Diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters (DGONC) - a molecularly defined glioneuronal CNS tumour class displaying recurrent monosomy 14.

AIMS: DNA methylation-based central nervous system (CNS) tumour classification has identified numerous molecularly distinct tumour types, and clinically relevant subgroups among known CNS tumour entities that were previously thought to represent homogeneous diseases. Our study aimed at characterizing a novel, molecularly defined variant of glioneuronal CNS tumour. PATIENTS AND METHODS: DNA methylation profiling was performed using the Infinium MethylationEPIC or 450 k BeadChip arrays (Illumina) and analysed using the 'conumee' package in R computing environment. Additional gene panel sequencing was also performed. Tumour samples were collected at the German Cancer Research Centre (DKFZ) and provided by multinational collaborators. Histological sections were also collected and independently reviewed. RESULTS: Genome-wide DNA methylation data from >25 000 CNS tumours were screened for clusters separated from established DNA methylation classes, revealing a novel group comprising 31 tumours, mainly found in paediatric patients. This DNA methylation-defined variant of low-grade CNS tumours with glioneuronal differentiation displays recurrent monosomy 14, nuclear clusters within a morphology that is otherwise reminiscent of oligodendroglioma and other established entities with clear cell histology, and a lack of genetic alterations commonly observed in other (paediatric) glioneuronal entities. CONCLUSIONS: DNA methylation-based tumour classification is an objective method of assessing tumour origins, which may aid in diagnosis, especially for atypical cases. With increasing sample size, methylation analysis allows for the identification of rare, putative new tumour entities, which are currently not recognized by the WHO classification. Our study revealed the existence of a DNA methylation-defined class of low-grade glioneuronal tumours with recurrent monosomy 14, oligodendroglioma-like features and nuclear clusters.

Imaging Characteristics of Wingless Pathway Subgroup Medulloblastomas: Results from the German HIT/SIOP-Trial Cohort.

BACKGROUND AND PURPOSE: In addition to the 4 histopathologically defined entities of medulloblastoma, 4 distinct genetically defined subgroups have been included in the World Health Organization classification of 2016. The smallest subgroup is the medulloblastoma with activated wingless pathway. The goal of this study was to identify a typical MR imaging morphology in a larger number of pediatric patients with wingless pathway medulloblastoma. MATERIALS AND METHODS: From January 2001 to October 2017, of 75 patients with histologically confirmed and molecularly subgrouped wingless pathway medulloblastomas recruited to the German Pediatric Brain Tumor (HIT) trials, 38 patients (median age, 12.8 ± 4.6 years at diagnosis; 24 [63.2%] female) had preoperative imaging that passed the entry criteria for this study. Images were rated by the local standardized imaging criteria of the National Reference Center of Neuroradiology. Additionally, a modified laterality score was used to determine tumor localization and extension. RESULTS: Twenty-eight of 38 (73.7%) were primary midline tumors but with a lateral tendency in 39.3%. One extensively eccentric midline tumor was rated by the laterality score as in an off-midline position. Five tumors were found in the cerebellopontine angle; 3, in the deep white matter; and 2, in a cerebellar hemisphere. Leptomeningeal dissemination was rare (11.5%). In 60.5%, intratumoral blood-degradation products were found, and 26.3% showed cysts with blood contents. CONCLUSIONS: According to our observations, wingless pathway medulloblastomas are not preferentially off-midline tumors as postulated in previous studies with smaller wingless pathway medulloblastoma cohorts. Dense intratumoral blood-degradation products and cysts with blood contents are frequently found and might help to differentiate wingless pathway medulloblastoma from other medulloblastoma subtypes.

Integrated molecular characterization of IDH-mutant glioblastomas.

