Absence of post-lesion reactive gliosis in elasmobranchs and turtles and its bearing on the evolution of astroglia.

In the mature mammalian and avian central nervous systems, neuronal destructions are followed by reactive gliosis, but data on other vertebrates are rather controversial. Mammals and birds belong to different amniote groups (Synapsida and Diapsida, respectively), but exhibit common general features in their glial architecture, mainly the predominance of astrocytes. Two vertebrate groups seem to be in special positions of glial evolution: turtles (Testudiniformes) and skates and rays (Batoidea). The purely ependymoglial system of turtles seems to be the simplest one among the extant amniotes. In skates and rays, true astrocytes are preponderant glial elements, in contrast to the other "anamniotes" (and even to reptiles). We investigated stab wounds by the immunohistochemical detection of GFAP in turtles (Trachemys-formerly Pseudemys-scripta elegans), a skate (Raja clavata) and rays (Dasyatis akajei and Torpedo marmorata). Sharks (Scyliorhinus canicula) as ependymoglia-predominated chondrichthyans, and-for positive controls-rats were also studied. In the elasmobranchs, other astroglial markers: glutamine synthetase and S100 protein were also applied. Neither turtles nor elasmobranchs presented considerable astroglial reactions. Critically surveying the former reports on different vertebrates, these results complete the picture that typical post-lesion reactive gliosis is confined to mammals and birds. Analysis of the astroglial systems from phylogenetic perspective suggests that the capability of forming glial demarcation and scar formation evolved independently in mammals and birds. Predominance of astrocytes is a necessary condition but not sufficient for reactive gliosis. The intense glial reactivity of mammals and birds may be attributed to their complex cerebralization.

Alterations of the perivascular dystrophin-dystroglycan complex following brain lesions: an immunohistochemical study in rats.

Dystroglycan is a laminin receptor, which with dystrophins and other components forms the dystrophin-dystroglycan complex. It has an important role in the formation of gliovascular connections, cerebral vascularisation and blood-brain barrier. Dystroglycan consists of two sub-units, α and ß. Previous studies demonstrated that the ß-dystroglycan immunoreactivity of cerebral vessels temporarily disappeared in the area adjacent to the lesion, whereas the vascular laminin which is not immunoreactive in the intact brain became detectable. The present study extends these investigations over other components of the complex: utrophin, α1-syntrophin and α1-dystrobrevin. The experiments were performed on adult rats. The lesions were stab wounds or cryogenic lesions in deep ketamine-xylasine narcosis. Following survival periods 2 to 30 days, the animals were perfused and floating brain sections were processed for fluorescent immunohistochemistry. The α1-dystrobrevin, like ß-dystroglycan, vanished temporarily around the lesion. The immunoreactivity of utrophin changed in a similar way to that of laminin. In intact brains they were confined to the entering segments of the vessels and to the circumventricular organs. Following lesions their immunoreactivity manifested in the vessels around the lesions. However, utrophin followed laminin with a delay: their peaks were about POD (postoperative days) 21 and 7, respectively. Only immunoreactivity of α1-syntrophin appeared in the reactive astrocytes, peaking at POD 14. Double-labeling proved its co-localization with GFAP. Cryogenic lesions had similar immunohistochemical effects, but provided more suitable samples for Western blot analysis, which proved the altered levels of α1-dystrobrevin and α1-syntrophin. The phenomena may help to monitor the post-lesion vascular processes and the alterations of the gliovascular connections.

Antitussive activity of Althaea officinalis L. polysaccharide rhamnogalacturonan and its changes in guinea pigs with ovalbumine-induced airways inflammation.

AIM: The presented studies were aimed on experimental confirmation of Althaea officinalis polysaccharide rhamnogalacturonan antitussive effect and its changes in conditions of allergic inflammation. METHODS: We have tested whether rhamnogalacturonan inhibits cough reflex and modulates airways reactivity of guinea pigs in vivo. The cough in guinea pigs was induced by 0.3 M citric acid (CA) aerosol for 3 min interval, in which total number of cough efforts (sudden enhancement of expiratory flow accompanied by cough movement and sound) was counted. Specific airway resistance and its changes induced by citric acid aerosol were considered as an indicator of the in vivo reactivity changes. RESULTS: 1) Althaea officinalis polysaccharide rhamnogalacturonan dose- dependently inhibits cough reflex in unsensitized guinea pigs. Simultaneously, plant polysaccharide shortened the duration of antitussive effect when it was been tested in inflammatory conditions. 2) Rhamnogalacturonan did not influence airways reactivity in vivo conditions expressed as specific resistance values neither sensitized nor unsensitized groups of animals. 3) The antitussive activity of codeine (dose 10 mg.kg(-1) b.w. orally) tested under the same condition was comparable to higher dose of rhamnogalacturonan in unsensitized animals. 4) The characteristic cellular pattern of allergic airways inflammation was confirmed by histopathological investigations. CONCLUSION: Rhamnogalacturonan isolated from Althaea officinalis mucilage possesses very high cough suppressive effect in guinea pigs test system, which is shortened in conditions of experimentally induced airways allergic inflammation (Tab. 1, Fig. 4, Ref. 25). Full Text in free PDF www.bmj.sk.

Optically adjustable light filaments generated by a compact laser convertor.

