5-aminolevulinic acid induced protoporphyrin IX fluorescence in high-grade glioma surgery: a one-year experience at a single institutuion.

OBJECTIVE: Among the factors determining prognosis in patients with malignant glioma, the extent of resection has long been controversial. However, recent data have shown that patients derive a survival benefit from extensive tumour resection. 5-aminolaevulinic acid (5-ALA)-induced fluorescence renders more complete resection possible in malignant glioma. We report on the feasibility of the method in daily clinical practice, the benefits for patients and surgeons, the technical limitations and the methods we have devised of overcoming these limitations. METHODS: We describe our initial experience in 74 cases undergoing gross total resection, partial resection and biopsy. Fluorescence intensity and histological data are analysed, specificity and sensitivity are calculated according to fluorescence intensity, and the pitfalls and limitations are defined. The fluorescence signal was quantified via digital video data and by single photon count. RESULTS: Solid fluorescence signals define tumours with a sensitivity of 0.98 and a specificity of 1.0. Vague fluorescence reduces sensitivity to 0.76 and specificity to 0.85. Limitations of 5-ALAassisted surgery are apparent within the inter-observer interpretation of solid or vague fluorescence, heterogeneity of gliomas, invasion beyond the resection cavity and intercell heterogeneity of porphyrin IX fluorescence. CONCLUSION: 5-ALA-induced PIX fluorescence improves the results in high-grade glioma surgery for gross total resection. Specificity and sensitivity in regions of solid fluorescence are very high. Quantitative analysis of fluorescence intensity corrects the reduced reliability of the method in areas of vague fluorescence and renders gross total resection more feasible without additional risk to the patient. PIX fluorescence is easy to implement in daily neurosurgical practice and side effects are very few. Heterogeneous tumours with lower grade elements and satellite lesions cannot be reliably resected using fluorescence-assisted surgery alone. In these cases the additional use of intra-operatively updated imaging-based neuronavigational methods (MR, ultrasound) is needed.

Effects of transforming growth factor-beta1 and basic fibroblast growth factor on proliferation of cell cultures derived from human vestibular nerve schwannoma.

The influence of transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) on growth of cell cultures derived from unilateral vestibular nerve schwannomas was investigated. Cell cultures were initiated from 9 schwannomas and characterized immunocytochemically with antibodies against S-100 and type IV collagen. The effects of TGF-beta1 and bFGF on DNA synthesis in chemically defined serum-free medium were assessed by measuring the incorporation of 5-bromo-2'-deoxy-uridine (BRDU) into cellular DNA. Cell proliferation was evaluated with an electronic cell counter. Reverse transcription polymerase chain reaction (RT-PCR) was performed using oligonucleotide primers specific for TGF-beta1 and TGF-beta2. TGF-beta1 stimulated DNA synthesis in a dose dependent manner. Maximal stimulation was observed at a concentration of 1 ng/ml, which induced a nearly 2-fold increase in DNA content. This effect was not seen when TGF-beta1 was added in the presence of neutralizing antibodies. In addition, antibodies against TGF-beta1 significantly reduced DNA synthesis in control cultures without supplemented exogenous growth factors. bFGF alone had no significant effects on DNA synthesis. In contrast, when TGF-beta1 and bFGF were added together, the mitogenic response was much greater than produced by TGF-beta1 alone. RT-PCR showed that the cultured cells expressed mRNA for both TGF-beta1 and TGF-beta2. We hypothesize that TGF-beta1 is an autocrine growth factor for human vestibular nerve schwannomas in culture. A similar mechanism might be involved in the growth of these tumors in situ.

Dexamethasone treatment decreases hyaluronan-formation by primate trabecular meshwork cells in vitro.

The functional significance of Hyaluronan (HA) present in the cribriform layer of Schlemm's canal is not known. It may contribute to the actual outflow resistance but the relatively inert molecule might also be necessary to prevent adherence of larger molecules to the cribriform network. Thus HA might rather prevent an increase in outflow resistance. It is well known that treatment with Dexamethasone (DM) in a number of patients leads to an increase in intraocular pressure presumably due to an increase in outflow resistance. To clarify whether an imbalance in HA formation might be involved in these changes we have treated confluent cultures of human trabecular cells as well as control cell lines (ciliary muscle cells, scleral fibroblasts) with 500 nM DM for 24 hr or 12 days and have measured HA-synthesis using incorporation assays with 0.05 m D-[6-3H] Glucosamine hydrochloride. In all six cell lines investigated there was a significant decrease in HA-synthesis following short and long term treatment with DM when compared with the untreated controls. This reaction of trabecular cells to DM treatment is different from the DM effect reported on the synthesis of many other components of the extracellular matrix like fibronectin and elastin which increase after DM treatment. If the DM-effect seen in cell cultures plays a role in vivo decreased formation in HA could result in impaired function of the outflow pathways.

alpha B-crystallin in the primate ciliary muscle and trabecular meshwork.

