In vitro culture of human acute lymphoblastic leukemia (ALL) cells in serum-free media; a comparison of native ALL blasts,ALL cell lines and virus-transformed B cell lines

The aim of this study was to standardize in vitro culture conditions for human acute lymphoblastic leukemia (ALL) cells. The cells were cultured in medium containing 10% fetal calf serum (FCS) and in the four serum-free media X-vivo 10, X-vivo 15, X-vivo 20 and Stem Span. Native ALL blasts could proliferate in all four serum-free media, but the strongest responses were usually observed with Stem Span. Native leukemia blasts were also cultured in the presence of various single cytokines or cytokine combinations. The highest proliferation was usually observed in the presence of Flt3-Ligand (Flt3-L) when single cytokines were examined, and these responses could be further increased especially by combining Flt3-L with interleukin 3 (IL3), IL7 or stem cell factor (SCF). Proliferation could also be increased when ALL blasts were cultured in the presence of two commercially available fibroblast cell lines (Hs27 and HFL1). Based on these results we suggest that in vitro culture conditions for native human ALL blasts can be standardized by using serum-free culture media supplemented with exogenous Flt3-L+IL3+SCF, and the use of accessory cells can also be standardized by using well-characterized fibroblast cell lines. Detectable ALL blast proliferation can then be observed for most patients. Our experimental model can thereby be used for in vitro evaluation of possible antileukemic treatment strategies, and it will then allow comparison of experimental results between different studies.     

Imaging of Experimental Rat Gliomas Using a Clinical MR Scanner

Background: Studies of brain tumor development in experimental animal models have to date mostly been based on post-mortem histological examinations. The use of magnetic resonance imaging (MRI) may provide a non-invasive technique for studying tumor growth and treatment effects in such animal models. However, most of these studies have been performed on purpose-dedicated small bore magnetic resonance (MR) systems, of high cost and limited availability. The purpose of this study was thus to obtain high-resolution images of experimental gliomas in the rat brain, using a clinical 1.5T MR scanner. Methods: Anesthesized rats bearing BT₄C brain tumors were positioned into a specially designed immobilizing device, and a small circular coil was positioned onto the skulls. Two T1 weighted series were acquired before and after subcutaneous contrast injections. A T2 weighted series was also obtained. The rats were then sacrified, the brains removed, and the histological tumor volumes were compared to the volumes obtained on MRI. Results: There were visible tumors in 10 of 13 animals scanned on MR. The rim of the tumors were visualized on T1 weighted series without contrast. On T1 images with contrast, the tumors were seen as high signal intensity areas. The T2 weighted images showed peritumoral edema. No necrosis or cystic parts of the tumors were detected. There was a consistency between the MR and the histology findings, showing a high degree of correlation between the two volume determination methods. Conclusions: High-resolution images of experimental rat gliomas can be obtained using a clinical MR scanner and a commercially available RF coil. This MRI technique may also be expanded to extraneural rat tumor models, for studies of tumor development and treatment.     

On the effect of biochemotherapy in metastatic malignant melanoma: an immunopathological evaluation

Although immunotherapy and biochemotherapy have shown promise, producing a subset of durable responses, for the majority of patients with metastatic melanoma the prognosis is still poor. Therefore there is a great need for predictive tests to identify patients with a high probability of responding. Furthermore, there is also a need for a better understanding of the mechanisms of action during treatment in order to be able to monitor the relevant antitumour reactivity during treatment and to optimize the efficacy of future immunotherapy and biochemotherapy. In the present study histopathological regression criteria were used to study the efficacy of biochemotherapy. Thirty-two patients with metastatic malignant melanoma (18 with regional disease and 14 with systemic disease) were treated with biochemotherapy (cisplatin 30 mg/m2 intravenously on days 1-3, dacarbazine 250 mg/m2 intravenously on days 1-3 and interferon-alpha2b 10 million IU subcutaneously 3 days a week, every 28 days). Pre-treatment fine needle aspirates were obtained from metastases to analyse the number of tumour-infiltrating CD4+ lymphocytes. Therapeutic efficacy was evaluated in metastases resected after treatment using histopathological criteria of tumour regression. Comparisons were also made with metastases from 17 untreated patients, all with regional disease. Regressive changes of 25% or more (of the section area) were found in two of the 17 untreated patients with regional disease compared with 13 of the 18 patients with regional disease and 10 of the 14 patients with systemic disease after biochemotherapy. Fifty per cent of the patients with regional disease showed a high degree of regressive changes (75-100% of the section area) after biochemotherapy. These results demonstrate the occurrence of an antitumour reactivity in the majority of patients. Patients with extensive regressive changes in 75-100% of the analysed biopsies were also found to have a longer overall survival (P = 0.019). In patients with regional disease there was a close correlation between a larger number of CD4+ lymphocytes pre-treatment and a higher degree of regressive changes post-treatment (P < 0.05). Thus, immunohistochemical analysis of tumour biopsies shortly after treatment seems to be a good surrogate endpoint. This technique also allows detailed analysis of antitumour reactivity and escape mechanisms.     

