Background charge noise in metallic single-electron tunneling devices.
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Journal Article↗
Biomedical subjects
H Wolf
Publications and source records attributed to H Wolf.
Sequence-specific resonance assignments of the 1H-NMR spectra and structural characterization in solution of the HIV-1 transframe protein p6.
The frameshift protein p6* encoded directly upstream of the protease in the human immunodeficiency virus type 1 (HIV-1) pol reading frame is thought to be a natural inhibitor of protease activation and to play a role in the polyprotein processing of Gag and Gag-Pol precursors. To allow structural characterization of the p6* transframe protein, the p6* coding region was cloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) under the control of the tac promoter. Thrombin cleavage of the construct resulted in a 70-amino-acid polypeptide which is extended by two additional residues at the N-terminus compared to the natural p6* sequence. The native purification procedure including an affinity and a size-exclusion chromatography step yielded sufficient amounts of highly pure protein suitable for NMR spectroscopy. Fluorescence, circular dichroism and 1H-NMR spectroscopy were applied to characterize the structure of protein. Two-dimensional NMR spectra provided essentially complete sequence-specific resonance assignments at pH 5.9. Although there is evidence for a helix-forming tendency in the N-terminus of the protein, the experiments indicate that p6* has no overall stable secondary or tertiary structure with the single tryptophan exposed in aqueous solution. However, the results reported herein open the way to characterize further the interaction of p6* with the HIV-1 protease in structural and functional in vitro studies.
Amino Acid Sequence↗
[Poor prognosis in children with ultrasonographically diagnosed abnormalities of the central nervous system].
OBJECTIVE: Long-term follow-up of children with a prenatally diagnosed malformation of the central nervous system. DESIGN: Descriptive study. SETTING: Department of prenatal diagnosis, Academic Medical Center, Amsterdam, the Netherlands. METHODS: Evaluation of all documented ultrasound examinations between 1985 and 1990 (6 years). These ultrasound examinations were performed in women with an increased risk for a foetal malformation, because of obstetrical complications or when a foetal malformation was suspected on ultrasound examination performed elsewhere. Paediatricians and general practitioners were asked to provide follow-up data on the children who were still alive. RESULTS: Seven out of 67 foetuses with a prenatally diagnosed malformation of the central nervous system lived longer than one month. Two out of these 7 children developed normally, one child died after several years and four children were severely retarded. The two children who developed normally both had mild hydrocephaly. CONCLUSION: Prognosis of children with a prenatally diagnosed malformation of the central nervous system is poor. Children with mild hydrocephaly may have a better prognosis.
Adult↗
The blood group ABO gene transcript is down-regulated in human bladder tumors and growth-stimulated urothelial cell lines.
The molecular mechanism that in human bladder tumors leads to the loss of blood group ABO glycosyltransferase activity and, thereby, the loss of ABO antigens was investigated. In 15 tumors and 3 normal biopsies from blood group AB individuals and 7 tumors and 3 normal biopsies from blood group O individuals, mRNA was detected by a reverse transcription PCR (RT-PCR) assay, and the ABO blood group structure was determined by immunohistology. The RT-PCR spanned several introns in the ABO gene to exclude DNA contamination, and the RT-PCR product was shown to reflect the ABO gene message by dideoxy sequencing. The ABO mRNA was present in normal urothelium and low-grade tumors but disappeared from high grade tumors. This correlation to tumor grade was significant (P<0.04). Immunohistochemistry with monoclonal anti-blood group antibodies showed a complete correlation between the presence of mRNA and the presence of AB carbohydrate structures on cell surfaces. In two urothelial cell lines, genotyped as A/- and A/A, growth stimulation with the cholera toxin B subunit led to a total loss of ABO mRNA, and epidermal growth factor stimulation had an identical effect on one of the cell lines. We conclude that the ABO glycosylation in normal and malignant urothelium is regulated at the mRNA level, and that a mechanism associated with cell proliferation may trigger down-regulation of ABO mRNA.
ABO Blood-Group System↗
The outcome of pregnancies with confined placental chromosome mosaicism in cytotrophoblast cells.
