Antigenic staining patterns of human glioma cultures: primary cultures, long-term cultures and cell lines

Summary The immunocytochemical staining patterns of cultured glioma cells were investigated. Fifty nine individual cases were stained at differentin vitro ages for glial fibrillary acidic protein, fibronectin, galactocerebroside, HNK-1/Leu 7, A2B5, vimentin, factor VIII and A4. Histologically, the cases were composed of eight low-grade astrocytomas, 11 high-grade astrocytomas, four low-grade oligodendrogliomas, seven high-grade oligodendrogliomas and 29 glioblastomas. The 45 cases were analysed within the first 3 weeks of culture, many of them as primary cultures. In 11 cases stainings were performed repeatedly at intervals of up to 6 months.Glial fibrillary acidic protein staining was positive in most of the early cultures of astrocytomas (low and high grade) and glioblastomas; expression in more than 50% of the cells was found in 1 of 5 low-grade astrocytomas, 5 of 11 high-grade astrocytomas and 14 of 29 glioblastomas. Two of the high-grade astrocytomas were stained once more after 6 weeks in culture and were found to be only 1% positive for glial fibrillary acidic protein but strongly positive for fibronectin. The same was true for five of the glioblastoma cases. Two of these cases remained glial fibrillary acid protein positive and developed into stable permanent cell lines. Only one case started with 1% of glial fibrillary acidic protein positive cells and later developed into a 99% glial fibrillary acidic protein positive cell line. Neither HNK-1/Leu 7 expression nor A2B5 staining appeared to have a relationship to the glial fibrillary acidic protein staining. It was observed that glial fibrillary acidic protein and HNK-1/Leu 7 were both 100% in some cases but that later one of the two antigens disappeared but not the other. The amount of glial fibrillary acidic protein staining does not allow the prediction of A2B5 staining.The study shows that initiation of primary cultures on an extracellular matrix yields more glial fibrillary acidic protein positive cells in primary cultures than have been found in other studies. It is concluded that only a rigid standardization of culture conditions will ensure the validity of comparisons ofin vitro data obtained in primary cultures.     

Comparison of two urinary antigen tests for establishment of pneumococcal etiology of adult community-acquired pneumonia

The Binax NOW immunochromatographic test (ICT) detecting the pneumococcal C polysaccharide and a serotype-specific latex agglutination (LA) test detecting 23 pneumococcal capsular antigens were evaluated for establishing pneumococcal etiology in community-acquired pneumonia (CAP) by use of nonconcentrated urine. ICT was considered to be strongly positive for result lines at least as intense as the control line and weakly positive for less intense result lines. When 215 adult CAP patients were tested, strong ICT, weak ICT, and LA positivity were found in 28, 24, and 16 patients, respectively; of these patients, 13 (46%), 6 (25%), and 13 (81%), respectively, had pneumococcal bacteremia and 27 (96%), 17 (71%), and 15 (94%), respectively, had Streptococcus pneumoniae isolated from blood, sputum, and/or nasopharynx. Among 108 controls tested, 2 (1.9%) were weakly ICT positive. When weak positivity was considered negative, the sensitivity of ICT decreased from 79% (19 of 24) to 54% (13 of 24), while the specificity increased from 83% (158 of 191) to 92% (176 of 191); no controls were false positive. The sensitivity and specificity of LA were 54% (13 of 24) and 98% (188 of 191), respectively. Eight of nine LA serotypes corresponded to culture serotypes. In conclusion, using nonconcentrated urine and dividing ICT-positive results into strongly and weakly positive results is a suitable way of performing ICT. While weak ICT positivity should be interpreted with caution, strong ICT positivity and LA positivity should be considered supportive of pneumococcal etiology in adult CAP. As such, these assays might have implications for antibiotic use in CAP. LA has promising potential for pneumococcal serotyping, although further evaluation is required.     

Allelic losses at 1p, 9q, 10q, 14q, and 22q in the progression of aggressive meningiomas and undifferentiated meningeal sarcomas

