First Report of Powdery Mildew of Platanus × acerifolia and P. occidentalis Caused by Erysiphe platani in Greece.

London planetrees (Platanus × acerifolia, syn. P. × hispanica), American sycamores (P. occidentalis), and oriental planes (P. orientalis) are widely planted as urban shade trees throughout Greece and many other countries. In June 2012, typical symptoms of a powdery mildew were detected on all sycamores (10 trees) along a central avenue of Heraklion (Crete, Greece), with the disease affecting approximately 80% of the leaves of all infected trees. In August 2013, similar symptoms were observed on 20% of the leaves of all three London planes in a small grove in the Vrysses area of Lasithi (Crete, Greece). In both cases, the disease was severe, with white superficial colonies developing amphigenously on leaves, twigs, floral peduncles, inflorescences, and fruits. The colonies were initially distinct and circular but gradually enlarged and often coalesced to cover the entire leaf blade. Young leaves appeared curled and chlorotic, occasionally leading to defoliation. For the morphological description of the pathogen, samples from seven infected P. occidentalis and three P. × acerifolia trees were microscopically characterized. In all samples, the pathogen's mycelium was branched, septate, and hyaline, with lobed appressoria; conidiophores were erect, cylindrical, unbranched, and consisted of three to four (to five) cells; and conidia were single or in short chains (two to four), ellipsoid or doliiform, with a truncated base and rounded apex. Their dimensions were 24.3 to 48.6 × 15.8 to 27.9 µm (averaging 39.2 × 21.2 µm; n = 100), and their surfaces appeared reticulate. The teleomorph was never observed. Total fungal DNA was extracted from conidia harvested from affected leaves of one infected plant of each of P. occidentalis and P. × acerifolia planes, and the ITS1-5.8S-ITS2 region was PCR-amplified with universal primers 18S-ITS1 and 28S-ITS2 (2) and sequenced (GenBank Accession Nos. KM068123 and KM068124, respectively). A BLASTn search of GenBank revealed 100% identity of both samples to Erysiphe platani strains described on P. orientalis in Greece (JQ365943) and P. occidentalis in Brazil (KF499270). Based on the morphological and molecular analyses, the pathogen was identified as E. platani (Howe) U. Braun & S. Takam. (formerly known as Microsphaera platani Howe) (1). To prove pathogenicity and fulfill Koch's postulates, 10 1-year-old seedlings of each of P. occidentalis and P. × acerifolia hosts were artificially inoculated with conidia obtained from naturally infected plants of the corresponding species, with two methods: (i) five plants of each host were dusted with conidia from diseased leaves, and (ii) the remaining five seedlings of each plane were sprayed with a conidial suspension of the fungus (107 conidia ml-1), while five additional control plants of each species were treated only with sterile distilled water. All plants were maintained in the greenhouse at 25 ± 3°C, with 90% humidity. Powdery mildew symptoms, which appeared 9 and 15 days after inoculation on all dusted and sprayed plants, respectively, were similar to those observed on naturally infected trees, whereas no symptoms were observed on control plants. Although E. platani is known to infect plane species in several parts of the world (1), including oriental planes (P. orientalis and P. orientalis var. cretica) in Greece (3), this is the first report of E. platani causing disease of P. occidentalis and P. × acerifolia in Greece, underlining the need for appropriate control measures to prevent significant losses to the local ornamental industry. References: (1) U. Braun and R. T. A. Cook. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11. CBS, Utrecht, 2012. (2) I. A. Papaioannou et al. Eur. J. Plant Pathol. 136:577, 2013. (3) D. J. Vakalounakis and E. Klironomou. EPPO Bull. 25:463, 1995.

First Report of Verticillium Wilt Caused by Verticillium dahliae on Avocado Trees in Greece.

Avocado (Persea americana) is an important crop for Chania, Crete, Greece, and is grown on more than 800 ha. In November 2013, 4-year-old trees in a new avocado grove of cv. Hass grafted onto the rootstock 'Bacon,' previously planted in citrus trees, showed symptoms of yellowing, leaf fall, twig and branch dieback and vascular tissue discoloration. Disease incidence was estimated at 2.3% (12 out of 530 trees affected). A fungus was consistently and readily isolated from symptomatic vascular tissue, previously surface-disinfested with 95% ethanol, on acidified potato dextrose agar (APDA). After 7 days, slow-growing colonies were transferred to PDA and the growth rate of the fungus was 2.9 mm/day at 24°C in the dark. Microscopic observations revealed hyaline hyphae with many irregular, dark microsclerotia measuring 40 to 200 × 30 to 75 µm (average 94.5 × 50.3 µm) developing after 21 days of growth. Hyaline, elliptical, single-celled conidia measuring 2.8 to 7.5 × 2.5 to 4.3 µm (average 4.8 × 3.1 µm) developed on verticillate conidiophores. For molecular characterization, Verticillium dahliae specific primer pair ITS1-F/ITS2-R that amplifies the rRNA internal transcribed spacer (ITS) region was used (2). Band of expected size was amplified, sequenced, and deposited in GenBank (Accession No. KJ818294). On the basis of morphological characteristics (3) and a BLAST search with 100% identity to the published ITS sequence of a V. dahliae isolate in GenBank (KC834733.1), the fungus was identified as V. dahliae. Five 1-year-old avocado plants of cv. Hass, grafted onto the rootstock 'Bacon,' were used for pathogenicity tests. Artificial inoculation was performed by making a 5.0 × 3.5 mm hole in the rootstock trunk, injecting approximately 40 µl of a 2.8 × 107 conidia/ml suspension into the vessels (spores were introduced passively), sealing with Vaseline, and covering with adhesive paper tape. Five control plants were mock inoculated with sterilized distilled water. Disease symptoms that appeared 18 days post artificial inoculation were similar to those observed under natural infection conditions. Thirty-five days post artificial inoculation, disease incidence was 80%, whereas the percentage of positive V. dahliae re-isolations from infected tissues was 95% (96.7 and 93.3% from rootstock and graft, respectively). The extent of vascular tissue discoloration from the point of inoculation ranged from 11 to 62 cm, whereas V. dahliae was successfully re-isolated even from the end of the graft (approximately 60 cm above the initial inoculation point), thus confirming Koch's postulates. Neither symptoms nor positive isolations were observed in control plants. The pathogenicity test was repeated twice with similar results. Verticillium wilt of avocado has been observed in several countries including Argentina, Chile, Ecuador, Israel, Mexico, Morocco, Spain, and the United States (1). To the best of our knowledge, this is the first report of Verticillium wilt on avocado in Greece. This disease could potentially be an increasing problem in areas where young avocado trees are established on land previously planted in vegetable crops. References: (1) J. C. Goud and J. A. Hiemstra. Chapter 3 in: A Compendium of Verticillium Wilt in Trees Species, 1998. (2) E. A. Markakis et al. Eur. J. Plant Pathol. 124:603, 2009. (3) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, Wallingford, UK, 2002.