Cytogenetic findings and outcome of pregnancy are reported in 108 cases in which confined placental mosaicism (CPM, n = 101) or generalized mosaicism (n = 7) was found at or after first-trimester chorionic villus sampling. In all samples, a (semi)direct cytogenetic analysis of cytotrophoblast cells was performed. Two pregnancies with CPM ended in a spontaneous abortion before 28 weeks (1.9 per cent). In 15 cases the pregnancy was terminated: eight cases were shown to be examples of CPM; seven cases can be considered as examples of generalized mosaicism. A normal cytogenetic result was obtained after follow-up amniocentesis in 88 of the remaining 91 cases. In three cases, no amniocentesis was performed but confirmation of a normal karyotype was obtained in other cells. One of the 91 pregnancies was nevertheless terminated for psychosocial reasons. One child died perinatally and another on the seventh day after birth. The birth weight is known for 89 children; the curve shows a normal distribution. In 11 of these children (12.3 per cent), the birth weight was found to be below the tenth centile. The outcome in a subgroup of eight pregnancies with CPM and involvement of chromosome 13, 16, or 22, however, revealed two fetal losses and four children with a birth weight below the tenth centile (75 per cent).
Abortion, Induced↗
Development of a rapid means of estimating the haemoglobin F content of candidate fetal cells isolated from maternal blood using HPLC.
Prenatal diagnosis of genetic disorders in nucleated fetal red blood cells present in maternal blood requires methods to detect and enrich for such cells. Here we describe a rapid high performance liquid chromatography (HPLC) method that allows one to determine as few as 100 cells containing haemoglobin F (HbF) in the presence of a vast excess of haemoglobin A (HbA)-producing cells. The HPLC separations of haemoglobins were performed with a weak cation exchange column-silica gel-bound asparaginic acid-and ammonium phosphate buffer as the mobile phase. Separations were carried out in 7 min. When applied to estimation of the recovery of fetal cells from maternal blood, the HPLC method indicates in a timely manner whether or not to proceed with further techniques (i.e., FISH or PCR). Several current techniques such as Ficoll gradients and fluorescence (FACS) or magnetic (MACS) activated cell sorting were thus evaluated. Unexpectedly, our method indicates high cell losses with both single gradient and triple density Ficoll pre-enrichment methods. Less than 20 per cent of the nucleated red blood cells can be recovered in the most optimal setting. Lysis of erythrocytes may be an alternative technique that leaves nucleated red blood cells of all maturation stages intact. Thus, any further improvements in the technology for fetal cell recovery may be aided by monitoring the yield with HPLC.
Cell Separation↗
Phenotypic and functional characterization of CD11c+ dendritic cell population in mouse Peyer's patches.
The antigen-presenting cell system in the gastrointestinal tract, one of three main sites (skin and lung being the others) of primary antigen contact, is poorly understood. Our study focused on dendritic cells (DC) as possible candidates for antigen uptake, processing and presentation in mucosal inductive sites, such as Peyer's patches (PP). To investigate the morphology, immunophenotype and stimulatory activity of intestinal DC, a procedure was developed to obtain a cell population by using collagenase digestion of PP, density centrifugation and cell sorting on the basis of CD11c expression. The resultant low-density cell fraction consisted of a nonadherent cell population expressing different intensities of CD11c that could at least be characterized by typical DC morphology (e.g. abundant cytoplasma with veil-like cytoplasmatic dendrites, irregularly shaped nuclei, multivesicular and multilamellar bodies), constitutive levels of surface MHC class II, the presence of macrophage-specific markers, such as F4/80, Mac-I and Fc receptors, respectively, on subpopulations of CD11c+ sorted cells and expression of adhesion and co-stimulatory receptors like ICAM-1 and CD44. The capability of this low-density CD11c+ fraction to stimulate T cell responses was demonstrated in primary allogeneic mixed-lymphocyte reactions (MLR). Herein, we show that the freshly isolated CD11c+ cells showed weak accessory function, but develop this capacity following short-term culture in vitro in the presence of granulocyte/macrophage colony-stimulating factor. Although the nature and functional capacity of the isolated CD11c+ needs further clarification, these preliminary results describing phenotype and accessory function provide some evidence that these cells isolated from the PP may be immature forms of DC and play a crucial role as antigen-presenting cells with important implications for understanding the complex network regulating intestinal antigen uptake, processing and presentation.
