional-dose neoadjuvant chemotherapy are administered prior to local therapy, and another three cycles thereafter. In an attempt to improve results, we increased the dosages and applied GM-CSF, which, besides being a hematopoietic growth factor, has become increasingly known for its immunostimulatory effects, which might enhance the antitumor effect. METHODS: Forty-two patients with stage IIIA or IIIB breast cancer were treated with doxorubicin (A) (90 mg/m2) and cyclophosphamide (C) (1,000 mg/m2) at three-weekly intervals. In the second and fourth cycle a 10% dose reduction of both agents was applied. On the second day GM-CSF 250 micrograms/m2/day was started and given for 10 days. Initially, some patients were treated with < or = four cycles, but as the study progressed and toxicity appeared tolerable, six cycles were given whenever possible. After the chemotherapy, patients underwent surgery and postoperative radiotherapy. RESULTS: The response rate for the whole group to AC was 98% (95% confidence interval 94%-100%), with a clinical complete response rate of 50% (95% confidence interval 35%-65%). Six patients had a pathological complete response. Median follow-up from the start of chemotherapy is 49 months (range 10-100). The disease-free survival (DFS) at three years is 57% and the overall survival (OS) at three years is 79%. There is a significant trend for improved DFS (p = 0.0000) and OS (p = 0.0002) with increasing number of cycles. CONCLUSION: The results of the present study with neoadjuvant dose-intensive AC chemotherapy and GM-CSF compare favorably with previous studies in patients with LABC. This is most apparent in patients who received six cycles of neoadjuvant chemotherapy. We hypothesize that these encouraging results are probably related to the prolonged presence of the primary tumor, and to the long-term administration of GM-CSF with the primary tumor and axillary lymph nodes in situ. Therefore, a randomized study is warranted. We already initiated an international randomized trial in patients with LABC in order to answer two questions. First, does prolonged neoadjuvant chemotherapy result in an improved DFS and OS in comparison with the conventional approach, and secondly, what is the effect of GM-CSF in this approach in comparison with G-CSF?

Adult↗

Complex familial rearrangement of chromosome 9p24.3 detected by FISH.

We describe a newborn male with minor facial anomalies, pyloric stenosis, and a chromosome rearrangement that involves deletion and addition of material at 9p24.3. Routine studies showed a 46, XY, add (9) (p24) karyotype. Fluorescence in situ hybridization (FISH) with two different whole chromosome probes for chromosome 9 failed to identify whether the additional material was derived from that chromosome. FISH with single copy YAC probes from 9p24 (D9S1858, D9S1813 and D9S54) showed a more complex rearrangement involving a deletion at D9S1858 but not at D9S1813 or D9S54. Parental chromosome studies demonstrated an apparently identical 9p abnormality in the patient's mother. This report describes a familial chromosome rearrangement in an abnormal child and his normal mother and demonstrates the use and limitations of FISH in characterizing chromosomal abnormalities.

Abnormalities, Multiple↗