Successful long-term erythrocytapheresis therapy in a patient with symptomatic sickle-cell disease using an arterio-venous fistula.

The use of long-term automated erythrocytapheresis via an arterio-venous fistula for the prevention of recurrent ischaemic stroke in a child with sickle-cell disease (SCD) has not been described previously. We report the successful use of this technique in a 13-year-old boy. A procedure was performed every 36 +/- 6 days, transfusing six units of donor packed red blood cells (RBCs) and discarding 1318 +/- 174 mL of exchanged erythrocytes (Hct 60%). After transfusion of 85 units over 17 months, there is no evidence for iron-overload, red cell alloimmunization, transfusion-transmitted infections, or other complications. Until now, no cerebrovascular ischaemia has been observed.

Detection of Epstein-Barr virus DNA in peripheral blood of paediatric patients with Hodgkin's disease by real-time polymerase chain reaction.

Hodgkin's disease (HD) is commonly associated with latent Epstein-Barr virus (EBV) infection. The aim of our study was a detailed molecular analysis of the EBV status in the peripheral blood of paediatric patients with HD. Blood samples from HD patients were examined before (n=28) and after treatment (n=12). The control group consisted of 20 healthy children and 10 immunosuppressed children with primary EBV infection. EBV load in plasma and peripheral blood mononuclear cells (PBMC) were determined by real time quantitative polymerase chain reaction (RQ-PCR) as recently described. Before treatment, EBV DNA was detected in the plasma of 13/24 EBV-seropositive HD patients, whereas in plasma of healthy controls no EBV DNA was detectable (P<0.001). After treatment, no EBV genomes were found in the plasma of 6 HD patients in stable and complete remission. In contrast, 2/5 HD patients with relapse of disease were positive for EBV DNA in the plasma. In PBMCs, no differences were found in EBV load measured in HD patients before or after treatment and healthy controls. A high EBV load was found in both the plasma and PBMCs of all immunosuppressed patients with primary EBV infection. Thus, EBV DNA detection in the plasma of paediatric HD patients might be of value for non-invasive diagnostic, prognostic and follow-up tests for HD.

Quantitative DNA fragment analysis for detecting low amounts of hepatitis B virus deletion mutants in highly viremic carriers.

Many variants of hepatitis B virus (HBV) with deletions in the viral genome have been identified. Some of these variants are indicator or even effector of a more severe course of hepatitis. These deletion mutants contribute a variable and sometimes very low proportion to the viral population. For early detection of small amounts of deletion mutants among a large number of wild-type genomes, we applied a new screening method designated quantitative fragment analysis (QFA). By QFA the whole viral genome can be scanned for the presence of deletions or insertions of >/=3 nucleotides representing more than 2% of the viral population. Using QFA we showed that an often described deletion of 8 nucleotides is packaged in viral capsids and not a polymerase chain reaction (PCR) artifact. QFA was applied to study the emergence of deletion mutants in a group of 18 pediatric patients who had been infected from a common source while being under multidrug cancer chemotherapy. All patients had developed a highly viremic asymptomatic HBV carrier state. In 3 of these patients 3 different kinds of HBV deletion mutants were found by QFA: 8 bp deletions within the core promoter, core gene deletions from 8 to 86 bp, and large deletions of up to 1,989 bp spanning the precore/core and the preS/S reading frames. PCR primers that specifically amplify deletion variants enabled the detection of additional patients harboring the investigated variant.

A new fingerprint method for sequence analysis of chromosomal translocations at the genomic DNA level.

Chromosomal rearrangements constitute a significant feature of leukemogenesis and malignant transformation in general. Nucleotide patterns in the immediate vicinity of the break point may provide important information about the underlying causalities, eg illegitimate recombination events mediated by topoisomerase II, Alu repeats, or VDJ recombinase. In order to facilitate the determination of those DNA patterns, we developed a new fingerprint approach. In a first step, two DNA fragments were independently amplified by long distance PCR: the genomic region carrying the break point and the normal nonrearranged counterpart. Subsequently, both PCR products were digested with restriction enzymes, end-labelled with a fluorescent dye, and subjected to high resolution polyacrylamide gel electrophoresis. By comparing the restriction patterns of the rearranged and the nonrearranged PCR fragments, the break points could be easily localized within a size range coverable by a single sequencing reaction. Finally, the exact DNA sequence across the break point was directly determined. The 'fingerprint' technique is fast, reliable and enables the assay of multiple samples in parallel.

Alcohol suppresses endothelium-dependent relaxation in rat mesenteric vascular beds.

The effects of prolonged infusions of ethanol on endothelium-dependent vasorelaxation induced by acetylcholine and adenosine triphosphate (ATP) and on endothelium-independent relaxation induced by papaverine were studied and compared in isolated perfused rat mesenteric artery preparations. Infusion of ethanol over 60 minutes at concentrations of 1.6, 4.7, 6.3, and 7.9 mg/ml caused concentration-related inhibition of norepinephrine-induced vasoconstriction. In preparations infused with 6.3 and 7.9 mg/ml, this effect reached a maximum after 10-20 minutes but had vanished by the end of the infusion; 1 hour after the end of the infusion, the effects of norepinephrine were potentiated by 71% and 108%, respectively. Acetylcholine-induced vasorelaxation (EC50 3.0 ng/ml in controls) was significantly reduced after 6.3 mg/ml ethanol infusion and totally abolished after 7.9 mg/ml ethanol infusion. ATP-induced vasorelaxation (EC50 180 ng/ml in controls) was also abolished after 7.9 mg/ml of ethanol infusion. By contrast, the vasorelaxant effects of papaverine were not affected by 7.9 mg/ml ethanol infusion. Light-microscopic examination revealed that the endothelial cells were present in ethanol-treated and in control mesenteric arterial beds. These observations indicate that ethanol suppresses endothelium-dependent vasorelaxation without apparent removal of the endothelial cells. The compromised relaxant capacity of the endothelium after ethanol and the resultant intensification of the vasoconstrictor response to norepinephrine may contribute to the development of vascular diseases such as hypertension and stroke.

Arthritis in MRL/LPR mice and in collagen II sensitized DBA-1 mice and their use in pharmacology.

For assessment of anti-arthritic drugs, we used mice of the autoimmune MRL/lpr strain and DBA-1 mice sensitized with collagen type II. Our studies showed that in these two mouse arthritis models, unlike the classical rat adjuvant arthritis, the nonsteroidal antiinflammatory compounds tested were either ineffective or minimally affected a chosen number of humoral parameters and the incidence and severity of arthritis. Interestingly, both arthritis models showed a distinct pharmacological pattern in response to the slow-acting anti-rheumatic drugs, whereas steroids and cyclophosphamide inhibited both arthritic processes. Therefore, these two mouse models are useful for studying the immunopathological events operating in chronic inflammation and could potentially serve the characterization of new antirheumatic drugs.

Trisomy 18 and cyclopia.

A stillborn male infant with cyclopia, holoprosencephaly, extracephalic malformations, and trisomy 18 is described. The importance of chromosome studies in infants with severe congenital malformations is discussed.