Loss of heterozygosity at locus F13B on chromosome 1q in human medulloblastoma

Medulloblastoma is a primitive neuroectodermal tumor of the cerebellum with poorly understood pathogenesis. Previous studies have reported loss of heterozygosity (LOH) on chromosome arms 17p, 11p and 9q and cytogenetic abnormalities of chromosome 1 in medulloblastoma. We have used the polymerase chain reaction to amplify 10 microsatellites on the short arm and B microsatellites on the long arm of chromosome 1 to assess allelic loss in 22 medulloblastomas. Loss of heterozygosity (LOH) on chromosome 1 was found in 9 cases. Eight medulloblastomas (36%) showed an interstitial LOH on chromosome 1q. The common region of overlap was mapped between DISI604 and DIS237 and included the locus F13B in the chromosomal region 1q31–q32.1. An additional tumor had LOH in a proximal region of 1p, but did not exhibit LOH on 1q. None of the medulloblastomas exhibited LOH of the telomeric portion of chromosome 1p, which has been associated with several other human malignancies. Our data suggest the presence of a putative tumor suppressor gene located near the locus F13B on chromosome arm 1q that appears to be involved in the pathogenesis of medulloblastoma. © 1996 Wiley-Liss, Inc.     

Cysteine protease activity is up‐regulated in inflamed ankle joints of rats with adjuvant‐induced arthritis and decreases with in vivo administration of a vinyl sulfone cysteine protease inhibitor

Objective

Cysteine proteases are postulated to play a role in tissue destruction in the joints of animals with arthritis. The purpose of the present study was to confirm the concept that cysteine proteases are enzymes involved in the pathology of rheumatoid arthritis (RA).

Methods

Arthritis was induced in Lewis rats by adjuvant injection (adjuvant‐induced arthritis [AIA] model) and scored for inflammation. At necropsy, the rear paws were either fixed in formalin and assigned a histologic score (based on synovial cell proliferation, cartilage erosion, bone erosion, and fibroproliferative pannus) or frozen, cryosectioned, and assayed for enzyme activity either by in situ cytochemical staining with a post‐azo‐coupling method using a chromogenic substrate (Z‐arg‐arg‐MNA) or by a novel assay placing the tissue section directly in a cuvette using the fluorogenic substrate Z‐arg‐arg‐AMC.

Results

Enzymatic activity, measured either in frozen sections in situ or in the cuvette assay, was positively correlated with joint destruction (r = 0.7) and inflammation (r = 0.8). Activity was not inhibited significantly by Pefabloc (a serine protease inhibitor), EDTA (a metalloprotease inhibitor), or pepstatin A (an aspartyl protease inhibitor) but was inhibited by E‐64 and vinyl sulfone irreversible inhibitors of cysteine proteases. The effect of one of the vinyl sulfone cysteine protease inhibitors, Mu‐Leu‐HomoPhe‐vinylsulfone, was tested in vivo by dietary administration at 2.2 mg/kg/day in the AIA model; this resulted in a significant decrease in inflammation and in the amount of cysteine protease activity measured in the joint tissue.

Conclusion

Cysteine protease activity levels increase in the diseased state and may be an important target for designing small molecule inhibitors to reduce the inflammation and tissue destruction associated with RA.

     

Higher spatial resolution improves the interpretation of the extent of ventricular trabeculation

The ventricular walls of the human heart comprise an outer compact layer and an inner trabecular layer. In the context of an increased pre‐test probability, diagnosis left ventricular noncompaction cardiomyopathy is given when the left ventricle is excessively trabeculated in volume (trabecular vol >25% of total LV wall volume) or thickness (trabecular/compact (T/C) >2.3). Here, we investigated whether higher spatial resolution affects the detection of trabeculation and thus the assessment of normal and excessively trabeculated wall morphology. First, we screened left ventricles in 1112 post‐natal autopsy hearts. We identified five excessively trabeculated hearts and this low prevalence of excessive trabeculation is in agreement with pathology reports but contrasts the prevalence of approximately 10% of the population found by in vivo non‐invasive imaging. Using macroscopy, histology and low‐ and high‐resolution MRI, the five excessively trabeculated hearts were compared with six normal hearts and seven abnormally trabeculated and excessive trabeculation‐negative hearts. Some abnormally trabeculated hearts could be considered excessively trabeculated macroscopically because of a trabecular outflow or an excessive number of trabeculations, but they were excessive trabeculation‐negative when assessed with MRI‐based measurements (T/C <2.3 and vol <25%). The number of detected trabeculations and T/C ratio were positively correlated with higher spatial resolution. Using measurements on high resolution MRI and with histological validation, we could not replicate the correlation between trabeculations of the left and right ventricle that has been previously reported. In conclusion, higher spatial resolution may affect the sensitivity of diagnostic measurements and in addition could allow for novel measurements such as counting of trabeculations.     