The presence of alpha B-crystallin, a protein with heat-shock protein-like properties, has been demonstrated in ciliary muscle and trabecular meshwork derived from human and monkey eyes using immunohistochemical and polymerase chain reaction methods. Both frozen sections and cultured cells have been analyzed. In the ciliary muscle, alpha B-crystallin staining is localized in the region of the dense bands, in the cytoplasm of the muscle cells and in the Schwann cells of the nerves supplying the muscle. In the trabecular meshwork, two cell types could be distinguished on the basis of alpha B-crystallin occurrence. Whereas the trabecular cells covering the lamellae were virtually devoid of the protein, the subendothelial or cribriform cells contained relatively large amounts in parallel with a higher alpha B-crystallin mRNA level.

Transforming growth factor-beta 1 induces alpha-smooth muscle-actin expression in cultured human and monkey trabecular meshwork.

Transforming growth factor-beta 1 (TGF-beta 1) induces alpha-smooth muscle (sm)-actin expression and a contractile myofibroblast-like phenotype in a considerable number of different cell types. Since alpha-sm-actin is expressed in some of the resident human trabecular meshwork (TM) cells in situ, and TGF-beta 1 is synthesized by TM in vitro, alpha-sm-actin expression in TM might also be under the influence of TGF-beta 1. To assess this question, TM cultures were initiated from the eyes of three human donors and three cynomolgus monkeys. Various doses of TGF-beta 1 (0.5-5 ng ml-1) were added to confluent cultures from third to fourth passages. Experiments were performed in the presence of fetal calf serum or under chemically defined serum-free conditions. Four days after treatment, cells were stained immunocytochemically for alpha-sm-actin, and the number of positively labelled cells was quantitatively evaluated. In addition, reverse transcription polymerase chain reaction (PCR) was performed using oligonucleotide primers specific for alpha-sm-actin. In control cultures supplemented with serum, 19.0 +/- 9.4% of human meshwork cells, and 10.2 +/- 4.5% of monkey meshwork cells expressed immunoreactivity for alpha-sm-actin. In human cultures, this number was significantly higher in serum-free conditions (34.1 +/- 7.7%). Treatment with TGF-beta 1 induced alpha-sm-actin expression in a dose-dependent manner. At 5 ng ml-1 TGF-beta 1, 75.5 +/- 7.1% of human meshwork cells expressed distinct stress fibers that stained positively for alpha-sm-actin (P < or = 0.01). A similar albeit smaller increase in alpha-sm-actin positive cells was observed in monkey cultures following treatment with TGF-beta 1. These effects were seen with and without serum, but not when TGF-beta 1 was supplemented in the presence of neutralizing antibodies. In PCR experiments, a distinct product was amplified with cDNA derived from cells treated with 0.1 ng and 1 ng ml-1 TGF-beta 1, but not in control cultures. We conclude that TGF-beta 1 may play a role in differentiating TM cells towards a myofibroblast-like cell type by modulating the expression of alpha-sm-actin.

The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum. Its gene structure relative to that of other polyketide synthases.

6-Methylsalicylic acid synthase (MSAS) from Penicillium patulum is a homomultimer of a single, multifunctional protein subunit. The enzyme is induced, at the transcriptional level, during the end of the logarithmic growth phase. After approximately 150-fold purification, a homogeneous enzyme preparation was obtained exhibiting, upon SDS gel electrophoresis, a subunit molecular mass of 188 kDa. By immunological screening of a genomic P. patulum DNA expression library, the MSAS gene together with its flanking sequences was isolated; 7131 base pairs of the cloned genomic DNA were sequenced. Within this sequence the MSAS gene was identified as a 5322-bp-long open reading frame coding for a protein of 1774 amino acids and 190,731 Da molecular mass. Transcriptional initiation and termination sites were determined both by primer extension studies and from cDNA sequences specially prepared for the 5' and 3' portions of the gene. The same cDNA sequences revealed the presence of a 69-bp intron within the N-terminal part of the MSAS gene. The intron contains the canonical GT and AG dinucleotides at its 5'- and 3'-splice junctions. An internal TACTGAC sequence, resembling the TACTAAC consensus element of Saccharomyces cerevisiae introns is suggested to represent the branch point of the lariat splicing intermediate. When compared to other known polyketide synthases, distinct amino acid sequence similarities of limited lengths were observed with some, though not all, of them. A comparatively low degree of similarity was detected to the yeast and Penicillium FAS or to the plant chalcone and resveratrol synthases. In contrast, a significantly higher sequence similarity was found between MSAS and the rat fatty acid synthase, especially at their transacylase, 2-oxoacyl reductase, 2-oxoacyl synthase and acyl carrier protein domains. Besides several dissimilar, interspersed regions probably coding for MSAS- and FAS-specific functions, the sequential order of the similar domains was colinear in both enzymes. The low similarity between the two P. patulum polyketide synthases, MSAS and FAS, possibly supports a convergent rather than a divergent evolution of both multienzyme proteins.