First Report of Stemphylium solani as the Causal Agent of a Leaf Spot on Greenhouse Cucumber.

During the 2011 to 2012 crop season, a severe leaf spot disease of cucumber (Cucumis sativus) cv. Cadiz was noticed on crops in some greenhouses in the Goudouras area, Lasithi, Crete, Greece. Symptoms appeared in late winter, mainly on the leaves of the middle and upper part of the plants. Initially, small necrotic pinpoint lesions with white centers, surrounded by chlorotic halos, 1 to 3 mm in diameter, appeared on the upper leaf surfaces, and these progressively enlarged to spots that could coalesce to form nearly circular lesions up to 2 cm or more in diameter. Stemphylium-like fructifications appeared on necrotic tissue of older lesions. Severely affected leaves became chlorotic and died. No other part of the plant was affected. Small tissue pieces from the edges of lesions were surface disinfected in 0.5% NaClO for 5 min, rinsed in sterile distilled water, plated on acidified potato dextrose agar and incubated at 22 ± 0.5°C with a 12-h photoperiod. Stemphylium sp. was consistently isolated from diseased samples. Colonies showed a typical septate mycelium with the young hyphae subhyaline and gradually became greyish green to dark brown with age. Conidiophores were subhyaline to light brown, 3- to 10-septate, up to 200 µm in length, and 4 to 7 µm in width, with apical cell slightly to distinctly swollen, bearing a single spore at the apex. Conidia were muriform, mostly oblong to ovoid, but occasionally nearly globose, subhyline to variant shades of brown, mostly constricted at the median septum, 22.6 ± 6.22 (11.9 to 36.9) µm in length, and 15.1 ± 2.85 (8.3 to 22.6) µm in width, with 1 to 8 transverse and 0 to 5 longitudinal septa. DNA from a representative single-spore isolate was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) was amplified using the universal primers ITS5 and ITS4. The PCR product was sequenced and deposited in GenBank (Accession No. JX481911). On the basis of morphological characteristics (3) and a BLAST search with 100% identity to the published ITS sequence of a S. solani isolate in GenBank (EF0767501), the fungus was identified as S. solani. Pathogenicity tests were performed by spraying a conidial suspension (105 conidia ml-1) on healthy cucumber (cv. Knossos), melon (C. melo, cv. Galia), watermelon (Citrullus lanatus cv. Crimson sweet), pumpkin (Cucurbita pepo, cv. Rigas), and sponge gourd (Luffa aegyptiaca, local variety) plants, at the 5-true-leaf stage. Disease symptoms appeared on cucumber and melon only, which were similar to those observed under natural infection conditions on cucumber. S. solani was consistently reisolated from artificially infected cucumber and melon tissues, thus confirming Koch's postulates. The pathogenicity test was repeated with similar results. In 1918, a report of a Stemphylium leaf spot of cucumber in Indiana and Ohio was attributed to Stemphylium cucurbitacearum Osner (4), but that pathogen has since been reclassified as Leandria momordicae Rangel (2). That disease was later reported from Florida (1) and net spot was suggested as a common name for that disease. For the disease reported here, we suggest the name Stemphylium leaf spot. This is the first report of a disease of cucumber caused by a species of Stemphylium. References: (1) C. H. Blazquez. Plant Dis. 67:534, 1983. (2) P. Holliday. Page 243 in: A Dictionary of Plant Pathology. Cambridge University Press, Cambridge, UK, 1998. (3) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (4) G. A. Osner. J. Agric. Res. 13:295, 1918.

First Report of Pink Rot of Phoenix and Washingtonia Species Caused by Nalanthamala vermoesenii in Greece.