AIMS: Mutations of isocitrate dehydrogenase (IDH)1/2 affect almost all astrocytomas of WHO grade II and III. A subset of IDH-mutant astrocytic tumours progresses to IDH-mutant glioblastoma or presents with the histology of a glioblastoma at first presentation. We set out here to assess the molecular spectrum of IDH-mutant glioblastomas. METHODS: We performed an integrated molecular analysis of a mono-centric cohort (n = 97); assessed through genome-wide DNA methylation analysis, copy-number profiling and targeted next generation sequencing using a neurooncology-tailored gene panel. RESULTS: Of these 97 IDH-mutant glioblastomas, 68 had a glioblastoma at first presentation ('de novo' IDH-mutant glioblastoma) and 29 emerged from a prior low-grade lesion ('evolved' IDH-mutant glioblastoma). Unsupervised hierarchical clustering of DNA methylation data disclosed that IDH-mutant glioblastoma ('de novo' and 'evolved') formed a distinct group separate from other diffuse glioma subtypes. Homozygous deletions of CDKN2A/B were found to be associated with shorter survival. CONCLUSIONS: This study demonstrates DNA methylation patterns in IDH-mutant glioblastoma to be distinct from lower-grade astrocytic counterparts but homogeneous within de novo and evolved IDH-mutant glioblastomas, and identifies CDKN2A as a marker for possible genetic sub-stratification.

Randomized clinical trial of stapler hepatectomy versus LigaSure™ transection in elective hepatic resection.

BACKGROUND: Previous studies have demonstrated stapler hepatectomy and use of various energy devices to be safe alternatives to the clamp-crushing technique in elective hepatic resection. In this randomized trial, the effectiveness and safety of stapler hepatectomy were compared with those of parenchymal transection with the LigaSure™ vessel sealing system. METHOD: Patients scheduled for elective liver resection at two tertiary-care centres were randomized during surgery to stapler hepatectomy or transection with the LigaSure™ device. Total intraoperative blood loss was the primary efficacy endpoint. Transection time, duration of operation, perioperative complications and length of hospital stay were recorded as secondary endpoints. RESULTS: A total of 138 patients were analysed, 69 in the LigaSure™ and 69 in the stapler hepatectomy group. Baseline characteristics were well balanced between the groups. Mean intraoperative blood loss was significantly higher in the LigaSure™ group than the stapler hepatectomy group: 1101 (95 per cent c.i. 915 to 1287) versus 961 (752 to 1170) ml (P = 0·028). The parenchymal transection time was significantly shorter in the stapler group (P = 0·005), as was the total duration of operation (P = 0·027). Surgical morbidity did not differ between the groups, nor did the grade of complications. CONCLUSION: Stapler hepatectomy was associated with reduced blood loss and a shorter duration of operation than the LigaSure™ device for parenchymal transection in elective partial hepatectomy. Registration number: NCT01858987 (http://www.clinicaltrials.gov).

[Nationwide survey of postgraduate medical training in clinical neurology]. / Deutschlandweite Evaluation der ärztlichen Weiterbildung in der klinischen Neurologie.

BACKGROUND: Currently, no data are available, which reflect the situation of medical doctors specializing in neurology in German hospitals. In order to secure the high standard of neurological patient care it is essential to evaluate the working conditions and the specialty training in neurology. OBJECTIVE: This nationwide survey was conducted throughout Germany with the aim to address problems and to give suggestions for improvements in neurological training curricula. MATERIAL AND METHODS: The survey was online from February to May 2017 and 953 neurologists undergoing further training participated. RESULTS: More than half of the young neurologists were satisfied with their medical training. One of the main problems that complicates clinical training is the workload. In addition, organizational obstacles within the clinic, such as poor structure of education or a lack of mentors, lead to dissatisfaction among participants. The size or type of the department, as well as the prevailing service system, exert only a minor influence on the quality of specialist training, although there were differences especially in the self-assessment of the participants in connection with the type of department (university hospital versus public or private hospital). CONCLUSION: Specialist training in neurology can be improved by simple arrangements, e. g., the introduction of a binding rotation scheme, internal mentoring and structured feedback. In addition, it will be necessary to relieve medical staff of administrative duties in order to create time for training and the learning of competencies.

