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Biomedical subjects

H Wolf

Publications and source records attributed to H Wolf.

At least 163 records · Page 9Linked to original sources
PubMed1998

Specific detection of monkeypox virus by polymerase chain reaction.

The open reading frame coding for the A-type inclusion body protein (ATI) of monkeypox virus (MPV) was identified and sequenced for two strains. Nucleotide sequence comparison revealed 72-95.3% homology with the reported open reading frame sequences of the ATIs of other orthopoxvirus species, such as variola, vaccinia, cowpox, ectromelia, and camelpox viruses. Each MPV strain contained an 8-bp deletion, which caused a frameshift that introduced a premature stop in the open reading frame at base 2091 relative to the ATI open reading frame of cowpox virus strain Brighton. The sequences enabled a primer pair to be designed that flanked the deletion and specifically amplified a 601-bp fragment that identified and differentiated 19 MPV strains examined from five other Old World orthopoxvirus species examined. The specificity was confirmed by cleavage of the 19 MPV strain amplicons with BglII, which produced three subfragments of expected sized, based on the determined MPV sequences.

Animals↗
PubMed1998

Maternal and perinatal outcome after expectant management of the HELLP syndrome compared with pre-eclampsia without HELLP syndrome.

OBJECTIVE: To compare maternal and perinatal outcome of pregnancies complicated by pregnancy induced hypertension and HELLP syndrome with the outcome of pregnancies complicated by pre-eclampsia only. DESIGN: It was a retrospective cohort study. Fifty one patients with pregnancy induced hypertension and HELLP syndrome were matched with 51 pre-eclamptic patients according to parity and gestational age on admission in hospital. Management was expectant, treatment only symptomatic and delivery was mainly effectuated because of fetal condition. RESULTS: There was no maternal mortality in either group; maternal morbidity was more frequent in the HELLP group. Immediate intervention within a few hours of admission because of fetal distress more often occurred in the HELLP group. In both groups 41 children (80%) are still alive, with one major handicapped child in each group. Logistic regression analysis identified gestational age on admission and antihypertensive treatment on admission as significant contributors to perinatal mortality or major handicap. Whether the patient belonged to the HELLP group or the pre-eclamptic group had no influence on outcome. CONCLUSION: Expectant management of pregnancy induced hypertension with HELLP syndrome and pre-eclampsia without HELLP syndrome results in similar maternal and perinatal outcome. Perinatal outcome is strongly influenced by gestational age and the severity of hypertension as expressed by the need of antihypertensive treatment, irrespective of the underlying syndrome.

Adolescent↗
PubMed1998

Irradiation induces G2/M cell cycle arrest and apoptosis in p53-deficient lymphoblastic leukemia cells without affecting Bcl-2 and Bax expression.

The tumor suppressor p53 has been implicated in gamma irradiation-induced apoptosis. To investigate possible consequences of wild-type p53 loss in leukemia, we studied the effect of a single dose of gamma irradiation upon p53-deficient human T-ALL (acute lymphoblastic leukemia) CCRF - CEM cells. Exposure to 3 - 96 Gy caused p53-independent cell death in a dose and time-dependent fashion. By electron microscopic and other criteria, this cell death was classified as apoptosis. At low to intermediate levels of irradiation, apoptosis was preceded by accumulation of cells in the G2/M phase of the cell division cycle. Expression of Bcl-2 and Bax were not detectably altered after irradiation. Expression of the temperature sensitive mouse p53 V135 mutant induced apoptosis on its own but only slightly increased the sensitivity of CCRF - CEM cells to gamma irradiation. Thus, in these, and perhaps other leukemia cells, a p53- and Bcl-2/Bax-independent mechanism is operative that efficiently senses irradiation effects and translates this signal into arrest in the G2/M phase of the cell cycle and subsequent apoptosis.

Apoptosis↗
PubMed1998

Long-term survival of patients with bladder tumours: the significance of risk factors.

OBJECTIVE: To evaluate the significance of known risk factors, accessible by simple endoscopic and histological/cytological examination, on the clinical course and long-term survival of patients with superficial urinary bladder tumours. PATIENTS AND METHODS: The study included 584 consecutive unselected patients, primarily admitted between 1976 and 1984 for newly diagnosed bladder tumour, which was superficial (Ta, T1, Tis) in 362. The patients were followed routinely in a control programme; causes of death were obtained by autopsy (44%), from hospital files (33%) or from death certificates (8%), the remaining patients being alive at the end of the study, up to 20 years after initial diagnosis. Known risk factors, e.g. tumour size, histological grade, multiplicity and positive urine cytology, and dysplasia as assessed by random or pre-selected site biopsies, were evaluated as predetermining factors for new occurrences and survival. RESULTS: Invasion of the lamina propria was the most significant prognostic factor detected in the multivariate analysis. While 14% of patients with Ta tumours had died from cancer after 15 years, 63% of the T1 tumours were eventually fatal, reaching the mortality of those with T2 disease. Other independent significant factors were tumour size and, to a lesser extent, histological grade. Multiplicity and concomitant epithelial changes, as assayed by voided urine cytology and pre-selected site biopsies, were relevant prognostic factors for Ta but not for T1 tumours. CONCLUSION: In the therapy and surveillance of superficial urinary bladder tumours, the presence of lamina propria invasion is very important.

