Determination of monoclonal antibody specificity by immunoadsorption and western blotting.

A rapid and simple method has been developed for identifying the specificity of monoclonal antibodies at an early stage in the production of hybridomas. The technique is a micro-method utilizing biotinylated crude antigen and the surface of microtiter plates as an immunoaffinity matrix. The monoclonal antibodies to be tested are adsorbed to the microtiter wells and incubated with the labeled antigen preparation. Non-specific binding can be reduced by blocking and repeated washing steps. The specific antigen is then eluted by SDS-containing buffers, subjected to SDS-PAGE, blotted onto nitrocellulose and detected by enzyme-labeled avidin in a Western blot assay. The amount of bound and removed antigen can be quantitated by developing eluted and non-eluted control wells by ELISA techniques. Since this ELISA can be performed rapidly, only samples which contain sufficient specific material can be selected for electrophoresis and blotting. The major advantages of the technique are (i) the use of a non-radioactive label resulting in an easy and time-saving procedure, (ii) the possibility of quantitating the amount of captured and detached antigen by ELISA, (iii) the need for only a minimal amount of antigen, (iv) the use of unpurified antibodies of all isotypes, (v) a high signal-to-noise ratio, and (vi) as with all immunoprecipitation techniques, the possibility of detecting SDS-sensitive epitopes and of using crude antigen preparations. Using this method we were able to characterize monoclonal antibodies against both soluble proteins (mouse and human C1q) and membrane determinants (human pan T cell CD5 and CD7).     

Pneumonien bei akuten Leukämien

Summary Acute leukaemia was complicated by pneumonia in 38 (34.8%) of 109 patients treated between 1979 and 1983; in 39.5% of the patients pneumonia occurred more than once. In 23 patients (60.5%) pneumonia occurred during cytostatic therapy, and 25 patients (65.8%) had less than 1000 mm² granulocytes. Antibiotic therapy had no or only little effect in 70%. A total of 21 patients (55.3%) died of pneumonia. In 15 patients a direct relationship could be seen between pneumonia and the bacterial spectrum in the sputum. A prevalence of gram-negative bacteria was found (24 of 40 bacteria isolated, especially Enterobacteriaceae (19). Fungi were cultivated in 10 cases. Each of the typical pneumonia bacteria was only seen once respectively. It is most important that therapy begin immediately, even before the bacteria have been identified. Only then is there hope that the survival time of patients with acute leukaemia can be influenced.

     

The importance of autopsy for quality assurance in medical care

For all persons who died in the city of Dresden and 7 surrounding counties from 1967 to 1978, documents relating to the clinical and pathologic diagnosis were examined and findings compared. The study covered nearly two thirds of all deaths which occurred in the district of Dresden during this time period. The autopsy rate overall was found to be 22.8%. In 57.3% the clinical diagnoses were found to have been correct, in 19.7% they corresponded in part, but in 23% there was no concordance whatsoever between the clinical and autopsy diagnoses. The influence of age, place of death and disease group on the agreement between clinical and underlying pathoanatomic diagnosis was also examined. The analysis highlighted the degree to which death statistics based on the death certificate are misleading. In up to 28.8% of cases there were formal errors in the underlying disease diagnosis listed by the certifying physician on the death certificate. The value of regular comparison between the diagnoses, and its usefulness for training and continuing medical education are emphasized. The results of the study underline the importance of making available more prospectors in the district of Dresden to meet the expanding tasks of the clinically active pathologist in autopsy and biopsy diagnostic efforts.     

Discriminative stimulus effects of combinations of drug and visual stimuli in pigeons

Six pigeons were studied to determine whether the brightness of a houselight interacted with the stimuli produced by methadone, and whether the nature of the interaction depended on the order of training of the two discriminations. Three pigeons were trained to peck the right key after methadone (2.0mg/kg) and the left key after saline, when the houselight was dim. The effects of a range of methadone doses (0.5, 1.0, 2.0mg/kg and saline) were tested. Three other pigeons were trained, in the absence of drug, to peck the right key when the houselight was dim and the left key when the houselight was bright. The effects of a range of houselight intensities were tested. Then, for both groups, right-key pecks were reinforced in the presence of methadone and the dim houselight, and left-key pecks were reinforced in the presence of saline and the bright houselight. Methadone doses were tested in the presence of both houselight brightnesses used in training. All pigeons pecked the methadone-appropriate key after high doses of methadone, regardless of houselight intensity. All pigeons trained to discriminate houselight brightness first, and one of the pigeons trained to discriminate methadone first, pecked according to the houselight condition when saline and lower doses were tested. In the other pigeon trained to discriminate methadone first, pecking was more related to drug dose. These data show that a drug stimulus can compete with external stimuli for behavioral control, that a drug stimulus can assume control over behavior originally controlled by external stimuli, and that discriminations based on external stimuli may be retained when saline or low doses of drug are administered.     