Animals↗
The association of human papillomavirus type 16 E6 and E7 antibodies with stage of cervical cancer.
OBJECTIVE: Previous investigators have reported higher HPV type 16 antibody positivity among cervical cancer patients than among healthy women. The objective of this study was to determine the association of HPV 16 antibody levels with the stage of cervical cancer. METHODS: Pretreatment tumor biopsies and sera were obtained from 137 newly diagnosed cervical cancer patients residing in Mexico. Using peptide ELISA and radioimmunoprecipitation assay (RIPA), HPV 16 E6- and E7-specific antibodies were measured. RESULTS: By ELISA, elevated antibody titers to HPV 16 E6 and E7 were detected in 16.8 and 32.8% of the women, respectively. While sera positivity did not differ by disease stage, the mean absorbance in the E7-positive sera was 0.42, 0.62, 0.91, and 0.81 for stages I to IV, respectively. Using RIPA, anti-E6 and E7 positivity was demonstrated in 46.7 and 38.7% of the females, respectively. Although no difference across disease stage was detected for E6, increasing proportions of positivity to E7 with stage of disease was detected. The rates for increasing disease stage were 0.14, 0.37, 0.40, and 0.67. Sera from the 6-month postradiation follow-up examinations of a small group of patients demonstrated a statistically significant decrease in antibody positivity from pretreatment positivity to HPV 16 E6 (n = 14; P = 0.01) and HPV 16 E7 (n = 20; P = 0.0001) using ELISA. CONCLUSIONS: These data suggest that HPV 16 E7 antibody positivity may be associated with stage of cervical cancer. Such immune parameters may be applicable to disease staging, monitoring of recurrence and, perhaps, diagnosis. Further investigation into the relationship of HPV 16 E6 and E7 antibodies with stage of cervical cancer and response to therapy is warranted.
Adult↗
Analysis of the initiation period of spontaneous autoimmune thyroiditis (SAT) in obese strain (OS) of chickens.
The early, predictable, onset of spontaneous autoimmune thyroiditis (SAT) in Obese strain (OS) chickens provides a unique opportunity to analyse the mechanisms initiating autoimmunity which is virtually impossible to obtain in humans. In this study we focused on the respective roles of viruses and macrophages in the initiation of SAT. To analyse viruses, leukosis virus-free OS chickens were bred over three generations and reared under gnotobiotic conditions. By 2 weeks of age there were no differences in the levels of thyroid mononuclear cell infiltration between these and control animals. The role of mononuclear phagocytes in SAT was determined by their depletion via injecting newborn OS chicks with silica or carrageenan and dichloro-methylene diphosphonate encapsulated into liposomes. Although this treatment did not substantially change the amount of macrophages in primary lymphoid organs or blood, there was destruction of splenic architecture and, most importantly, mononuclear cell infiltration of the thyroids was significantly lower compared to controls. The role of activated macrophages in SAT is discussed.
Animals↗
Specific and sensitive two-step polymerase chain reaction assay for the detection of Salmonella species.
A polymerase chain reaction (PCR) assay was applied for the selective amplification of a characteristic sequence within a Salmonella-specific chromosomal fragment. A two-temperature PCR cycle enhanced both the speed and overall sensitivity of the amplification procedure. Twenty-one well-characterized Salmonella strains and a number of non-Salmonella strains were tested. With the exception of the rarely isolated Salmonella arizonae strain, the PCR-based approach enabled the specific identification of Salmonella with a detection limit of 10(3) organisms. In combination with a nested PCR assay, as few as ten organisms were detectable. Specificity was demonstrated as no distinct amplification products were detectable with any of the tested non-Salmonella strains. With a pre-enrichment step using paramagnetic anti-Salmonella beads, an increase in sensitivity was observed in the case of clinical samples while the amplification process was not influenced.