Adult↗
PubMed1998

Development and characterization of a murine monoclonal antibody reactive with a 64 kDa somatic antigen of Burkholderia cepacia.

Monoclonal antibodies (MAbs) to Burkholderia cepacia were produced from mice immunized with inactivated whole-cell antigen. For screening of resulting MAbs an enzyme-linked immunosorbent assay (ELISA) was used. A stable hybridoma cell line (BC-2) producing specific antibodies to a 64 kDa somatic antigen from B. cepacia was established. In ELISA and immunoblotting analysis the MAb BC-2 recognized all tested strains of B. cepacia whereas no cross-reaction with 32 Pseudomonas aeruginosa strains was found. From a wide range of other bacteria only strains of the species Burkholderia mallei, Burkholderia pseudomallei, and Burkholderia gladioli showed cross-reactions. The MAb BC-2 will be used to develop a diagnostic assay for the identification of B. cepacia and B. gladioli, important agents of nosocomial infections in immunocompromised patients suffering especially from cystic fibrosis (CF).

Animals↗
PubMed1998

Low molecular weight heparin prevents the pulmonary hemodynamic and pathomorphologic effects of endotoxin in a porcine acute lung injury model.

Tumor necrosis factor alpha (TNF-alpha) activity, platelet and neutrophil degranulation and margination, and increased vascular permeability are central to the pathophysiology of endotoxin-mediated acute lung injury. Nonanticoagulant activities of low molecular weight heparin (LMWH) include solubilization of the TNF-alpha receptor protein, inhibition of neutrophil adhesion, and regulation of thromboxane B2 (TXB2) biosynthesis. In this study, we evaluated the ability of LMWH to modulate TNF-alpha and TXB2 activity during endotoxemia and the subsequent effects on pulmonary hemodynamics. Domestic pigs 8-10 weeks old were anesthetized and catheterized for standard cardiopulmonary measurements and the lungs harvested for cuff:vessel ratio, myeloperoxidase activity, and permeability index. Pigs were randomly assigned to one of four groups: lipopolysaccharide (LPS) (n = 6), given .5 microg/kg/h Escherichia coli LPS intravenously for 6 h; saline control (n = 5); LMWH (n = 5), given .5 mg/kg LMWH for 30 min, followed by .5 mg/kg/h; and LMWH + LPS (same dosages, n = 6). Administration of LPS resulted in increased plasma TNF-alpha and TXB2 activity; increased pulmonary arterial pressure, pulmonary vascular resistance, and alveolar-arterial oxygen tension; decreased systemic arterial oxygen tension; and pulmonary edema. The cardiopulmonary parameters for the LMWH-treated pigs did not differ from those of the saline-treated control pigs. Pretreatment with LMWH attenuated the LPS-mediated TNF-alpha and TXB2 activity and attenuated LPS-mediated pulmonary hypertension, hypoxemia and neutrophil emigration, and edema formation. In conclusion, the data show that the protective effects of LMWH in this model of acute lung injury are associated with altered neutrophil adhesion and TNF-alpha and thromboxane activity.

Animals↗
PubMed1998

Tree-pollen allergy is efficiently treated by short-term immunotherapy (STI) with seven preseasonal injections of molecular standardized allergens.

The efficacy and tolerance of short-term immunotherapy (STI) by seven preseasonal injections of tree-pollen allergens (ALK7 Frühblühermischung) was investigated in a double-blind, placebo-controlled, multicenter study with 111 rhinoconjunctivitis patients. Nasal and bronchial symptoms simultaneously analyzed, and nasal symptoms as a single end point, but not the overall score of nasal, bronchial, and conjunctival symptoms, showed a significantly lower increase with STI during birch-pollen exposure (both P=0.033, n=105, Mann-Whitney U-test). However, a selective analysis with patients from centers with high recruitment figures (n> or =10 patients, n=29 STI, n=32 placebo) showed a significantly lower increase of nasal, bronchial, and overall symptom score (STI 11.0 vs placebo 18.0, P=0.001, U-test). STI had equidirected effects on conjunctival, nasal, and bronchial symptoms analyzed as multiple end points, although conjunctival symptoms were not significantly different as a single end point. The seasonal increase in drug use was reduced by 62% in the STI group compared with placebo (P=0.032, t-test). Specific IgG4 increased only after STI (P<0.001); IgE was not significantly different. Eosinophil cationic protein remained unchanged with STI, but significantly increased with placebo in the pollen season (P=0.003). STI was well tolerated. In conclusion, STI was shown to be efficacious and safe for the treatment of patients with tree-pollen rhinoconjunctivitis.