No Significant Cytotoxic Effect of the EZH2 Inhibitor Tazemetostat (EPZ-6438) on Pediatric Glioma Cells with Wildtype Histone 3 or Mutated Histone 3.3.

BACKGROUND: Glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG) belong to the most aggressive cancers in children with poor prognosis and limited therapeutic options. Therapeutic targeting of epigenetic proteins may offer new treatment options. Preclinical studies identified Enhancer of Zeste Homolog 2 (EZH2) within polycomb repressor complex 2 (PRC2) as a potential epigenetic anti-tumor target in adult GBM cells but similar inhibition studies in pediatric GBM/DIPG were still missing. Moreover, approximately 30% of pediatric high grade gliomas (pedHGG) including GBM and DIPG harbor a lysine 27 mutation (K27M) in histone 3.3 (H3.3) which is correlated with poor outcome and was shown to influence EZH2 function. PATIENTS, MATERIALS AND METHODS: The present study investigated the correlation of expression of EZH2 and other PRC2 genes (EZH1, SUZ12, EED) with overall survival of pediatric GBM patients and the cytotoxic impact of EZH2 inhibition by the novel agent Tazemetostat in pediatric GBM/DIPG cells harboring either a H3.3 mutation or a H3 wildtype. RESULTS: EZH2 gene expression does not correlate with survival of pedHGG patients, and EZH2 inhibition does not induce significant cytotoxicity in pedHGG cells independently of H3.3 mutations. DISCUSSION AND CONCLUSION: We suggest that EZH2 inhibition might not offer an effective single agent treatment option for paedHGG patients. However, the therapeutic efficacy in combination with cytotoxic and/or other epigenetically active agents still has to be elucidated.

Traction force during vacuum extraction: a prospective observational study.

OBJECTIVE: To investigate the traction force employed during vacuum extractions. DESIGN: Observational cross-sectional study. SETTING: Obstetric Department, Karolinska University Hospital, Sweden, and the Swedish National Congress of Obstetrics and Gynaecology, 2013. POPULATION: Two hundred women with vacuum extraction at term and 130 obstetricians participating in a simulated setting. METHODS: In a normal clinical setting, we used a specially adapted device to measure and record the force used to undertake vacuum extraction. In a subsequent part of the study, the force employed for vacuum extraction by a group of obstetricians in a fictive setting was estimated and objectively measured. MAIN OUTCOME MEASURES: Applied force during vacuum extraction in relation to the estimated level of difficulty in the delivery; perinatal diagnoses of asphyxia or head trauma; estimated force compared with objectively measured force employed in the fictive setting. RESULTS: The median (minimum-maximum) peak forces for minimum, average and excessive vacuum extraction in the clinical setting were 176 N (5-360 N), 225 N (115-436 N), and 241 N (164-452 N), respectively. In 34% of cases a force in excess of 216 N was employed. There was no correlation between the umbilical arterial pH at delivery and the traction force employed during extraction. Four cases of mild hypoxic ischaemic encephalopathy were observed, three of which were associated with a delivery whereby excessive traction force was employed during the vacuum extraction. In the fictive setting, the actual exerted force was twice the quantitative estimation. The measured forces in the clinical setting were four times higher than that estimated in the fictive setting. CONCLUSIONS: Higher than expected levels of traction force were used for vacuum extraction delivery. As obstetricians tend to underestimate the force applied during vacuum extraction, objective measurement with instantaneous feedback may be valuable in raising awareness.

Transitioning from genotypes to epigenotypes: why the time has come for medulloblastoma epigenomics.