DNA, Bacterial↗
Radiotherapy with iodine-131 in recurrent malignant struma ovarii.
Malignant struma ovarii is a very rare disease and, therefore, there is neither common agreement on treatment regimens nor sufficient follow-up experience. We present a case of a 49-year-old woman with malignant struma ovarii of the follicular type, who received ablative radioiodine treatment after thyroidectomy and surgical removal of the primary tumour. During follow-up examinations an increasing thyroglobulin level was found, caused by a tumour relapse with suspected urinary bladder infiltration on CT and proven uptake of radioiodine on whole-body scanning with iodine-131. After administration of 6GBq 131I, complete tumour regression was achieved with no evidence of a new relapse during a 30-month follow-up period. Correspondingly, repeated thyroglobulin measurements were all negative. This case demonstrates the benefit of combined surgical and radioiodine treatment of malignant struma ovarii for both monitoring and therapy of relapse or metastases; thus, the same therapeutic regimen as is employed in primary differentiated thyroid carcinoma may be recommended.
Biomarkers, Tumor↗
Expression and purification of an Epstein-Barr virus encoded 23-kDa protein and characterization of its immunological properties.
Serodiagnosis of Epstein-Barr virus (EBV) infection is currently based on the detection of antibodies to distinct EBV antigens by immunofluorescence and enzyme-linked immunosorbent assay-based tests, or in part on the detection of heterophile antibodies by the Paul-Bunnell-Davidson heterophile assay. In the past few years, the specificity and the sensitivity of serodiagnostic assay systems has been improved considerably by the use of purified recombinant EBV antigens. Screening of EBV-positive sera for antigenic reactivities by immunoprecipitation with extracts of EBV-positive cells revealed a 23-kDa protein (p23) that was recognized by antibodies from all EBV carriers tested. Open reading frame BLRF2 was identified as the coding region for this protein. After cloning and high-level expression of the BLRF2 open reading frame as DHFR fusion protein in Escherichia coli, the recombinant protein was purified to near homogeneity with the help of continuous elution electrophoresis. Sera from both EBV-positive and -negative donors were screened by immunoblot analysis and enzyme-linked immunosorbent assay for IgM and IgG antibodies against the EBV-encoded protein p23. Since anti-p23 antibodies were not detectable in 30 of 30 EBV-negative sera, and 294 of 302 EBV-positive sera had either IgM and/or IgG antibody responses to this protein, recombinant p23 seems to be a useful diagnostic marker for EBV-infection.
Antibodies, Viral↗
Humoral and cellular immune responses in the murine respiratory tract following oral immunization with cholera toxin or Escherichia coli heat-labile enterotoxin.
Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are the strongest mucosal immunogens identified to date and are also good adjuvants when given orally together in combination with unrelated antigens. We used these potent immunogens to monitor local and systemic immune responses following oral immunization of BALB/c mice, and compared their action on the following: (a) immunoglobulin production rates (IgG, IgM and IgA) in mucosal inductive (Peyer's patches-PPs), effector (intestinal lamina propria-LP, respiratory tract) and systemic (spleen) sites; (b) analysis of systemic antigen-specific antibodies (IgG subclasses, IgA and IgE); (c) time monitoring of fecal anti-CT and anti-LT antibodies, and (d) in vivo relevance of interleukin-6 (IL-6) to mucosal responses. Both mucosal immunogens elicited specific antibody responses (IgA, IgG) not only in the gastrointestinal tract (PP's and intestinal LP), but also in the respiratory tract and spleens of orally immunized mice. These mucosal responses were accompained by elevated secretion of IL-6 in all investigated tissues, indicating involvement of this cytokine in B-cell maturation processes. Furthermore, oral immunization with CT and LT induced elevated serum titers of IgG1 followed by IgG2a, IgG2b, IgG3 and IgA, while high antigen-specific IgA and IgG1 responses were found in fecal extracts. These findings illustrate the action of orally administered CT and LT, respectively, on several humoral and cellular immune responses not only at the gastrointestinal tract, the application site, but also in distant mucosal effector sites such as the respiratory tract. These data suggest the potential use of these mucosal adjuvants in oral immunization strategies to improve the local immune response in remote mucosal tissues, in accordance with the concept of a common mucosa-associated immune system.