Adolescent↗
PubMed1998

[Variability of Trichophyton cerrucosum isolates from vaccinated herds with cattle ringworm].

Twenty-seven strains of Trichophyton verrucosum from 14 cattle herds in the Federal States of Thuringia and Mecklenburg-Vorpommern were examined by culture morphological and molecular biological (PCR fingerprinting, AFLP analysis sequencing of ITS region) methods. Six reference strains of the same species, among them the so-called album and ochraceum varieties, were also included. Despite great variability in terms of culture morphology, which suggested their possible classification into 4 different colony types, all T. verrucosum isolates were genotypically almost identical. Even the 2 field isolates growing with yellow pigment, which could possibly be regarded as belonging to the ochraceum variety, could not be differentiated using molecular biological methods. The results do not provide indications of a separate taxonomic position of the 3 T. verrucosum varieties. Furthermore, there is no evidence confirming the suspected infection of cattle herds with ochraceum strains as the cause of the failure of immune prophylaxis using various T. verrucosum vaccines. The frequent occurrence of animals not responding to vaccination could not be explained either. It should be assumed that the main factors responsible for this situation include poor handling of the vaccine strains and errors in application, especially the absence of continuous and systematic immune prophylaxis in the herds.

Animals↗
PubMed1998

Clinical evaluation of the automated COBAS AMPLICOR MTB assay for testing respiratory and nonrespiratory specimens.

We evaluated the COBAS AMPLICOR PCR system (Roche Diagnostics) for the routine detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. Diagnostic culture, considered as the reference method, was performed with BACTEC, Löwenstein-Jensen, Stonebrink, and Kirchner media. Occasionally MB-Redox, ESP, or MGIT medium was also used. A total of 643 respiratory and 506 nonrespiratory specimens collected from 807 patients were investigated. Of the 95 culture-positive specimens, 80 were COBAS AMPLICOR MTB positive, and of the 1,054 culture-negative specimens, 1,044 were COBAS AMPLICOR MTB negative. After resolving discrepancies by review of the medical history, the overall sensitivity, specificity, and positive and negative predictive values for the COBAS AMPLICOR MTB assay, respectively, were 83.5, 98.8, 86.7, and 98.6% compared to those of diagnostic culture. In smear-positive specimens, the sensitivity of the COBAS AMPLICOR MTB assay was 96%, versus 48% for smear-negative specimens. No significant differences in the test performance between respiratory and nonrespiratory specimens were observed. The overall inhibition rate was less than 2%, excluding stool specimens. The clear advantages of the COBAS AMPLICOR PCR system are standardized procedures and reagents for specimen processing as well as an internal control for reliable monitoring of PCR inhibitors. By simplifying the work flow through a completely automated amplification and amplicon detection procedure, the COBAS AMPLICOR PCR system proved itself as a very useful component for routine diagnostic procedures.

Automation↗
PubMed1998

Characterization of cis-acting and NS1 protein-responsive elements in the p6 promoter of parvovirus B19.

Parvovirus B19 infections are associated with diverse clinical manifestations, ranging from no symptoms to severe symptoms. The virus shows an extreme tropism for replication in erythroid progenitor cells, possibly due to the activity of the only functional promoter (p6) of the B19 virus genome in combination with both cell- and cell cycle-specific factors and the trans-activator protein NS1. As presented here, p6 promoter sequences derived from several B19 virus isolates proved to be highly conserved. Furthermore, mutations did not affect any of the potential binding sites for transcription factors. One variation of the base at position 223 was identified only in B19 virus isolates derived from patients with persistent infection or chronic arthritis. To determine promoter activity and to characterize regulatory elements, sequences spanning the total p6 promoter and subfragments of them were introduced into a eukaryotic expression vector upstream of the luciferase gene (from Photinus pyralis). After transfection into HeLa, CEM, BJAB, and K562 cells, the p6 promoter was found to be highly active. When introduced into the erythroid cell line K562, p6-controlled transcription exceeded that of the simian virus 40 promoter-enhancer used as a control by more than 25-fold. Sequence elements relevant for promoter activity mapped to the regions from nucleotides (nt) 100 to 190 and 233 to 298. Also, the segment from nt 343 to 400 downstream of the TATA box was important for transcriptional activity in HeLa and K562 cells. By transfecting the promoter-luciferase constructs into a HeLa cell line stably carrying the viral NS1 gene under the control of an inducible promoter, transcriptional activity mediated by the p6 promoter rose significantly after induction of NS1 expression. The region from nt 100 to 160 proved to be essential for NS1-mediated transcriptional activation. Furthermore, NS1-mediated transactivation was dependent on the presence of two GC-rich elements arranged in tandem upstream of the TATA box. These data indicate that NS1-mediated p6 transactivation is dependent on a multicomponent complex combining NS1 with ATF, NF-kappaB/c-Rel, and GC-box binding cellular factors.

