Preparation and immunochemical characterization of meningococcal group C polysaccharide-tetanus toxoid conjugates as a new generation of vaccines.

Neisseria meningitidis group C polysaccharide-tetanus toxoid conjugates have been prepared by using high-molecular-weight polysaccharide and purified tetanus toxoid and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide as a coupling reagent. The influence of three conditions of preparation was studied. Biochemical assays, the enzyme-linked immunosorbent assay, and isopycnic CsCl gradient ultracentrifugation have been used to characterize the conjugates. The polysaccharide-to-protein ratios of the various conjugate preparations showed differences. The ability of the group C polysaccharide component to react with specific antibodies was reduced, whereas most of the tetanus toxoid seemed to be hidden by the polysaccharide. The composition of the conjugate was not homogeneous, and at least 10% of free polysaccharide was present. Thermostability in lyophilized condition in the presence of lactose was excellent.     

Haemodynamic dose-response effects of intravenous oxprenolol in coronary heart disease

The haemodynamic dose-response effects of intravenous oxprenolol over the range of 2--64 mg were measured in 28 patients with coronary heart disease. At rest there were statistically significant dose-response trends, linear or quadratic, between the cumulative doses of oxprenolol and reductions in systolic pressure, heart rate, and cardiac output and increases in stroke volume and pulmonary wedge pressure. The magnitude of the changes was uninfluenced by the degree of left ventricular functional disability as judged by the level of the pulmonary wedge pressure. During dynamic exercise the haemodynamic changes induced by oxprenolol were significantly greater in patients with more marked left ventricular disability than in those less severely affected. These observations define the immediate effects of intravenous oxprenolol over a relatively wide dose range and confirm its relative haemodynamic safety in patients with stable coronary heart disease.     

Intraoperative electrocortical stimulation of Brodman area 4: a 10-year analysis of 255 cases

Background

Brain tumor surgery is limited by the risk of postoperative neurological deficits. Intraoperative neurophysiological examination techniques, which are based on the electrical excitability of the human brain cortex, are thus still indispensable for surgery in eloquent areas such as the primary motor cortex (Brodman Area 4).

Methods

This study analyzed the data obtained from a total of 255 cerebral interventions for lesions with direct contact to (121) or immediately adjacent to (134) Brodman Area 4 in order to optimize stimulation parameters and to search for direct correlation between intraoperative potential changes and specific surgical maneuvers when using monopolar cortex stimulation (MCS) for electrocortical mapping and continuous intraoperative neurophysiological monitoring.

Results

Compound muscle action potentials (CMAPs) were recorded from the thenar muscles and forearm flexors in accordance with the large representational area of the hand and forearm in Brodman Area 4. By optimizing the stimulation parameters in two steps (step 1: stimulation frequency and step 2: train sequence) MCS was successful in 91% (232/255) of the cases. Statistical analysis of the parameters latency, potential width and amplitude showed spontaneous latency prolongations and abrupt amplitude reductions as a reliable warning signal for direct involvement of the motor cortex or motor pathways.

Conclusion

MCS must be considered a stimulation technique that enables reliable qualitative analysis of the recorded potentials, which may thus be regarded as directly predictive. Nevertheless, like other intraoperative neurophysiological examination techniques, MCS has technical, anatomical and neurophysiological limitations. A variety of surgical and non-surgical influences can be reason for false positive or false negative measurements.     

p53 and recombination intermediates: role of tetramerization at DNA junctions in complex formation and exonucleolytic degradation

Heteroduplex joints represent intermediates of Rad51-dependent recombination processes, which are recognized by p53 with extremely high affinities, in a manner independent of the DNA sequence content. To determine the structural elements required for complex formation, we monitored DNA-binding by protection against restriction endonuclease cleavage. We show that wild-type (wt) p53 interacts with heteroduplex joints in the proximity of the flexible junction. Association of p53 within this junction region was also observed with preformed Rad51-heteroduplex complexes, whereas SSB counteracted p53 binding. At a distance of 31 bp from the junction p53 established very few contacts with the heteroduplex, despite the presence of an A-G mismatch. Consistently, p53-dependent exonucleolytic degradation decreased when we raised the distance between the junction and the heteroduplex terminus by 27 bp. Different from the cancer-related mutant p53(273H), which did not recognize the junction, tetramerization defective p53-1262 was protection competent but displayed reduced complex stability in gel shifts. Moreover, p53-1262 performed exonucleolytic activities towards ssDNA like wtp53, but reduced degradation of heteroduplex joints. These results suggest that during recombination wild-type p53, as a tetramer, stably binds to strand transfer regions, enabling the protein to exonucleolytically correct heteroduplex intermediates early after strand invasion.     