Recent advances in genomic technologies have allowed for tremendous progress in our understanding of the biology underlying medulloblastoma, a malignant childhood brain tumor. Consensus molecular subgroups have been put forth by the pediatric neuro-oncology community and next-generation genomic studies have led to an improved description of driver genes and pathways somatically altered in these subgroups. In contrast to the impressive pace at which advances have been made at the level of the medulloblastoma genome, comparable studies of the epigenome have lagged behind. Complementary data yielded from genomic sequencing and copy number profiling have verified frequent targeting of chromatin modifiers in medulloblastoma, highly suggestive of prominent epigenetic deregulation in the disease. Past studies of DNA methylation-dependent gene silencing and microRNA expression analyses further support the concept of medulloblastoma as an epigenetic disease. In this Review, we aim to summarize the key findings of past reports pertaining to medulloblastoma epigenetics as well as recent and ongoing genomic efforts linking somatic alterations of the genome with inferred deregulation of the epigenome. In addition, we predict what is on the horizon for medulloblastoma epigenetics and how aberrant changes in the medulloblastoma epigenome might serve as an attractive target for future therapies.

From IEDs to AIDS? Detection of HIV in human corpses by rapid screening tests after suspected intentional transmission in terrorist attacks.

OBJECTIVES: We evaluated the feasibility of intentional transmission of HIV by means of suicide bombing and rape as a terrorist tactic in asymmetric conflicts by evaluating the recognised optimum conditions for biological warfare. We also estimated the suitability of a fourth-generation rapid test for HIV detection in the blood of dead terrorists killed in the completion of their mission. METHODS: The feasibility of deliberate transmission of HIV for terroristic ends was evaluated on the basis of published experience from passive biological warfare research. In addition, blood from four recently deceased HIV-positive patients and four HIV-negative control corpses, stored at 4°C in a mortuary, was analysed at 12, 24, 36 and 48 h postmortem by rapid serological testing. RESULTS: The feasibility of HIV infection for terroristic purposes was established. The fourth-generation HIV rapid test we evaluated identified all HIV-positive samples and was negative for all HIV-negative samples. CONCLUSIONS: Rapid HIV testing from the remains of dead terrorists in the deployed military environment is possible. Samples should be acquired quickly, basic sample preparation is advisable and consequent decisions concerning postexposure prophylaxis should take into account the diagnostic gap in early infections.

Perioperative PAI-1 values in surgically treated colorectal carcinoma patients under low molecular weight heparin thromboprophylaxis.

AIM: We have monitored the perioperative changes of plasmatic values of plasminogen activator inhibitor -1 (PAI-1) in colorectal carcinoma patients depending on the stage of disease and the use of prophylactic low molecular weight heparin (LMWH). METHODS: One hundred 100 colorectal carcinoma patients (64 men and 36 women) with average age of 60, in two randomized groups. All patients were surgically treated using the same technique and in all cases adenocarcinoma was confirmed. Two hours before the surgery, the first group (48 patients) received LMWH-nadroparin calcium in doses of 0.3 or 0.4 mL sc, while the second group (52 patients) received it eight hours after the surgery. Following the surgery, Nadroparin calcium was administered daily using the same dose as before. Blood samples were collected: before the surgery, 10 minutes after the first surgical incision, 8 hours after the surgery, and on the 3rd, 5th and 10 th postoperative days. The staging of the disease (according to the Dukes classification) was compared with the patients' blood plasma concentration of PAI-1. Statistical analysis using the c2 test, the LSD test, Wilcoxon and Mann-Whitney test was performed. RESULTS: Adenocarcinoma was patohistologicaly confirmed in all 100 subjects. According to the Dukes classification, 6 patients had stage A, 51 had stage B, and 43 had stage C. PAI-1 measurements showed that baseline measurements were within normal boundaries for both groups. Ten minutes after the first incision a sharp decline of PAI-1 values in the plasma of both patient groups was observed, which could be explained by the use due to the effect on t-PA secreted from the damaged endothelium. PAI-1 values in both groups of subjects have returned to starting (baseline) values, and remained within these values through the third, fourth and fifth measurement in both groups of patients. There was no difference between the two randomized groups which leads to the conclusion that the application of LMWH does not affect PAI-1 values. A statistically significant difference of the tested parameters according to the Dukes classification was obtained only for parameter PAI-1 for pairs Dukes A:B as well as Dukes A:C. A statistically significant correlation was found for plasma values of fibrinolysis inhibitor PAI-1 according to the Dukes classification, but it does not correlate to the tumor invasion through intestinal wall structures. The reason for the statistically significant increase of PAI-1 values in the Dukes A stage remains unclear. CONCLUSION: By activating t-PA and blocking PAI-1, Nadroparin calcium perioperatively makes the haemostasis system stable and balanced.