Neonatal lethal dwarfism with distinct skeletal malformations--a separate entity?

We describe a case of neonatal lethal dwarfism characterised by short trunk, short, stick-like tubular bones, deficient ossification of the axial skeleton and broad, sclerotic horizontal ribs. Two similar cases have previously been reported as examples of the Neu-Laxova syndrome. However, the radiological findings of the Neu-Laxova syndrome, as reported in 16 out of 40 documented cases, show a heterogeneous pattern of minor features, which differ distinctively from those found in the previous two cases and by us. A literature research did not reveal similar cases, and we therefore suggest that our case, together with the two previous cases, may represent a new distinctive form of neonatal lethal dwarfism. Received: 6 March 2000 Revised: 3 October 2000 Accepted: 15 March 2001     

Tissue engineering in plastic reconstructive surgery

Tissue engineering (TE) is a new interdisciplinary field of applied research combining engineering and biosciences together with clinical application, mainly in surgical specialities, to develop living substitutes for tissues and organs. Tissue engineering approaches can be categorized into substitutive approaches, where the aim is the ex vivo construction of a living tissue or organ similar to a transplant, vs. histioconductive or histioinductive concepts in vivo. The main successful approaches in developing tissue substitutes to date have been progresses in the understanding of cell-cell interactions, the selection of appropriate matrices (cell-matrix interaction) and chemical signalling (cytokines, growth factors) for stimulation of cell proliferation and migration within a tissue-engineered construct. So far virtually all mammalian cells can be cultured under specific culture conditions and in tissue specific matrices. Future progress in cell biology may permit the use of pluripotent stem cells for TE. The blueprint for tissue differentiation is the genome: for this it is reasonable to combine tissue engineering with gene therapy. The key to the progress of tissue engineering is an understanding between basic scientists, biochemical engineers, clinicians, and industry.     

On glomerular structural alterations in type-1 diabetes

Glomerular structural modifications were measured in kidney biopsies from two follow-up studies in type-1 diabetic patients with microalbuminuria and in kidney donors. Stereologic methods were used to obtain data on glomerular composition and absolute quantities per glomerulus to supplement data on diabetic glomerulopathy previously published. Diabetic patients at baseline (n=37) showed significant changes compared with controls (n=11). The volume fraction of tuft/glomerulus was increased, the proportion of capillary surface facing peripheral basement membrane was decreased (0.72+/-0.04 vs 0.77+/-0.03, P=0.0008), the ratio of mesangial surfaces, urinary/capillary, was decreased (0.67+/-0.17 vs 1.11+/-0.28, P<10(-4)), and the average capillary diameter was increased (8.9+/-0.9 microm vs 7.5+/-1.0 microm, P=0.0002). The total volume of mesangial extracellular material per glomerulus was increased (P=0.01), whereas glomerular volume was not significantly different from controls. Follow-up biopsies after antihypertensive treatment with ACE-inhibitor (n=7) or beta-blocker (n=6; 36-48 months) and after intensive insulin treatment (n=7; 24-33 months) showed no change. In a conventionally treated group (n=9), the glomerular volume, the volume of extracellular material/glomerulus, and the capillary length increased. The mean capillary diameter did not correlate with the glomerular volume. In conclusion, the development of diabetic glomerulopathy entails structural modifications of the glomerular tuft. Antihypertensive and intensified insulin treatment seem to slow the progression of ultrastructural changes.     