A disease resembling pink rot was first observed on Phoenix dactylifera in Heraklion (Crete, Greece) in the summer of 2007, and was later found to be relatively common in the same district on additional species (P. canariensis, P. theophrasti, Washingtonia filifera, W. robusta). Symptoms included chlorotic and necrotic leaves (dead tips), light-brown spots (1 to 2 mm in diameter) on the leaves and rachis, rot of the rachis, sheath, and trunk, and eventual death of infected plants. A pinkish-orange layer formed both on the surface and within the infected tissues. A hyphomycete was isolated from symptomatic petioles and the pinkish-orange layer of the sheath. Sixteen isolates were examined on potato dextrose agar (PDA). All formed salmon to grayish-red colonies with sparse aerial mycelium, hyaline conidiophores with penicillate branches and terminal phialides, and ovoid, single-celled conidia in long chains. Mean conidial dimensions were 3.5 (± 0.1) × 5.5 (± 0.1) µm (n = 60 each), for 1-week-old cultures of two single-spore isolates recovered from W. filifera. A BLASTn search of GenBank with sequences of rDNA ITS (JX456472 to JX456474) revealed 100% identity of three isolates to that of Nalanthamala vermoesenii (Biourge) Schroers, comb. nov. [syn. Penicillium vermoesenii Biourge; Gliocladium vermoesenii (Biourge) Thom] originating from several palm species in Spain, the Czech Republic, Australia, and the United States (GenBank AY554212 to AY554217). Therefore, our examination of morphological and molecular characteristics suggested that the fungus recovered from symptomatic trees was N. vermoesenii (3,4). Pathogenicity tests were performed on wounds (shallow cuts 0.5 to 1.0 cm wide, made parallel to the surface with a sterile scalpel) of petioles of mature leaves of eight 2-year-old seedlings each of P. canariensis, P. theophrasti, and W. filifera. A 6-mm agar plug from a 1-week-old PDA culture was placed on the artificial wound of each inoculated plant. For non-inoculated controls, sterile PDA plugs were placed on the artificial wounds of four seedlings per host. All plants were maintained in the greenhouse at 16 ± 5°C, with 95% humidity and a 12-h photoperiod. Petiole and stem rot, leaf necrosis, and production of pinkish-orange spore masses appeared at 5 weeks post-inoculation. Average lesion length was 2.75 ± 0.15, 3.28 ± 0.21, and 6.14 ± 0.53 cm for P. canariensis, P. theophrasti, and W. filifera, respectively, suggesting that the latter is more susceptible. The fungus was consistently reisolated from all three inoculated palm species, whereas no symptoms appeared on control plants. To our knowledge, this is the first report of N. vermoesenii infecting palms in Greece. The invasion of the plants by the fungus is probably favored by wounds, such as those caused by pruning or by feeding of the red palm wheevil Rhynchophorus ferrugineus Olivier, which is widespread in Greece (1). References: (1) D. C. Kontodimas et al. Entomol. Hellenica 16:11, 2006. (2) M. P. Pantou et al. Mycol. Res. 109:889, 2005. (3) H.-J. Schroers et al. Mycologia 97:375, 2005. (4) J. Y. Uchida. Page 25 in: Compendium of Ornamental Palm Diseases and Disorders, APS Press, St. Paul, MN, USA, 2004.

First Report of Leaf Spot of Phoenix theophrasti Caused by Paraconiothyrium variabile in Greece.

In the spring of 2011, a severe leaf spot disease of Phoenix theophrasti was observed in the vicinity of Heraklion (Crete), Greece. Initial symptoms were small, round-ovoid spots of varying shades of brown on the leaves, later being transformed into oblong streaks (average dimensions 7.3 ± 1.0 × 3.3 ± 0.5 mm), surrounded by dark brown rings. As the disease progressed, the expanding streaks often coalesced to form enlarged necrotic lesions. Similar symptoms were also detected on petioles and leaf bases. Extended spotting and blighting occasionally resulted in leaf death. A filamentous fungus was consistently isolated onto potato dextrose agar plates from the periphery of the characteristic lesions, with cultures invariably producing brick to cinnamon colonies with sparse aerial mycelium, subglobose and dark brown superficial pycnidial conidiomata on pine needles, 1- to 3-celled hyaline conidiophores, and hyaline, subcylindrical to ellipsoidal, 1-celled, smooth- and thin-walled conidia, with average dimensions of 3.5 ± 0.6 × 1.7 ± 0.4 µm (n = 100). Total DNA of two isolates was extracted and used for PCR amplification and sequencing of the ITS1-5.8S-ITS2 region, together with parts of the flanking 18S and 28S rRNA genes (4). Both sequences (GenBank Accession Nos. JX456476 and JX456477) were 100% identical to deposited Paraconiothyrium variabile ITS sequences (EU295640 to 48, JN983440 and 41, and JF934920), and were clustered together as a single group with these sequences with good support by phylogenetic analysis that included representatives of the relative P. brasiliense and P. africanum species. Based on the morphological, molecular, and phylogenetic analyses, the pathogen was identified as P. variabile Riccioni, Damm, Verkley & Crous (2). To prove pathogenicity, 10 P. theophrasti 2-year-old seedlings were sprayed with a conidial suspension of the fungus (107 conidia ml-1, 10 ml per plant), while five additional control plants were treated with sterile distilled water. All plants were maintained in the greenhouse at 15 ± 5°C, with 90% humidity. Characteristic leaf spots were evident 4 weeks post inoculation on the older leaves, and P. variabile was consistently reisolated from all inoculated plants. No symptoms were observed on control plants. Paraconiothyrium variabile has been isolated from various woody host plants such as Prunus persica, P. salicina, and Malus sp. in South Africa (1,2), Actinidia chinensis and A. deliciosa in Italy (2), Laurus nobilis in Turkey (2), and Salix matsudana in China (3). To our knowledge, this is the first report of P. variabile naturally infecting and causing a leaf spot disease on a palm species. Palms are extensively used as ornamentals throughout Greece and the occurrence of P. variabile can potentially result in economic loss to the local ornamental industry. References: (1) M. Cloete et al. Phytopathol. Mediterr. 50:S176, 2011. (2) U. Damm et al. Persoonia 20:9, 2008. (3) H. Gao et al. Afr. J. Biotechnol. 10:4166, 2011. (4) M. P. Pantou et al. Mycol. Res. 109:889, 2005.