Oral glucose tolerance test and HbA1c for diagnosis of diabetes in patients undergoing coronary angiography: [corrected] the Silent Diabetes Study.

AIMS/HYPOTHESIS: The primary aim of this study was to compare the results of HbA(1c) measurements with those of an OGTT for early diagnosis of 'silent diabetes' in patients with coronary artery disease (CAD) undergoing angiography without prediagnosed diabetes. A secondary aim was to investigate the correlation between the extent of CAD and the glycaemic status of the patient. METHODS: Data from 1,015 patients admitted for acute (n = 149) or elective (n = 866) coronary angiography were analysed. Patients with known diabetes were excluded from the study. Using the OGTT results, patients were classified as having normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT) or diabetes. According to the results of the HbA(1c) measurements, patients were classified into three groups: normal (HbA(1c) <5.7% [<39 mmol/mol]), borderline (HbA(1c) 5.7-6.4% [39-47 mmol/mol]) and diabetes (HbA(1c) ≥6.5% [≥48 mmol/mol]). RESULTS: Based on the OGTT, 513 patients (51%) were classified with NGT, 10 (1%) with IFG, 349 (34%) with IGT and 149 (14%) were diagnosed with diabetes. According to HbA(1c) measurements, 588 patients (58%) were classified as normal, 385 (38%) as borderline and 42 (4%) were diagnosed with diabetes. The proportion of patients with IGT and diabetes increased with the extent of CAD (IGT ρ = 0.14, p < 0.001, diabetes ρ = 0.09, p = 0.01). No differences in HbA(1c) were seen among the groups with different extents of CAD (p = 0.652). CONCLUSIONS/INTERPRETATION: An OGTT should be performed routinely for diagnosis of diabetes in patients with CAD undergoing coronary angiography, since HbA(1c) measurement alone appears to miss a substantial proportion of patients with silent diabetes. A limitation of the study is that the OGTT was not performed before the angiography.

Two cases of improvement of smooth pursuit eye movements after selective posterior rhizotomy.

OBJECTIVE: Selective posterior rhizotomy (SPR) represents a standard neurosurgical approach in the treatment of spasticity in children with cerebral palsy (CP). Beside the reduction of spasticity in lower limbs, SPR may have suprasegmental effects, considerably above the surgery site. In this communication, we report on the improvement of smooth pursuit eye movements (SPEM) in two children after SPR. MATERIAL AND METHODS: Four children with CP underwent SPR. Eye movements were registered by infrared video-oculography before and after the surgery. RESULTS: The analysis of SPEM showed the improvement of the correlation coefficient of the eye response to the stimulus after SPR in two subjects. Improvement of SPEM performance was largely due to suppression of spontaneous fixation nystagmus. CONCLUSION: SPR may lead to the improvement of SPEM in children with CP. The influence of SPEM improvement on quality of life in a group of severely disabled nonambulant children with CP remains to be assessed.

[Family doctor-based health care]. / Hausarztbasierte Versorgung.

The idea of family doctor-based health care corresponds to a social necessity for economical health care and a basic requirement for family doctors. However, this request is turned into the opposite by the legal description of a "particularly qualified family doctor". Economic interests dominate, dissect the family doctor level and influence long-standing family doctor-patient relationships and put the performance of social goals into question. Against that, the Family Doctor Association is setting the concept for family doctor-based integrated care, which also places quality demands, but does not exclude physicians basically or by selection of the compulsory health insurance fund.