Base Sequence↗
PubMed1998

E1B 55-kilodalton-associated protein: a cellular protein with RNA-binding activity implicated in nucleocytoplasmic transport of adenovirus and cellular mRNAs.

The adenovirus type 5 (Ad5) early 1B 55-kDa protein (E1B-55kDa) is a multifunctional phosphoprotein that regulates viral DNA replication and nucleocytoplasmic RNA transport in lytically infected cells. In addition, E1B-55kDa provides functions required for complete oncogenic transformation of rodent cells in cooperation with the E1A proteins. Using the far-Western technique, we have isolated human genes encoding E1B-55kDa-associated proteins (E1B-APs). The E1B-AP5 gene encodes a novel nuclear RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that is highly related to hnRNP-U/SAF-A. Immunoprecipitation experiments indicate that two distinct segments in the 55-kDa polypeptide which partly overlap regions responsible for p53 binding are required for complex formation with E1B-AP5 in Ad-infected cells and that this protein interaction is modulated by the adenovirus E4orf6 protein. Expression of E1B-AP5 efficiently interferes with Ad5 E1A/E1B-mediated transformation of primary rat cells. Furthermore, stable expression of E1B-AP5 in Ad-infected cells overcomes the E1B-dependent inhibition of cytoplasmic host mRNA accumulation. These data suggest that E1B-AP5 might play a role in RNA transport and that this function is modulated by E1B-55kDa in Ad-infected cells.

Adenoviridae↗
PubMed1998

Immediate-early transactivator Rta of Epstein-Barr virus (EBV) shows multiple epitopes recognized by EBV-specific cytotoxic T lymphocytes.

We analyzed the immediate-early transactivator Rta of Epstein-Barr virus (EBV) for its role as a target for specific cytotoxic T lymphocytes (CTL). Panels of overlapping peptides covering the entire amino acid sequence of Rta were synthesized and used to induce and analyze specific CTL responses in EBV-positive donors. Using peptide-pulsed target cells, we found nine different CTL epitopes that are distributed over the entire protein sequence. One epitope restricted by HLA-A24 could be mapped to the decameric sequence DYCNVLNKEF between amino acid positions 28 and 37 of the Rta protein. A second epitope could be assigned to the same region of Rta (residues 25 to 39) and was shown to be restricted by HLA-B18. Another, minimal epitope could be mapped to the nonameric sequence ATIGTAMYK between amino acid positions 134 and 142; this peptide was restricted by HLA-A11. Another four epitopes were proven to be restricted by HLA-A2, -A3, -B61, and -Cw4 and were located between Rta residues 225 and 239, 145 and 159, 529 and 543, and 393 and 407, respectively. For two other epitopes, only the location within the Rta protein is known so far (residues 121 to 135 and 441 to 455); their exact HLA restriction patterns have not yet been identified. Using target cells infected with recombinant vaccinia virus containing the gene for Rta, we showed that six of eight Rta-specific CTL lines recognized the corresponding peptides also after endogenous processing. These data suggest that Rta comprises an important target for EBV-specific cellular cytotoxicity. Together with recent findings of other immediate-early and early proteins also acting as CTL targets, they reveal the role of proteins of the lytic cycle in the immune recognition of EBV-infected cells.

Amino Acid Sequence↗
PubMed1998

Epstein-Barr virus small RNA (EBER) genes: differential regulation during lytic viral replication.

In every latently Epstein-Barr virus-infected cell the viral genes EBER-1 and EBER-2 are transcribed by polymerase III. In lytically infected cells in vivo the EBER genes could not be detected. However, in cell culture downregulation could not be confirmed, and hence the relevance of this shutdown to the replication of the virus was not clear. We assayed the transcriptional activity of the EBER genes by nuclear run-on assays with enriched lytically infected cells and demonstrated that EBER-1 and EBER-2 are differentially downregulated on the transcriptional level during the switch to lytic viral replication. This downregulation was an early event during the lytic replication of the virus.