First Report of Petiole (Rachis) Blight of Washingtonia filifera Caused by Phoma glomerata in Greece.

In July 2007, a severe petiole (rachis) blight disease was observed on several California fan palms (Washingtonia filifera) in the vicinity of Heraklion (Crete), Greece. Typical symptoms included discolored (brown to reddish-brown), reversed V-shaped lesions on the petiole bases of the oldest (lowest) leaves, and elongated yellow to dark-brown stripes along the petiole. The lesions progressively expanded and penetrated the petioles, resulting in gradual discoloration (from tan to brown-black) of the internal petiole tissues, including the vascular tissue. The bases of infected petioles occasionally became fragile and burst open, while the corresponding leaf blades were characterized initially by yellowing and one-sided or uneven wilt and, later, desiccation and death with the entire leaves curving downwards. The disease gradually moved upward to younger leaves, severely debilitating but rarely killing the infected trees. A filamentous fungus was consistently isolated onto potato dextrose agar (PDA) plates from sections of diseased petioles, forming dense, dark green colonies with abundant light to dark brown, subglobose pycnidia (diameter ranging between 36.4 to 177.4 µm, and averaging 99.4 µm, n = 50) on the agar surface or immersed in the medium. Chlamydospores and numerous dictyochlamydospores were also observed, with the latter being initially light to dark brown and later becoming black. The numerous conidia were hyaline, ovoid to ellipsoid, and single-celled. Their dimensions were 5.3 to 7.3 × 2.4 to 4.9 µm, averaging 6.5 × 3.2 µm (n = 100). The ITS1-5.8S-ITS2 region, together with parts of the flanking 18S and 28S rRNA genes (3), were amplified with PCR from total DNA extracted from two representative isolates, and sequenced (GenBank Accession Nos. KC802086 to KC802087). Using BLASTn, both sequences were 100% identical to Phoma glomerata ITS sequences (FJ427018, FJ427011, AF126816). Based on morphological and molecular analyses, the pathogen was identified as Phoma glomerata (Corda) Wollenw. & Hochapfel, also known as Peyronellaea glomerata (Corda) Goid. ex Togliani or Coniothyrium glomeratum Corda (1,2). To prove pathogenicity and fulfill Koch's postulates, petioles of the older leaves of eight W. filifera 2-year-old seedlings were wounded with a sterile scalpel (shallow cuts 0.5 to 1.0 cm wide, made parallel to the surface), inoculated with agar discs from a 2-week-old PDA culture of the fungus, and sealed with Parafilm. For controls, sterile PDA plugs were placed on the artificial wounds of five more seedlings. All plants were maintained in the greenhouse at 15 ± 5°C, with 90% humidity. Petiole blight and leaf necrosis symptoms-identical to those observed in the infected plants-were evident 5 weeks post-inoculation, and P. glomerata was consistently reisolated from all inoculated plants. No symptoms were observed on control plants. This is the first report of petiole blight of a palm species caused by P. glomerata in Greece. Due to the extensive use of palms as ornamentals in Greece, the occurrence of P. glomerata can potentially cause economic loss to the local ornamental industry. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:1, 2010. (2) R. M. Hosford, Jr. Phytopathology 65:1236, 1975. (3) M. P. Pantou et al. Mycol. Res. 109:889, 2005.

First Report of Palm Rot of Phoenix spp. Caused by Neodeightonia phoenicum in Greece.