Administration, Oral↗
Importance of simultaneous active cytomegalovirus and Epstein-Barr virus infection in renal transplantation.
BACKGROUND: Although being the most common infective complication after transplantation, cytomegalovirus (CMV) infection does not always produce disease symptoms in immunosuppressed patients. Development of CMV disease may depend on different factors such as virulence of particular CMV strains and impairment of CMV-specific immune reactions. OBJECTIVE: Demonstration of the importance of simultaneous Epstein-Barr virus (EBV) activation for development of symptomatic CMV infections. STUDY DESIGN: 208 renal transplantation patients were monitored for 3 years with respect to (i) CMV and EBV replications, and (ii) clinical symptoms associated with combined and single infections, respectively. RESULTS: CMV and EBV replications were observed in 22% and 19% of the patients, respectively. Many of these active virus infections were found to overlap in time (59% and 74% of all active CMV and EBV infections, respectively). The increased detection of combined CMV and EBV infections probably does not result from higher initial immunosuppression in these patients, since the percentage of patients receiving OKT3 or ATG was almost identical in the groups of single and combined infections. In 18 cases of combined infections, CMV replication preceeded EBV replication, while EBV replication prior to CMV replication was observed in one case only, indicating that activation of latent EBV infection may be induced during active CMV infection. CONCLUSIONS: Simultaneous replication of both viruses seems to be clinically important, since severe clinical symptoms were observed only in the group of combined CMV and EBV infections. Symptoms were similar to the clinical pictures of CMV disease. Thus, simultaneous EBV replication may be an important co-factor for the development of CMV disease, possibly by further decreasing the number of functional CD4 T cells or enhancing the CD8-positive cytolytic/suppressor T-cell subset as reflected by the comparatively stronger decrease of CD4/CD8 ratio during simultaneous CMV and EBV replication, particularly in the case of symptomatic infections.
Journal Article↗
Fetal distress due to placental insufficiency at 26 through 31 weeks: a comparison between an active and a more conservative management.
OBJECTIVE: To compare perinatal mortality and short-term morbidity in extremely preterm infants with fetal distress due to placental insufficiency in two centers with different management attitude. DESIGN: Retrospective cohort study in two university hospitals of all infants with fetal growth retardation due to placental insufficiency resulting in signs of fetal distress at 26 through 31 weeks gestational age, during the years 1984 through 1989. Center A followed a conservative management: in some cases the risk of major handicaps or mortality was estimated so high, based on antenatally estimated fetal weight and gestational age, that the decision was taken to abstain from treatment. In all other cases cesarean section took place, but only if fetal distress was obvious. Center B used a more active management: cesarean section was performed in all cases, sometimes with only minor changes in fetal heart rate variability. RESULTS: Overall survival differed significantly: 55% (center A) versus 72% (center B), largely due to antenatal mortality in center A. Discharge survival rate of liveborn infants was 81% in center A and 72% in center B. More than half of the postnatal mortality was attributed to respiratory causes in both centres. An active management showed a tendency to a higher incidence of short-term morbidity. CONCLUSION: Selection by antenatal prediction of postnatal mortality using estimated fetal weight fails. Even in the group with the lowest birthweight postnatal mortality did not surpass 50%. Early intervention may be associated with higher short-term morbidity. Long-term follow-up of these children is needed to discriminate between both policies with regard to further development of surviving infants.
Bronchopulmonary Dysplasia↗
Pathogenesis of chronic Epstein-Barr virus infection: detection of a virus strain with a high rate of lytic replication.