Plaque removal efficacy of two rotating/oscillating electric toothbrushes.

This study compared the plaque removal efficacy of the Philips Jordan HP510 and Braun Oral-B D9 electric toothbrushes in two clinical trials. In Trial I, a single-blind randomized split-mouth study, 24 subjects received an oral prophylaxis and abstained from all oral hygiene procedures for 24 hours before each quadrant was professionally brushed for 30 seconds. Plaque was scored before and after brushing using the Turesky-modified Quigley and Hein index (Q&H) and the approximal plaque index (API). Trial II was a two-week, single-blind balanced cross-over study comprising 54 subjects. At baseline, the subjects received an oral prophylaxis and instruction in the use of the assigned brush. Two weeks later, they returned to the clinic with 24-hour plaque accumulation. Plaque (Q&H, API) was scored before and after supervised brushing (30 sec/quadrant). After a prophylaxis, the subjects received the other brush with instructions on use, and the procedure was repeated. In Trial I, plaque was removed almost equally well by both brushes, with the HP510 and the D9 reducing Q&H from 1.46 and 1.47 (baseline) to 0.53 and 0.58, respectively, on average over all teeth. For the premolars and molars in particular, use of the HP510 resulted in statistically significantly lower Q&H scores than the D9 (0.70 vs. 0.81). In Trial II, the HP510 resulted in significantly better plaque scores than the D9 on Q&H (0.55 vs. 0.67) and API (23% vs. 28%) for all teeth. The buccal surfaces contributed the largest difference in Q&H. Statistically significant differences were also found in scores of the posterior teeth for Q&H (0.75 vs. 0.86) and API (31% vs. 38%). In conclusion, both rotating/oscillating electric toothbrushes were highly effective in plaque removal. The HP510 removed more plaque than the D9, particularly from the posterior teeth. It appeared that the smaller height and the Active Tip of HP510's brush head could have resulted in better posterior and approximal access.

Influence of a controlled pressure system on toothbrushing behavior.

The use of excessive brushing force has been shown to be a major cause of gingival abrasion. To aid in preventing over-vigorous brushing, the Philips/Jordan electric toothbrush incorporates a Controlled Pressure system (CPS) that causes the brush head to flex back when a toothbrushing force (TBF) in excess of a pre-determined threshold is exerted against the teeth or soft tissues. Two studies (I/II) were conducted to determine whether this mechanical feedback system is sufficiently sensitive to enable users to control their brushing behavior. In Study I, the learning pattern of brushing behavior as a response to the feedback system was evaluated. Seventeen subjects were asked to brush their teeth under observation at least twice a day for a two-week period. During these observations, the number of clicks, as well as the time the brush was pushed "through the click" were recorded. Ten of seventeen volunteers demonstrated a clear learning behavior; the mean number of clicks/minute (for all subjects) was reduced from 10 to 4 after 10 sessions of brushing, and then to 2 or 3 clicks at the end of two weeks. In Study II, 46 subjects used the electric toothbrush with the CPS click force set at various levels between 150-420 g (at 30 g intervals). After a 4-week learning period, the mean TBF was determined in each subject. TBF was most strongly influenced at pre-set click forces between 180 and 270 g. The mean TBF was lowest (about 80 g) when the threshold was set at 210 or 240 g; it then increased (to about 130-140 g TBF) both for smaller and larger values of the threshold setting. Hence, both studies indicate that the Controlled Pressure system is a functional feature that can be used to control the habitual brushing force in a learning period of less than 2 weeks.

Plaque removal efficacy of an electric and a manual toothbrush with additional interdental tufts.