In July 2007, a severe rot was observed on Phoenix dactylifera and P. canariensis palms in the vicinity of Heraklion (Crete), Greece. Initial symptoms were pale, elongated spots that gradually turned to dark brown streaks extending along the leaf base and rachis. In early stages, the upper parts of the leaves usually remained unaffected. Eventually decay and premature death of leaves occurred, followed by terminal bud necrosis. Shoot blights and stalk rots were also observed. A filamentous fungus was consistently isolated onto potato dextrose agar (PDA) from leaf base necrotic lesions. Immersed pycnidial conidiomata on pine needles in culture were multiloculate and dark brown to black. Pycnidial paraphyses were absent. Conidiogenous cells were hyaline, cylindrical, and swollen at base. Conidia were thick-walled, ovoid to ellipsoid, with rounded apex and base; initially hyaline and aseptate, 15.2 ± 0.4 × 11.7 ± 0.3 µm, later becoming dark brown and 1-septate, 21.3 ± 0.4 × 11.8 ± 0.3 µm, with a striate appearance. Total DNA was extracted and used for PCR amplification and sequencing of the ITS1-5.8S-ITS2 region, together with parts of the flanking 18S and 28S rRNA genes (1). The sequence (GenBank Accession No. JX456475) was found 99% identical to Neodeightonia phoenicum ITS sequences (GenBank Accession Nos. EU673338 to EU673340), and was clustered together as a single group with the above sequences with good support by phylogenetic analysis that included representatives of other Neodeightonia species and several other Botryosphaeriaceae members. Based on the morphological, molecular, and phylogenetic analyses, the pathogen was identified as N. phoenicum A. J. L. Phillips & Crous (2) (syn. Diplodia phoenicum (Saccardo) H. S. Fawcett & Klotz), formerly also known as Macrophoma phoenicum Saccardo and Strionemadiplodia phoenicum (Saccardo) Zambettakis. To prove pathogenicity, the petioles of the older leaves of seven 2-year-old seedlings of each of three palms, P. canariensis, P. theophrasti, and Washingtonia filifera were wounded with a sterile scalpel (shallow cuts 0.5 to 1.0 cm wide, made parallel to the surface) and inoculated with agar discs from a 1-week-old PDA culture of the fungus. For controls, PDA discs without fungal mycelium were placed on the wounds of four seedlings of each host. Petiole rot, blight, and leaf necrosis were evident on all inoculated plants 6 weeks post inoculation and the pathogen was consistently reisolated from all three inoculated palm species, whereas no symptoms were observed on control plants. N. phoenicum has repeatedly and globally been reported on P. dactylifera (3). To the best of our knowledge, this is the first report of the occurrence of N. phoenicum infecting Phoenix species in Greece. Palms are extensively used as ornamental trees throughout Greece. A potential spread of palm rot caused by N. phoenicum might have a substantial economic impact and should be urgently addressed through appropriate disease management programs. References: (1) M. P. Pantou et al. Mycol. Res. 109:889, 2005. (2) A. J. L. Phillips et al. Persoonia 21:29, 2008. (3) A. Zaid et al. Chapter XII in: Date palm cultivation, FAO Plant Production and Protection Paper 156 Rev. 1, 2002.

Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS.

During development of the mammalian brain, both neurons and glia are generated from multipotent neural stem cells. Although neurogenesis ceases in most areas at birth, stem cells continue to generate neurons within the subventricular zone and hippocampal dentate gyrus throughout adult life. In this work, we provide the first demonstration that precursors native to regions of the adult brain that generate only glia can also generate neurons after exposure to FGF-2 in vitro. When progenitors isolated from hippocampal tissue were directly compared with cells isolated from the neocortex, both populations were able to initiate a program of proliferative neurogenesis. Genetic marking and lineage analysis showed that a majority of the cells able to generate neurons were multipotent precursors; however, progeny from these precursors acquired the competence to differentiate into neurons only after exposure to FGF-2. The recruitment of similar FGF-2-responsive cells from the adult optic nerve, a structure well isolated from the neurogenic zones within the brain, confirmed that neuron-competent precursors naturally exist in widely divergent tissues of the adult brain.

Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles.

The subgranule zone of the dentate gyrus in rats has been shown to be proliferative into adulthood and senescence. However, the connectivity of newly generated, identified neurons in the adult has not been definitively described. In the present study, 9 weeks after a series of intraperitoneal injections of 5-bromo-2'-deoxyuridine (BrdU), animals received stereotaxic iontophoretic injections of Fluoro-Gold (FG) into field CA3. Three weeks after FG injections, sections were analyzed for BrdU immunoreactivity (proliferative label), FG retrograde label, and either calbindin-D28k or synaptophysin immunohistochemistry. A large proportion (up to 44%) of BrdU-labeled cells in the dentate gyrus within regions of FG retrograde label incorporated FG. All of the doubly labeled (BrdU-FG) neurons also immunolabeled with the antibody to calbindin-D28k. Many doubly labeled (BrdU-FG) cells were also surrounded in three planes by synaptophysin immunoreactivity. We conclude that newly generated neurons in the dentate gyrus have the correct immunohistochemical profile, send appropriate axonal projections to field CA3, and are surrounded by profiles containing synaptic vesicle proteins.

Evaluation of intracranial pressure from transcranial Doppler studies in cerebral disease.

The extent to which estimations of intracranial pressure can be derived from intracranial flow patterns was studied. The blood flow velocity in the middle cerebral artery was recorded with the EME TC 2-64 transcranial Doppler (TCD) device in 26 patients suffering from various severe cerebral diseases. Simultaneously the mean intracranial pressure (ICP) was measured by means of an epidural device. Arterial carbon-dioxide tensions were monitored by blood gas analysis. In all patients it was observed that the middle cerebral artery flow patterns changed distinctly when the ICP increased; these changes were distinguished by a decrease of the mean flow velocity and an increase of the Pourcelot index. A good correlation between the ICP and the flow parameters (especially the product mean systemic arterial pressure x Pourcelot index/mean flow velocity) was found in a select group of 13 patients, in whom comparable initial conditions existed and in whom additional parameters influencing the TCD recordings could be kept constant (r = 0.873; P less than 0.001).

Cisternal S100 protein and neuron-specific enolase are elevated and site-specific markers in intractable temporal lobe epilepsy.