In rare cases Epstein-Barr virus (EBV) leads to chronic active infection (CAEBV) which is characterized by persistant symptoms of infectious mononucleosis. Previously we described a case of persisting polyclonal B-cell lymphocytosis (PPBL) that was associated with CAEBV. Using reverse transcription and polymerase chain reaction we showed that in late passages of a spontaneous cell line, SM, latent EB viral genes such as EBNA1, EBNA2, EBNA3A/3B/ 3C, LMP1 and LMP2A were active. The master gene of the lytic cycle, BZLF1, was silent. This indicated that there was no general defect in immortalization and establishing latency by this CAEBV isolate SM. We obtained virus from the standard immortalizing strain B95-8 and the CAEBV strain SM from latently infected LCL quantified the number of virus particles by competitive PCR and demonstrated that the impaired capacity to immortalize umbilical cord blood lymphocytes was a virus strain-specific property, and was not due to an incapability to infect purified CD19+ B lymphocytes. Transcription of latency- and immortalization-associated genes such as EBNA1, EBNA2 and LMP2A was reduced, in contrast to a strongly enhanced activity of the master gene of the lytic cycle, BZLF1. A scenario for an antagonistic regulation of lytic and latent cycle genes is presented and a role for the pathogenesis of CAEBV is discussed.
B-Lymphocytes↗
Anticoagulant effects and tissue factor pathway inhibitor after intrapulmonary low-molecular-weight heparin.
The present study was designed to investigate the anticoagulant action of inhaled low-molecular-weight (LMW) heparin on the release of tissue factor pathway inhibitor (TFPI) and on antifactor Xa activity, Heptest, activated partial thromboplastin time (APTT) and thrombin clotting time (TCT) in healthy volunteers. 3000 IU (group 1), 9000 IU (group 2), 27,000 IU (group 3) or 54,000 IU (group 4) LMW-heparin were given to 20 healthy volunteers each at 4 weeks intervals by inhalation. For safety reasons a dose escalating design was chosen. APTT and TCT did not change after inhalation of any dose of LMW-heparin as well as all parameters in group 1. In group 2, Heptest coagulation times were prolonged from 18.7 +/- 2.0 s before to 26.1 +/- 5.2 s 6 h and 20.5 +/- 1.9 s 24 h after inhalation and the other parameters remained uneffected. In group 3, S2222 method and the protamine assay increased from 0.01 to about 0.1 IU/ml 6 h after inhalation and returned to normal values after 24 h. TFPI antigen increased from 74.1 +/- 13.9 to 80.5 +/- 14.2 ng/ml 3 h after inhalation. TFPI activity remained unchanged. Heptest coagulation values were prolonged to 42 +/- 7.6 s after 6 h and returned to normal within 72 h after inhalation. In group 4, the following changes were observed: antifactor Xa activity increased to 0.343 +/- 0.196 U/ml after 6 h and normalized after 72 h. The protamine assay detected 0.2 +/- 0.18 U LMWH/ml after 6 h, TFPI antigen increased to 103 +/- 17.9 ng/ml and TFPI activity to 1.14 +/- 0.23 U 3 h after inhalation. All tests were normal after 24 h. Heptest coagulation values increased to 77.5 +/- 11.8 s 6 h after inhalation and normalized after 144 h. The area under the activity time curve of the S2222 method and of the Heptest assay increased with increasing doses (r = 0.677 and r = 0.571), respectively. The calculated elimination half-life of the aXa-effect was 7.5 h using S2222-, Heptest- and protamine assays. The data demonstrate a resorption of LMW-heparin by intrapulmonary route in man. The dose to produce antifactor Xa levels, prolongations of Heptest coagulation values and in releasing TFPI is about ten-fold higher than after subcutaneous administration.
Administration, Inhalation↗
Somatostatin receptor scintigraphy in the staging of small cell lung cancer.
The aim of this study was to evaluate somatostatin receptor scintigraphy (SRS) in the staging of patients with small cell lung cancer. Prior to chemotherapy, 20 patients were investigated up to 24 h following an injection of 200 MBq 111In-octreotide. Following chemotherapy and restaging, four patients were re-evaluated. Primary tumour was detected in 18 of 23 studies, which exhibited increasing target-to-back-ground ratios over time. Lymph node metastases and distant metastases were detected in 7 of 27 and 8 of 31 sites, respectively. Thus, the overall sensitivity for detecting metastases was less than 26%. SRS did not result in any upstaging of patients. We conclude that in patients with small cell lung cancer, functional imaging by SRS has no impact on clinical decision making.
Aged↗