Ictal spiking patterns recorded from temporal depth electrodes predict good outcome after anterior temporal lobectomy

PURPOSE: Investigators have shown that the presence of ictal spiking (IS) recorded from temporal depth electrodes is associated with mesial temporal sclerosis (MTS). We investigated the relation of IS to seizure control and pathology after anterior temporal lobectomy (ATL). METHODS: All patients undergoing intracranial ictal monitoring from a single institution since 1989 were identified. Those who did not undergo ATL or had postoperative follow-up of <1 year were excluded. All received at a minimum bilateral temporal depth electrodes. Ictal recordings were reviewed for the presence of IS, and the proportion of seizures with IS was determined for each patient. Outcome was determined by using Engel's classification. Surgical specimens were reviewed for pathology. Statistics used were chi2, Fisher exact test, and Wilcoxon rank sum. RESULTS: Forty patients with 571 seizures were reviewed. In 292 seizures from 32 patients, IS was seen. Outcomes were 24 class I (22 with IS), five class II (four with IS), three class III (one with IS), seven class IV (four with IS), and one lost to follow-up (with IS). Pathologic review revealed 25 with MTS, 22 of whom had IS. The presence of IS was associated with class I outcomes (p = 0.04), but not MTS (p = 0.06). Patients with class I outcomes had a significantly greater proportion of seizures with IS (mean, 0.58 +/- 0.3) compared with other outcomes (mean, 0.30 +/- 0.3, p = 0.02). CONCLUSIONS: The presence of IS and higher proportion of seizures with IS correlated with good seizure outcome after ATL. This information may be used in preoperative counseling.     

In vitro hematopoietic and endothelial potential of flk-1(-/-) embryonic stem cells and embryos

Mice deficient in the Flk-1 receptor tyrosine kinase are known to die in utero because of defective vascular and hematopoietic development. Here, we show that flk-1(-/-) embryonic stem cells are nevertheless able to differentiate into hematopoietic and endothelial cells in vitro, although they give rise to a greatly reduced number of blast colonies, a measure of hemangioblast potential. Furthermore, normal numbers of hematopoietic progenitors are found in 7.5-day postcoitum flk-1(-/-) embryos, even though 8. 5-day postcoitum flk-1(-/-) embryos are known to be deficient in such cells. Our results suggest that hematopoietic/endothelial progenitors arise independently of Flk-1, but that their subsequent migration and expansion require a Flk-1-mediated signal.     

Cognitive dysfunction in children with brain tumors at diagnosis

Background

Survivors of brain tumors have a high risk for a wide range of cognitive problems. These dysfunctions are caused by the lesion itself and its surgical removal, as well as subsequent treatments (chemo‐ and/or radiation therapy). Multiple recent studies have indicated that children with brain tumors (BT) might already exhibit cognitive problems at diagnosis, i.e., before the start of any medical treatment. The aim of the present study was to investigate the baseline neuropsychological profile in children with BT compared to children with an oncological diagnosis not involving the central nervous system (CNS).

Methods

Twenty children with BT and 27 children with an oncological disease without involvement of the CNS (age range: 6.1–16.9 years) were evaluated with an extensive battery of neuropsychological tests tailored to the patient's age. Furthermore, the child and his/her parent(s) completed self‐report questionnaires about emotional functioning and quality of life. In both groups, tests were administered before any therapeutic intervention such as surgery, chemotherapy, or irradiation. Groups were comparable with regard to age, gender, and socioeconomic status.

Results

Compared to the control group, patients with BTs performed significantly worse in tests of working memory, verbal memory, and attention (effect sizes between 0.28 and 0.47). In contrast, the areas of perceptual reasoning, processing speed, and verbal comprehension were preserved at the time of measurement.

Conclusion

Our results highlight the need for cognitive interventions early in the treatment process in order to minimize or prevent academic difficulties as patients return to school. Pediatr Blood Cancer 2015;62:1805–1812. © 2015 The Authors. Pediatric Blood & Cancer, published by Wiley Periodicals, Inc.