In the brain, S100 protein and neuron-specific enolase (NSE) are mainly found in glial cells and neurons, respectively. We investigated concentrations of S100 protein and NSE in cisternal cerebrospinal fluid obtained during implantation of foramen ovale electrodes in eight patients with temporal lobe epilepsy (TLE). In addition, the meningeal markers cystatin-C and beta-trace as well as total protein were measured. Patients with trigeminal neuralgia (TN) undergoing glycerol rhizotomy served as controls. S100 protein and NSE levels ipsilateral to the site of seizure onset were significantly higher than in TN. Contralateral TLE values were also markedly but not significantly elevated. The meningeal markers cystatin-C and beta-trace protein as well as total protein did not differ in TLE and TN. We conclude that interictal temporal lobe dysfunction corresponds with neuronal and glial marker elevations in the extracellular space and that site-specific elevations may predict the site of seizure origin biochemically.

Brain metastases in neurosurgery: indications, surgical procedures and outcome.

Between July 1987 and June 1995, 1897 patients underwent operative therapeutic procedures for resection of tumours of the central nervous system in our department. 252 patients (13.3%) suffered from a metastatic disease in the brain or spinal cord. They comprised 113 females and 139 males. The age distribution ranged between 14 and 86 years with a median age of 58 years. The histology of resected tumours was distributed among lung-(54), breast-(33), melanoma-(27), kidney-(23) and prostate carcinoma (19). Other metastizing tumour sites were present in 96 cases. The age distribution of breast carcinoma ranged between 32 and 78 years with the median located at 54 years. 5 patients underwent a surgical resection of a local recurrence with a median latency of 9 months. Median survival time was estimated as 13 months with a one year survival of 63%. The aim of the study was to reflect on incidences, therapeutic procedures and outcome of brain metastatis.

Expression of CD 73 (ecto-5'-nucleotidase) in 165 glioblastomas by immunohistochemistry and electronmicroscopic histochemistry.

BACKGROUND: CD 73 (5'-nucleotidase) is an ectoenzyme, which is expressed on normal and neoplastic glial plasma membranes. The enzyme binds to intracellular filamentous actin and the extracellular matrix proteins laminin and fibronectin. CD 73 is a signalling pathway metabolite in the immune response of lymphocytes. The ectoenzyme catalyzes the conversion of purine and pyrimidine ribo- and deoxyribo-nucleoside monophosphates (AMP, GMP, IMP) and leads to elevation of the corresponding nucleosides (adenosine) in the extracellular space and might therefore modulate neuronal signalling and vascular perfusion. CD 73 has also been called a cellular motility factor. There is an increasing amount of evidence for the modulatory role of PKC-mediated CD 73 activity in ischemia, regeneration and repair, glioma cell proliferation and a possible invasion promoting feature of the ectoenzyme. The aim of the present study was to investigate the expression patterns of CD 73 together with the labelling of PKC and EGFR. The latter is known as a marker for primary glioblastomas. PATIENTS AND METHODS: We investigated the expression of CD 73 in 165 glioblastoma specimens together with the expression patterns of PKC and EGFR by immunocytochemistry on cryosections with a 4-step grading evaluation by two independent observers. CD 73 was further investigated morphologically by electron-microscopic histochemistry in cell cultures of glioblastoma specimens. RESULTS: With these methods it was possible to demonstrate a dense labelling pattern of glioblastoma specimens with anti-CD 73. 95.7% of the glioblastomas were identified with staining products, 63% with labelling grades 2 and 3. The dense staining of the endoplasmatic reticulum, vesicles, caveolar structures and glial membranes was demonstrated by electron-microscopic histochemistry. Some free enzymatic activity was located bound to the ECM components. We observed a significant coexpressions of CD 73 with PKC (p = 0.001) and CD 73 with EGFR (p = 0.022), which is a prospective marker for a high rate of early recurrency. CONCLUSIONS: The CD 73 activity was densely distributed on the membranes of glioblastoma cells in vivo and in cell cultures. The electron-microscopic histochemical studies could demonstrate enzymatic activity at the cell membranes and in vesicular structures and caveolae. Free staining deposits located on ECM components may result in a migration- and infiltration-promoting activity. The CD 73 expression could be correlated with the expression grades of PKC and EGFR. The latter has been identified as a prognostic factor which is expressed mainly on primary glioblastomas. PKC is a known tumour metabolite in several proliferation promoting pathways of EGF receptor signalling.

Expression of nitric oxide synthase isozymes (NOS I-III) by immunohistochemistry and DNA in situ hybridization. Correlation with macrophage presence, vascular endothelial growth factor (VEGF) and oedema volumetric data in 220 glioblastomas.