Meningiomas are usually benign tumors; however, they can recur after surgical resection and occasionally show histological progression to a higher malignancy grade. Five such rare cases of aggressively recurring meningiomas were present in our departmental cohort of 923 primary meningeal neoplasms operated over a 17-year period. Four other aggressively recurring meningeal tumors with a very similar clinical and histomorphological appearance (three undifferentiated meningeal sarcomas, one hemangiopericytoma) were also included in this study. We investigated whether disease progression can be traced by genetic alterations and whether a pattern of genetic alterations is specific for meningiomas. A total of 40 specimens from primary tumors and multiple recurrences of the nine patients were analyzed with 26 polymorphic allelic markers for deletions on 1p, 1q, 9q, 10q, 14q, and 22q. Loss of heterozygosity (LOH) at 22q was observed in all meningiomas cases at the earliest time point analyzed. Allelic loss at 1p was seen in the original tumor in two cases and upon meningioma recurrence in two others. Deletion on 10q occurred during tumor progression in two cases, and on 9q and 14q in one case. While allelic loss at 22q appears to be an early event in aggressive meningioma disease, there is a clear correlation of further deletions on chromosome arms 1p, 9q, 10q, and 14q with histopathological and clinical progression, as shown in these intraindividual trackings. None of these genetic findings were present in the non-meningiomatous meningeal tumors, indicating that meningothelial cells have their own lineage-specific genetic pathways towards clinical malignancy.     

Expression of platelet-derived growth factor-AA is associated with tumor progression in osteosarcoma.

Platelet-derived growth factors are secreted by mesenchymal cells. The homodimer platelet-derived growth factor-AA especially stimulates bone cells through interaction with the platelet-derived growth factor-alpha receptor homodimer. In this study we wanted to determine the expression of the receptor and its ligand in human osteosarcomas and to correlate the expression of platelet-derived growth factor-AA and -alpha receptor with clinicopathological parameters. Fifty-seven osteosarcomas were immunohistochemically analyzed for expression of platelet-derived growth factor-AA and platelet-derived growth factor-alpha receptor. Spearman's correlation coefficient revealed a strong correlation between the expression of platelet-derived growth factor-AA and platelet-derived growth factor-alpha receptor (r = 0.867). No differences were observed relative to gender, age, tumor stage, tumor location, and response to neoadjuvant chemotherapy between high or low platelet-derived growth factor-AA and platelet-derived growth factor-alpha receptor expression. High platelet-derived growth factor-AA expression correlated with tumor progression in univariate analysis (P = .0415; log-rank test), whereas platelet-derived growth factor-alpha receptor expression showed a trend toward a shorter disease-free survival, which failed to reach significance (P = .0627, log-rank test). In multivariate analysis, platelet-derived growth factor-AA expression remained a significant independent predictor of tumor progression (P = .021, Cox regression). Immunohistochemical analysis of platelet-derived growth factor-AA expression in osteosarcoma may be a useful marker of prognosis and may be considered as a possible target for novel therapeutic strategies.     

Identification of a frequent variant in ALG6, the cause of Congenital Disorder of Glycosylation-Ic

Congenital Disorder of Glycosylation (CDG) type Ic is caused by mutations in ALG6. This gene encodes an alpha1,3 glucosyltransferase used for synthesis of the lipid linked oligosaccharide (LLO) precursor of the protein N-glycosylation pathway. CDG-Ic patients have moderate to severe psychomotor retardation, seizures, hypotonia, strabismus, and feeding difficulties. We previously identified a typical patient with a heterozygous point mutation, c.391T>C (p.Tyr131His) in ALG6. Using complementation analysis of ALG6-deficient yeast, we show that this alteration is as severe as the most common disease-causing mutation, c998C>T (p. Ala333Val), which occurs in over half of all known CDG-Ic patients. The frequency of c.391T>C (p.Tyr131His) in the US population, is 0.0214, suggesting that homozygotes would occur at a rate of& tilde;1:2,200. We identified one patient with typical CDG-Ic symptoms and a homozygous p.Tyr131His alteration in ALG6. However, in contrast to most CDG patients, her LLO and plasma transferrin glycosylation appeared normal. Thus, it is unclear whether c.391T>C causes CDG-Ic or contributes to the symptoms. Genotyping additional patients with CDG-like symptoms will be required to resolve this issue.     

Relations between chronic stimulation-induced changes in contractile properties and the Ca2+-sequestering system of rat and rabbit fast-twitch muscles

This study compares changes in contractile properties, Parvalbumin content, and Ca²⁺-uptake by the sarcoplasmic reticulum (SR) of low-frequency stimulated rat and rabbit tibialis anterior (TA) muscles. Time to peak tension increased 1.8-fold in 35-day stimulated rabbit TA, while no change occurred in rat TA. Isometric twitch tension increased 2-fold in rabbit TA, but was unaltered in rat TA. Parvalbumin (PA) content was more than 90% reduced in rabbit TA, but only 60% in rat TA after 35 days. At this time, PA content of the stimulated rat TA was still higher than that of normal rabbit TA. Taking into account the suggested role of PA as a cytosolic Ca²⁺ buffer, its decrease could lead to an impaired free Ca²⁺-decay with a prolonged active state and a higher tension output during a single twitch. This would explain why chronic stimulation led to an increase in isometric twitch tension in rabbit TA, but not in rat TA. The 1.6-fold rise in half-relaxation time of 35-day stimulated rat and rabbit TA most likely resulted from a 50% reduced Ca²⁺-uptake by the SR, due to a still unknown modification of the Ca²⁺-transport ATPase.     