Base Sequence↗
PubMed1998

Flow-cytometric evaluation of oxidative burst in phagocytic cells of children with cystic fibrosis.

OBJECTIVES: The objective of this study was to assess the dye 2', 7'-dichlorofluorescein (DCF) assay in screening for alterations in polymorphonuclear cell (PMN) and monocyte (MC) oxidative burst of cystic fibrosis (CF) patients. STUDY DESIGN: 56 CF patients aged between 2 and 20 years were investigated. Purified cells were stimulated with phorbolmyristate acetate (PMA) and zymosan (ZX). A range for DCF fluorescence for PMA- and ZX-stimulated and non-stimulated cells was established based on data from 60 healthy controls. RESULTS: PMNs showed both enhancement and impairment. A deficient oxidative burst was detected in a total of 14 CF patients caused by abnormally high mean fluorescence intensity (MFI) of resting cells. Enhanced oxidative burst was seen in 6 CF patients. CF patients responded differently to PMA or ZX stimulation. Pseudomonas aeruginosa colonization significantly enhanced (p<0.005) the MFI of resting PMNs. MCs of CF patients showed a significantly (p<0.05) enhanced oxidative burst after stimulation with PMA compared to healthy controls, but no differences could be observed after stimulation with ZX. Serum concentrations of interleukin-6 were elevated in all CF patients, in particular in those with activation of both PMNs and MCs. CONCLUSION: The DCF assay shows for the first time the heterogeneity of the oxidative burst reaction in CF patients. In our opinion, the DCF assay is a reliable method for detecting pathological oxidative burst in CF patients.

Adolescent↗
PubMed1998

Epstein-Barr viral gene expression in B-lymphocytes.

The strategy of the Epstein-Barr virus to persist lifelong in the host depends on establishing a reservoir, which cannot be detected by the immune system but allows reactivation of the virus for shedding and transmission to a new host. Epithelial cells and B-cells play a major role in this viral strategy of EBV, since differentiating epithelial tissues were shown to be permissive for lytic replication in vivo, whereas the B-lymphocytes become predominantly latently infected. However, which cells are the reservoir and which the sites of lytic replication are not quite clear. With the technique of reverse transcription, PCR and immunohistochemistry, we demonstrated that the B-cells of the peripheral blood are a major site of virus production during the primary infection during infectious mononucleosis. These permissive B-cells were also detected after convalescence, however, the absence of any lytic transcripts suggested an efficient immunological control very early in the viral lytic cycle. Serological data on reactivation of EBV correlated with the detection of lytic cycle transcripts in the blood and thus demonstrated that the site of virus production during infectious mononucleosis must be different from that of the persistent state. In those cases, where the infection takes a chronic active course, control of lytic replication is insufficient, either on the level of immune surveillance or of viral gene regulation. We have demonstrated a virus strain with a lytic phenotype in an individual suffering chronic active infection. The impaired capability of this virus to immortalise B-cells correlated with an enhanced expression of the lytic switch gene BZLF-1 and down-regulation of latent regulatory genes in the early phase of infection.

B-Lymphocytes↗
PubMed1998

The control of lytic replication of Epstein-Barr virus in B lymphocytes (Review).

Uncontrolled replication of a virus, which is harmful to the host is also disadvantageous to the virus. Most viruses cannot compete with the various immune mechanisms and become eliminated in the course of infection. Therefore, only the time between infection and eradication remains for these viruses to proliferate. A few viruses, like the Herpesviruses or the papillomaviruses, however, have developed a sophisticated strategy for persisting lifelong, usually asymptomatically in the host, hiding from the immune system and producing infectious progeny at the same time. This strategy depends on a separation of latency and the lytic replication, either by time due to differentiation-dependent mechanisms or by spatial separation as the result of different host cell types. Both are true for the Epstein-Barr virus (EBV). B cells and epithelial cells have a pivotal role in the life cycle of the virus. The former can become latently infected and are thought to be the virus reservoir in vivo, whereas the latter were shown to be permissive for lytic replication. However, replication of EBV in vivo is controlled primarily by host immune mechanisms selecting for cells that are not permissive for viral replication as the result of a particular set of transcription factors. These factors control the activity of the regulatory immediate-early genes and, in addition, lytic and latent cycle regulatory genes negatively interfere with each other and thus link cellular and viral gene regulatory mechanisms. Disturbance of both the immune surveillance as well as viral gene regulation may result in EBV-associated disease.

Animals↗