BACKGROUND: Nitric oxide (NO) is synthesized from arginine by three different isozymes of nitric oxide synthase (NOS I-III). NO has been identified as a powerful metabolite of vascular smooth muscle cell function, cerebral blood circulation and oedema induction. NOS induction by different cytokines has been shown previously in glioblastoma cell cultures and NOS III expression due to astrocytoma grading has been shown in several tumors recently. The aim of the present study was to study the coexpression of NOS I-III, macrophage and capillary presence with VEGF, EGF and their receptors and to investigate a possible mechanism in peritumoral oedema generation. MATERIALS AND METHODS: We have investigated the expression (4-grade values, blinded assay by two observers) of NOS I-III together with those of VEGF, VEGF- R (Flt-1), EGF-R1, von-Willebrand-factor (VWF) and a pan-macrophage marker (Ki-M1P) immunohistochemically in tumor specimens from 220 patients and performed tumor volume morphometry by image analysis in a subgroup of 32 cases to test for any correlation with the peritumoral oedema volumes. Inducible NOS II was further investigated by in situ labelling with a DNA oligonucleotide probe cocktail. RESULTS: All of the specimens revealed some NOS expression, NOS II was expressed in macrophages, microglia and endothelial cells, NOS III and I was localized in glioblastoma cells, NOS III in endothelial cells as well. The highest degrees of expression were observed in 46% (NOS I), 22% (NOS II) and 75% (NOS III) of all specimens. Inducible NOS II in any expression grade was observed in 47.5% of the specimens. Significant correlations were observed for the expression of the macrophage marker Ki-M1P with NOS II (p = 0.024), endothelial NOS III with NOS I (p = 0.0003), VEGF-R1 with NOS II (p = 0.0008) and NOS III (p = 0.011) The oedema volumes could not be correlated significantly with NOS or VEGF-R1 expression values but with those of endothelial staining (p = 0.02). We observed a trend towards higher Ki-M1P expression values together with higher oedema volume extensions. In situ hybridization demonstrated reaction products in endothelial and perivascular regions and sometimes scattered throughout the specimens revealing the labelling of macrophages. CONCLUSIONS: The main source of NO is NOS I and NOS III. The latter is located in endothelial cells and glioblastoma cells. The expression of NOS II in glioblastomas is restricted to infiltrating macrophages. NOS II and III expressions were observed significantly together with that of VEGF-R1. Neither NOS I-III nor VEGF-R expression could be correlated with the extension of the peritumoral oedema.

Oedema extension in cerebral metastasis and correlation with the expression of nitric oxide synthase isozymes (NOS I-III).

BACKGROUND: The development of a peritumoral oedema is a common radiological sign in preoperative CT- and MRI scans of patients with cerebral metastasis. Large tumours can be accompanied by a marginally extended oedema and vice versa. Several cytokines (VEGF) have been identified as mediators of vascular induction and permeability. Transmitters such as nitric oxide (NO) have been identified as specific mediators of vascular dilation and tumour blood flow in primary brain tumours in which different NOS isozymes (NOS I and III) are induced as a result of the latent hypoxic metabolic scenery. Other authors have considered NO as an endothelial stabilising metabolite. Inducible NOS II is expressed by microglia and macrophages invading during tumour growth. At present, no data exist on NO synthesising enzymes in cerebral metastasis. MATERIALS AND PATIENTS: Cryosections (N = 96) of metastatic resections were investigated immunohistologically using a 4-step grading evaluation for the expression of NOS I-III, VEGF-receptor FLT-1, a pan-macrophage marker Ki-M1P, and capillary vessel presence by endothelial Von-Willebrand-Factor staining. The tumour and oedema extension was measured in preoperative MRI scans by an image processing device (Kontron) and calculated for the ratios of oedema volumes to total tumour volumes. The data were analysed statistically (Pearson Chi2 and Kruskal-Wallis analysis of variances) and correlated with the clinical data. Inducible NOS II was further investigated by in situ hybridization with a (4x30 mer) DNA oligoprobe cocktail. RESULTS: Between 1987 and 1996 289 patients in our department suffered from a metastatic disease in the brain or spinal cord. In 96 cases resected tumour material was processed for the immunohistological investigation. The age distribution ranged from 14 to 85 years with a median age of 58 years. The mean duration of symptoms before diagnosis was estimated as 53 days. The expression of NO synthase was frequently observed. NOS I was detected in 83.6%, gradings 2 and 3 in 40.5% of them. NOS III, the endothelial isoform, was observed in 39.4% (gradings 2 and 3), inducible NOS II in 29.4% (grading 2 and 3) of the specimens. The VEGF receptor FLT-1 could be detected in 70% of them, 24% in higher expression 2 and 3. The pan macrophage marker Ki-M1P was observed in 72% of all cases. Fifty seven percent of the specimens exhibited strong labelling with antibodies against VWF. Coexpressions were statistically significant for the VEGF receptor and NOS I-III (p < 0.01), Ki-M1P and NOS I and II (p < 0.05). A negative correlation was detected for the oedema index (oedema volume/total volume) and the labelling data for NOS III (r = -0.44, p = 0.13) and VEGF-R (r = -0.42, p = 0.022). No correlation existed for Ki-M1P, VWF and NOS I. CONCLUSIONS: The objective of the study was to investigate oedema morphometry, expression of NOS I-III and VEGF-R, presence of capillary vessels and macrophages in cerebral metastasis. A further aim was to investigate a putative oedema induction by NO producing isozymes. Nitric oxide synthase expression was statistically significantly correlated with the expression of the VEGF receptor and the presence of macrophages and microglia. There was a negative correlation between oedema extension and the presence of NOS III and VEGF-R. The results seem to indicate a specific oedema modulating role of NO in cerebral metastasis.

Using transcranial duplex sonography for monitoring vessel patency during surgery for intracranial aneurysms.