This clinical study compares the plaque removal efficacy of the Philips/Jordan 2-Action Plaque Remover electric toothbrush with that of the Oral-B 35 Advantage manual toothbrush. In this single-blind randomized split-mouth study, subjects brushed under supervision after having abstained from all oral hygiene for 24 hours. Plaque scored after brushing was used to compare the efficacy of the toothbrushes. Both toothbrushes have special tufts to promote interdental cleaning. It was found that these tufts improve interdental cleaning. Overall, the Philips/Jordan brush removed significantly more plaque than the Oral-B manual brush.

Clinical course after total parathyroidectomy without autotransplantation in patients with end-stage renal failure.

In patients with chronic renal failure, hyperparathyroidism is a common problem and surgical parathyroidectomy (PTX) is frequently required. The three different surgical approaches are subtotal PTX, total PTX with autotransplantation, and total PTX without autotransplantation. Recurrence of hyperparathyroidism varies from 5% to 80% in different studies for the first two surgical approaches. To minimize the risk for recurrence, and because we fear severe relapses with calciphylaxia, we perform total PTX without autotransplantation. From October 1993 to October 1997, 20 patients (9 men and 11 women) underwent total PTX without autotransplantation (median age, 52 years; range, 23 to 74 years; median dialysis time before PTX, 6.5 years; range, 1 to 22 years). All patients were supplemented with vitamin D analogues postoperatively. Patients were followed up for 1 to 48 months (median, 20 months). Bone pain, when present, disappeared within the first week after total PTX. Postoperatively, most patients had temporary hypocalcemia. In the long term, five patients had asymptomatic hypocalcemia. One patient, however, repeatedly had hypocalcemic seizures. Five patients developed asymptomatic hypercalcemia when supplemented with calcitriol. At the end of the individual's observation time, parathyroid hormone (PTH) levels were less than normal in six patients, normal in seven patients, and increased in seven patients despite total PTX. We conclude that total PTX should be reconsidered an option for the treatment of hyperparathyroidism secondary to renal failure. There was no evidence of clinical bone disease after total PTX. Apparently, remaining ectopic parathyroid tissue accounts for PTH levels after total PTX.

Isolation and characterization of human monoclonal antibodies against hepatitis C virus envelope glycoproteins.

The isolation and characterization of human monoclonal antibodies (humAbs) against the hepatitis C Virus (HCV) glycoproteins E1 and E2 are described. B-cells from blood donors with anti-HCV were transformed with Epstein-Barr virus. The supernatants of the resulting lymphoblastoid clones were screened by ELISA with an extract of cells infected with a recombinant vaccinia virus RMPA95 expressing the envelope proteins E1 and E2 of an HCV genotype 1a virus (H strain). Positive clones were fused to the heteromyeloma cell line K6H6/B5. Fifteen heterohybridoma cell lines have been established. The specificity of the isolated humAbs was determined both by ELISA and Western blot assays. Several recombinant extracts expressing either the E1 or E2 protein or truncated forms were used in an attempt to map the epitopes on the viral glycoproteins. Some of the humAbs were used successfully for immunofluorescence investigation of transfected cells. Seven specific anti-E2 humAbs, which react with the envelope protein 2 of genotype 1a and 1b isolates, were characterized.

Anti-HCV envelope prevalence in blood donors from Baden-Württemberg.

We assess the diagnostic value of the recombinant antigens E1/E2 (heterodimer) and E2 for the screening of blood donors. For this purpose an ELISA assay was developed which used these recombinant glycoproteins as solid antigens on the microtiter plates. The anti-E1/E2 assay was able to detect 80% of the positive samples by Matrix and 87% of the PCR-positive samples. Anti-E2 was less sensitive. Fifteen percent of the indeterminate samples were anti-E1/E2 and 8% anti-E2 positive. In the case of the indeterminate samples there was no coincidence of results between these assays and the PCR. Fifty percent of seroconverters showed an anti-E1/E2-positive result with a previous indeterminate donation. We conclude that the recombinant envelope proteins, mainly anti-E1/E2, might be used in future in the diagnostics of HCV infection to increase the sensitivity of the currently used assays.