High bad and bcl-xL gene expression and combined bad,bcl-xL,bax and bcl-2 mRNA levels: molecular predictors for survival of stage 2 soft tissue sarcoma patients

The role of the bcl-2 gene family members in promoting or antagonizing apoptosis in malignant tumors, including soft tissue sarcomas (STS), is well known. However, the impact of mRNA expression of bcl-2 family genes on prognosis has not been thoroughly investigated in STS. Samples from 82 STS patients were analyzed for mRNA expression of bad, bax, bcl-xL and bcl-2 by a high-throughput quantitative RT-PCR approach, using validated assays based on TaqMan technology. The mRNA data, related to glyceraldehyde-3-phosphate dehydrogenase expression measured in the same sample, were analysed for their correlation to tumor stage and overall survival of patients. In a Kaplan-Meier analysis none of the mRNA levels investigated differed significantly with regard to their impact on survival (log-rank test). However, after including the tumor stage in the statistical analysis, a borderline significance was observed for bad mRNA expression (p=0.068) indicating a stage-specific impact of mRNA expression on prognosis. Considering STS patients of tumor stage 2, multivariate Cox analysis revealed that bad mRNA values > or = 10 (p=0.0039; RR=9.08), bcl-xL > or = 1.5 (p=0.067; RR=4.59), bax > or = 0.005 (p=0.1; RR=2.84) and bcl-2 < 3 (p=0.42; RR=1.7) were associated with a poor prognosis. Combined high bad/bcl-xL mRNA expression levels revealed a 20-fold increase in the relative risk of tumor-related death (p=0.016) when comparing the poor and good prognosis groups. There was a 14.5-fold and 6.5-fold increase in the risk for the combinations of high bax/bcl-xL mRNA (p=0.018) and bax/bcl-2 mRNA expression (p=0.017), respectively. In conclusion, high bad mRNA levels and combined values of bad/bcl-xL bax/bcl-xL and bax/bcl-2 appear to be independent prognostic factors at least for stage 2 STS patients. In the combinations of mRNA levels there was more than an additive effect pointing to different pathways of prognostic relevance.     

In vivo recording of monophasic action potentials in awake dogs – new applications for experimental electrophysiology

Introduction Despite enormous developments in the field of clinical and experimental electrophysiology there is still a gap in evaluating repolarization in the awake animal. Numerous previous studies have used monophasic action potentials (MAP) to assess repolarization in vitro and in vivo in anesthetized animal models. However, an approach for recording MAP in awake dogs without interference of anesthesia has not yet been developed. Methods and results We developed an experimental technique to record MAP in conscious dogs by means of conventional rubber introducers which were implanted into the internal jugular vein. In seven awake dogs, atrial as well as ventricular MAP were simultaneously measured without complications. Pacing thresholds were low and stable over time ranging from 0.2 to 4.0 mA. The MAP amplitudes ranged from 10 to 30 mV for ventricular and from 5 to 15 mV for artial MAP. Contrinuous MAP recordings of stable amplitude could be made from the same endicardial site for periods of up to one hour. Antegrade and retrograde AV-nodal conduction properties could be assessed. Programmed stimulation was performed to simultaneously determine local refractory periods and MAP duration at cycle length from 500 to 200 ms. Conclusion In awake, unsedated dogs the measurement of MAP via rubber introducers permits safe, long-term recording of MAP. Such recordings may be useful for safety pharmacological studies in evaluating cardiovascular and non-cardiovascular drugs with regard to their effects on repolarization. In various canine in vivo models including in vivo models of long QT syndrome, heart failure or sudden cardiac death, the present technique permits electrophysiological measurements without interference of anesthesia. Received: 20 July 2000, Returned for revision: 23 August 2000, Revision received: 29 August 2000, Accepted: 30 August 2000     

Successful mobilization of peripheral blood stem cells with the DHAP regimen (dexamethasone, cytarabine, cisplatinum) plus granulocyte colony-stimulating factor in patients with relapsed Hodgkin's disease

High-dose chemotherapy followed by autologous stem cell transplantation can improve the outcome of relapsed and refractory Hodgkin's disease (HD) patients. The objective of the trial was to determine the mobilizing potential of the DHAP salvage regimen (dexamethasone, cytarabine, cisplatin) for the collection of peripheral blood stem cells (PBSC) in patients with relapsed HD. The target yield of harvesting CD34 + cells was ≥ 2 × 10⁶/kg in order to support the subsequent myeloablative chemotherapy. Most of the 105 patients included were intensively pre-treated with different combination chemotherapy regimens prior to mobilization. The use of DHAP followed by granulocyte colony-stimulating factor (G-CSF; 10 μg/kg) resulted in the successful collection of adequate numbers of PBSC in 97.1% of patients (102 of 105) with a median harvest of CD34 + cells of 13 × 10⁶/kg (range 2.6 - 85.1). More than 2.0 × 10⁶ CD34 + cells/kg were achieved in 65 of 103 (63%) patients after 1 apheresis, the maximum number of aphereses for all patients was 3. It was found that the optimal time of PBSC harvest was at days 13 - 16 after initiating the mobilization regimen.

These results demonstrate that the salvage chemotherapy regimen, such as DHAP combined with G-CSF, can be successfully used to mobilize PBSC in HD patients.