This article reports a method for reliable intraoperative monitoring of blood flow velocities in the basal cerebral arteries during clipping of intracerebral aneurysms. Transcranial color-coded duplex sonography provides practical integration of transcranial Doppler technology with real-time imaging capabilities through the intact human skull. With a computerized sonography system equipped with a 2.5-MHz probe in 50 healthy volunteers, the contralateral internal carotid artery, A1 and A2, as well as M1 and P1 vessels were identified and measured in most patients. In 13 patients undergoing dipping of intracranial aneurysms, the technique successfully imaged 12; it allowed definitive identification of vessels potentially threatened by clipping and not fully visible to the surgeon. Data were easily comparable to preoperative data. This noninvasive, repeatable neuroimaging technique provides useful intraoperative information about intracranial hemodynamics during dipping of intracranial aneurysms.

Quantitative proton magnetic resonance spectroscopy of focal brain lesions.

The diagnostic value of single-voxel proton magnetic resonance spectroscopy (2 T, stimulated echo acquisition mode, TR = 6,000 ms, TE = 20 ms, 4-5 mL volumes-of-interest) was assessed for a differentiation of focal brain lesions of unknown etiology in 17 patients 1-14 years of age. Absolute metabolite concentrations were compared with age-matched control subjects and an individual control region. Most of the brain tumors were characterized by strongly reduced total N-acetylaspartyl compounds and marked increases of myo-inositol and choline-containing compounds, consistent with a lack of neuroaxonal tissue and a proliferation of glial cells. Lactate was elevated in only four patients. When using this pattern for a metabolic discrimination of brain tumors from other focal lesions, proton spectroscopy correctly identified 14 of 17 abnormalities, as confirmed by histologic examination after neurosurgical intervention. One false-positive tumor diagnosis was a severe reactive gliosis mimicking a typical tumor spectrum. Two inconclusive cases comprised an astrocytoma with moderately elevated myo-inositol but reduced choline-containing compounds and a patient with an abscess leading to a marked reduction of all metabolites but strong contributions from mobile lipids. In summary, quantitative proton spectroscopy has considerable clinical value for preoperative characterization of focal brain lesions.

Postoperative intracranial abscess--clinical aspects in the differential diagnosis to early recurrence of malignant glioma.

Out of 2941 patients who received a clean, major craniotomy, 39 patients (1.3%) developed the complication of an intracranial deep infection, i.e. abscess or empyema. A total of 14 patients with a postoperative abscess were initially operated upon intracerebral malignant glioma (WHO III or IV) and could be compared to a matched group of patients with recurrent malignancy concerning clinical and radiological aspects. A statistically significant elevation of median values was seen for erythrocyte sedimentation rate (ESR), fibrinogen and body temperature in the study group. C-reactive protein (CRP) was not investigated in the control group and could not be compared, but it was elevated in all abscess patients when measured. CT-scan did not allow a safe differentiation between infection and recurrent glioma. Local signs like suppuration of the wound could be observed in 71% of patients with intracranial infection. Postoperative abscesses had been diagnosed in all cases within 3 months, whereas none of the early recurrences of intracerebral malignoma became symptomatic before 12 weeks after initial operation. Therefore, the course of time seems to be another important factor in this differential diagnosis.

Immunoscintigraphy for the detection of brain abscesses.

The aim of this prospective study was to assess the diagnostic usefulness of a 99Tcm-anti-granulocyte antibody in the early differentiation of the aetiology of a ring-enhancing structure on computed tomography (CT) scans following neurosurgical intervention. In 26 patients (15 males, 11 females) aged 20-82 years with suspected intracranial infection, 29 SPET scans of the head were obtained 4-6 h following the intravenous injection of 555 MBq 99Tcm-anti-granulocyte antibody. The patients had antibiotic, antimycotic or corticosteroid therapy. The diagnosis was confirmed by surgery (19 cases) or subsequent CT/MRI (magnetic resonance imaging) scans and clinical follow-up (10 cases). The immunoscan was true-positive (abscess) in 6 (sensitivity = 100%), true-negative in 19 and false-positive in 4 (specificity 83%) cases. There was no obvious detrimental effect on the results due to the antibiotic, antimycotic or corticosteroid therapy. In conclusion, despite false-positive results, the 99Tcm-anti-granulocyte antibody is a useful tool in the early detection and exclusion of intracranial abscess after neurosurgical interventions.

c-myc oncogene family expression in glioblastoma and survival.

BACKGROUND: In gliomas, c-myc proto-oncogene expression has been found to correlate with the grade of malignancy, with low expression in Grade I and II and high expression in Grade III and IV tumors. We aimed to discover if myc expression is of prognostic significance in glioblastomas. METHODS: Expression of the c-myc, N-myc, and L-myc proto-oncogenes and of the max gene was investigated in 46 supratentorial glioblastomas from adult patients using in situ hybridization. RESULTS: Seventy-eight percent of the tumors expressed c-myc m-RNA, 84% max m-RNA, 57% N-myc m-RNA, and 57% L-myc m-RNA. The postoperative survival of patients over 60 years of age and that of patients under 60 years of age were analyzed separately, since advancing age was found to be negatively correlated with the duration of postoperative survival (p = 0.004). There was no significant difference in postoperative survival in either age group between patients whose tumors expressed either c-myc, N-myc, or L-myc, respectively, and those whose tumors did not exhibit this characteristic. A difference in postoperative survival, however, was found in the over 60-year age group between patients whose tumors expressed max to an equal or lesser extent than c-myc and those whose tumors expressed max to a greater extent than c-myc or neither max nor c-myc. CONCLUSION: The biologic behavior of glioblastomas in older patients may depend on the relative, but not on the absolute content of the c-myc protein and interacting proteins.