Yeasts in periodontal pockets

OBJECTIVES: The presence of yeasts in periodontal pockets has been described in a few studies. The association between yeasts and putative periodontal pathogens is not well described. This study aims at assessing the prevalence of yeasts in periodontal pockets and possible associations with the clinical conditions of the sampled sites and other micro-organisms present. MATERIAL AND METHODS: 2 subject groups form the basis for this study. The 1st comprises results from microbiological samples from periodontal pockets of 128 subjects. The 2nd originates from 126 periodontal patients with untreated pockets. Microbiological identification was performed after cultivation on blood and Sabouraud agar plates, and "checkerboard" DNA-DNA hybridisation. RESULTS: The prevalence of subjects with yeasts in the pockets was 15.6% and 17.5% in the 2 groups respectively and was inconsistent according to gender. No correlation was found between age and the presence of yeasts. Eubacterium saburreum was weakly correlated with presence of yeasts (r=0.194 p=0.03). Yeasts were rarely found in both samples from the same individual. CONCLUSIONS: Our results indicate that yeasts can be expected to be present in periodontal pockets in one out of 6 periodontal patients independent of gender and age. Eubacterium saburreum seems to occur frequently together with yeasts.     

Umbilical cord plasma interleukin-6 and fetal growth restriction in preeclampsia: a prospective study in Norway

OBJECTIVE: To study the association between umbilical plasma levels of interleukin-6 (IL-6) in relation to fetal growth in subgroups of preeclampsia, and in control pregnancies. METHODS: Umbilical cord plasma was collected from 12,804 consecutive births. A total of 271 singleton cases of preeclampsia were identified, and classified as mild or severe, and as disease with early or late onset. As controls, 611 singleton pregnancies without preeclampsia were selected, and the ratio between observed and expected birth weight was used as a measure of fetal growth. In the analysis, we also included maternal smoking during pregnancy. Umbilical cord plasma IL-6 concentration was measured with an IL-6 bioassay. Comparing controls with subgroups of preeclampsia (severe and early onset), this study had a statistical power of 90% to detect a difference in cord IL-6 of 10 pg/mL. RESULTS: In severe preeclampsia, cord plasma IL-6 concentration was lower than among controls (P <.001), and there was a sharp decrease in cord plasma IL-6 with decreasing birth weight ratio (P trend <.001). By further dividing the preeclampsia group into early or late onset, the strong association between low IL-6 levels and low birth weight ratio appeared to be present mainly in early-onset disease. These results were not confounded by maternal smoking. CONCLUSION: Restricted fetal growth related to preeclampsia is associated with reduced umbilical cord plasma IL-6 concentration in cases with early-onset disease. In these cases, fetal growth restriction could be mediated by impaired trophoblast function.     

Mobility provocation radiostereometry in anterior cervical spine fusions

This study aimed to evaluate the use of mobility-provocation radiostereometry (RSA) in anterior cervical spine fusions and compare the results to deformation studies on the same patients and plain flexion-extension radiographs. Mobility-provocation RSA was used to evaluate anterior cervical spine fusions in 45 patients. The motions recorded at 3 and 12 months postoperatively were compared to RSA measurements of deformation of the fusion over time and to plain flexion-extension radiographs in the same patients taken 3 months postoperatively. Studies of rotations from right to left revealed ten cases with significant motion at 3 months, and three at 12 months. With motion from flexion to extension, ten cases showed significant motion at 3 months and three at 12 months. In only three cases was the mobility-provocation RSA considered to add any information on the stability of the fusions compared to that obtained with the deformation studies. In 37 patients mobility-provocation radiography in flexion-extension using conventional technique was done to evaluate the accuracy. The mean difference between angular motions recorded on plain radiographs and rotations around the transverse axis in flexion to extension recorded with RSA was 1.6° (range 0.04°–8.04°, SD 2.1°). The corresponding 95% and 99% confidence limits for the difference between the two methods were 5.8° and 7.2°. The study showed that the use of mobility-provocation RSA did not add any information over that obtained by deformation RSA studies. Conventional radiography is too inaccurate to measure inducible displacement in this patient population.