Expression of interleukin‐21 receptor in epidermis from patients with systemic sclerosis

Objective

To examine the expression and regulation of interleukin‐21 (IL‐21) and IL‐21 receptor (IL‐21R) in patients with systemic sclerosis (SSc; scleroderma).

Methods

Skin biopsy specimens were obtained from 23 patients with SSc and 15 healthy controls. IL‐21/IL‐21R messenger RNA (mRNA) was quantified using real‐time polymerase chain reaction (PCR). The expression pattern of IL‐21/IL‐21R was analyzed by in situ hybridization and Western blotting. Stimulation experiments were performed with cultured dermal fibroblasts from patients with SSc and healthy controls as well as with keratinocytes, using IL‐1β, platelet‐derived growth factor BB, monocyte chemoattractant protein 1, transforming growth factor β, and IL‐21. The SCID‐hu skin mouse model was used for in vivo experiments.

Results

IL‐21R mRNA was detected in all biopsy specimens from patients with SSc and controls, with a 4.7‐fold increase observed in SSc samples. In situ hybridization and immunohistochemical analysis showed an up‐regulation of IL‐21R in samples of epidermis from SSc patients, whereas no signal was detected in skin specimens from healthy controls. These results were confirmed in vitro, in that cultured keratinocytes expressed significant levels of IL‐21R, whereas no signal was observed in fibroblasts. Interestingly, mRNA for IL‐21 could not be detected by real‐time PCR and in situ hybridization. Various concentrations of key cytokines in the pathogenesis of SSc did not stimulate the expression of IL‐21R mRNA in cultured keratinocytes and dermal fibroblasts. In the SCID mouse transplantation model, the overexpression of IL‐21R mRNA in SSc keratinocytes remained unchanged after transplantation.

Conclusion

The up‐regulation of IL‐21R in keratinocytes indicates that, similar to fibroblasts and endothelial cells, the expression pattern is altered in SSc. Moreover, the expression of IL‐21R appears to be independent of key cytokines that are operant in SSc.

     

<Superscript>31</Superscript>P cardiac magnetic resonance spectroscopy during leg exercise at 3 Tesla

Investigation of phosphorus (³¹P) magnetic resonance spectroscopy under stress conditions provides a non-invasive tool to examine alterations in cardiac high-energy phosphate metabolism that may not be evident at rest. Our aim was to establish cardiac ³¹P MR spectroscopy during leg exercise at 3T. The increased field strength should provide a higher signal to noise ratio than at lower field strengths. Furthermore, relatively high temporal resolution at a sufficiently fine spatial resolution should be feasible. ³¹P MR spectra were obtained with a 3D acquisition weighted chemical shift imaging sequence in 20 healthy volunteers at rest, during dynamic physiological leg exercise and after recovery at 3T. Haemodynamic measurements were made throughout and the rate pressure product calculated. With exercise, the mean heart rate increased by 73%, achieving a mean increase in rate pressure product of 115%. The corrected PCr/ATP ratio for subjects at rest was 2.02 ± 0.43, exercise 2.14 ± 0.67 (P = 0.54 vs. rest) and at recovery 2.03 ± 0.52 (P = 0.91 vs. rest, P = 0.62 vs. exercise). A cardiac ³¹P MR spectroscopy physiological exercise-recovery protocol is feasible at 3T. There was no significant change in high-energy cardiac phosphate metabolite concentrations in healthy volunteers at rest, during physiological leg exercise or during recovery. When applied to patients with heart disease, this protocol should provide insights into physiological and pathological cardiac metabolism.     

Electron microscopic evidence for microglial phagocytic activity and cholinergic cell death after administration of the immunotoxin 192IgG-saporin in rat

192IgG-saporin represents a novel cholinergic immunotoxin which selectively and specifically destroys cholinergic cells in rat basal forebrain. Activated microglial cells are known to play an important role in phagocytosis in regions of neuronal loss. To study the immunotoxin-induced phagocytic events in the basal forebrain activated microglial cells were visualized by lectin cytochemistry using Griffonia simplicifolia agglutinin and analyzed by electron microscopy. Three and 7 days following an intracerebro-ventricular injection of 4 μg 192IgG-saporin, increased numbers of activated microglial cells were observed at both survival times, but the number was strikingly increased at day 7 postlesion. Three days after immunotoxin application microglial cells displayed features similar to those of resting microglia. Only translucent vacuole-like hollows were found intracellularly beneath the plasma membrane of microglial cells and in the adjoining extracellular space. Most neurons in the vicinity of microglial cells did not show any signs of degeneration. However, 7 days after injection of the immunotoxin microglial cells revealed different stages of phagocytosis. The majority of microglial cells were localized in perineuronal positions attached by processes to large areas of neuronal soma or dendrites, which in general showed signs of severe degeneration. The present study provides electron microscopic evidence for phagocytic microglial reactions in the rat basal forebrain after cholinergic lesion by 192IgG-saporin. J. Neurosci. Res. 48:465–476, 1997. © 1997 Wiley-Liss, Inc.