Use of 1-(m-aminophenyl)flavazoles for the preparation of immunogens with oligosaccharide determinant groups

A method is described for the preparation of immunogens with oligosaccharide determinant groups. The method, which consists of conversion of the sugar into its 1-(maminophenyl)flavazole and subsequent azo-coupling to protein, has been elaborated with various oligosaccharides of the isomaltose-, maltose-, and cellobiose-series. It requires unsubstituted hydroxyl groups on positions 2 and 3 adjacent to the reducing end of the sugar and appears to be especially suited for oligosaccharides having a polymerization degree between 3 and 8. The procedure does not necessitate protection of the sugar hydroxyl groups. Oligosaccharide-flavazole-azo-edestin conjugates have been tested for immunogenicity in rabbits. Specific anti-oligosaccharide antibodies were formed in all instances. High titres of dextranspecific antibodies were obtained upon immunization with an isomaltoheptaose-flavazole-azo-edestin conjugate. Further applications of the method will be discussed.     

Fatty acid synthesis in the arrested rabbit heart during ischemia

In rabbit hearts arrested by a carnosine-buffered cardioplegic solution the incorporation of [1-¹⁴C]acetate into lipids was investigated. After 10–20 and 60 min of ischemia the radioactivities of phospholipids, mono-, di-, and triacylglycerols, acyl-CoA, acylcarnitine, and free fatty acids were determined. In the first period of ischemia mainly acylcarnitine was labelled (ca. 50%) and only 25% of [¹⁴C]-activity was found in phospholipids which showed the lowest specific activity of all lipid classes. After 60 min of ischemia the percentage of total radioactivity of acylcarnitine and phospholipids was decreased, whereas that of neutral lipids was increased to more than 50%. During the increase of total radioactivity the relative specific activity of all lipids decreased except that of triacylglycerols. Only fatty acids up to chain lengths of 16 carbon atoms were labelled. Lauric and myristic acid had high specific activities. These results indicated de novo synthesis of fatty acids accumulating in triacylglycerols during ischemia of the arrested heart.     

Glial cell line-derived neurotrophic factor receptor alpha1 availability regulates glial cell line-derived neurotrophic factor signaling: evidence from mice carrying one or two mutated alleles

Glial cell line-derived neurotrophic factor receptor alpha1 (GFRalpha1, also known as GDNFR-alpha) is a glycolipid-anchored membrane protein of the GFRalpha family, which binds glial cell line-derived neurotrophic factor [Jing S. et al. (1996) Cell 85, 1113-1124; Treanor J. J. et al. (1996) Nature 382, 80-83], a survival factor for several populations of central and peripheral neurons, including midbrain dopamine neurons [Lin L. F. et al. (1993) Science 260, 1130-1132], and mediates its ligand-induced cell response via a tyrosine kinase receptor called Ret [Takahashi M. et al. (1988) Oncogene 3, 571-578; Takahashi M. and Cooper G. M. (1987) Molec. Cell Biol. 7, 1378-1385]. In this paper, we show that mice with a null mutation of the GFRalpha1 gene manifest epithelial-mesenchymal interaction deficits in kidney and severe disturbances of intestinal tract development similar to those seen with glial cell line-derived neurotrophic factor or Ret null mutations. There is a marked renal dysgenesis or agenesis and the intrinsic enteric nervous system fails completely to develop. We also show that newborn GFRalpha1-deficient mice display no or minimal changes in dorsal root and sympathetic ganglia. This is in contrast to the deficits reported in these neuronal populations in glial cell line-derived neurotrophic factor and Ret null mutations. Mesencephalic dopaminergic neurons in the substantia nigra and ventral tegmental area appear intact at the time of birth of the mutated mice. Mice homozygous for the GFRalpha1 null mutation die within 24 h of birth because of uremia. Heterozygous animals, however, live to adulthood. There is a significantly reduced neuroprotective effect of glial cell line-derived neurotrophic factor in such heterozygous animals, compared with wild-type littermates, after cerebral ischemia. Taken together with previous data on glial cell line-derived neurotrophic factor and Ret, our results strongly suggest that GFRalpha1 is the essential GFRalpha receptor for signaling in the glial cell line-derived neurotrophic factor-Ret pathway in the kidney and enteric nervous system development, and that GFRalpha2 or GFRalpha3 cannot substitute for the absence of GFRalpha1. Moreover, neuroprotective actions of exogenous glial cell line-derived neurotrophic factor also require full GFRalpha1 receptor expression.