In the paper, the geometrical parameters and energetics of the extremely narrow pseudo-nondiffracting beams with the spot size of several micrometers are examined. The main attention is focused on design, realization and testing of the set-up enabling conversion of the laser diode beam or the fiber mode to the narrow Bessel-Gauss beam whose spot can be continuously relocated across the plane perpendicular to the beam propagation direction. Application of the laser convertor to the optical manipulation is demonstrated on experiments enabling transport of microparticles along a desired trajectory.

Disappearance of the post-lesional laminin immunopositivity of brain vessels is parallel with the formation of gliovascular junctions and common basal lamina. A double-labelling immunohistochemical study.

Previous studies revealed that during development the laminin immunopositivity gradually disappeared from the brain vessels, but temporarily re-appeared in them around lesions. The question of the present study was the correlation between the post-lesional vascular immunopositivity to laminin and the glial reaction. Following stab wounds, double fluorescent immunohistochemical labelling was performed against laminin (using a polyclonal antiserum against laminin 1) and glial fibrillary acidic protein. A number of vessels exhibited intense immunopositivity to laminin within the lesioned tissue. Where these laminin immunopositive vessels entered the perilesional brain substance, the astroglia formed contacts on them, and the separate vascular and glial basal laminae fused. The disappearance of the post-lesional laminin immunopositivity seemed to coincide with these phenomena. When monoclonal antibodies were applied against the beta1 and gamma1 laminin chains, vessels proved to be immunopositive at the lesion, but none in the intact brain tissue. No immunoreactivity was detected in the cases of alpha2 and beta2 chains. The results suggest that the disappearance of laminin immunopositivity may be attributed to that the epitopes become inaccessible for antibodies owing to the formation of gliovascular junctions and common basal lamina between astroglia and vessel. The possible role of an alteration in the laminin composition and the effect of fixation are discussed.

Reactive glia support and guide axon growth in the rat thalamus during the first postnatal week. A sharply timed transition from permissive to non-permissive stage.

The present study demonstrates a supportive and guiding effect of the reactive glia on the postlesional axon growth in vivo, and offers a model system to compare permissive and non-permissive forms of the glial reaction. After stab wounds in early postnatal (P2-P9) rats, the reactive glia and the nerve fibers were detected by the immunohistochemical staining of glial fibrillary acidic protein (GFAP) and neurofilament protein, respectively. In the thalamus of the animals lesioned at P5 or earlier, an extraordinary bundle of fibers immunoreactive to neurofilament protein was found, corresponding to the lesion track marked by reactive glia. This bundle persisted up to 2 months, as shown by electron microscopy. When the animals were lesioned at P7 or later, the lesion track was immunonegative to neurofilament protein. Following P6 lesions, an intermediate situation was found, the strip of immunoreactive neurofilament protein was missing, or short and weak. GFAP immunostaining demonstrated a typical reactive glia in every case. As a result of the same operation, reactive glia plus a deficiency of neurofilament protein immunostaining was found in every animal in the cortex and the corpus callosum, independently from the age at lesion. The results demonstrate that the permissive nature of the glial reaction depends on the lesioned area as well, and changes to a non-permissive effect in a short time interval.

Immunohistochemical investigation of actin-anchoring proteins vinculin, talin and paxillin in rat brain following lesion: a moderate reaction, confined to the astroglia of brain tracts.

The present study investigated the immunoreactivity of vinculin, talin and paxillin following stab wounds in the cortex and the underlying white matter of adult rats. These proteins participate in the anchoring of actin filaments to the cell membrane at the focal adhesion plaques, and they are of essential importance in cell motility. Without lesion, vinculin, talin and paxillin immunopositive astrocytes were not recognizable in the cortex and only scarcely in the white matter, if at all. Following lesion, several astrocytes immunopositive to vinculin, talin and paxillin appeared in the white matter, whereas none of them was found in the overlying cortex. Glial fibrillary acidic protein (GFAP) immunopositivity was intense in both areas. The distribution of the actin-anchoring proteins following lesions was similar to that found of the intermediate-filament associated protein plectin, in our previous study.

GFAP-immunopositive structures in spiny dogfish, Squalus acanthias, and little skate, Raia erinacea, brains: differences have evolutionary implications.

GFAP expression patterns were compared between the brains of a spiny dogfish (Squalus acanthias) and a little skate (Raia erinacea). After anesthesia, the animals were perfused with paraformaldehyde. Serial vibratome sections were immunostained against GFAP using the avidin-biotin method. Spiny dogfish brain contained mainly uniformly-distributed, radially arranged ependymoglia. From GFAP distribution, the layered organization in both the telencephalon and the tectum were visible. In the cerebellum, the molecular and granular layers displayed conspicuously different glial structures; in the former a Bergmann glia-like population was found. No true astrocytes (i.e., stellate-shaped cells) were found. Radial glial endfeet lined all meningeal surfaces. Radial fibers also seemed to form endfeet and en passant contacts on the vessels. Plexuses of fine perivascular glial fibers also contributed to the perivascular glia. Compared with spiny dogfish brain, GFAP expression in the little skate brain was confined. Radial glia were limited to a few areas, e.g., segments of the ventricular surface of the telencephalon, and the midline of the diencephalon and mesencephalon. Scarce astrocytes occurred in every brain part, but only the optic chiasm, and the junction of the tegmentum and optic tectum contained large numbers of astrocytes. Astrocytes formed the meningeal glia limitans and the perivascular glia. No GFAP-immunopositive Bergmann glia-like structure was found. Astrocytes seen in the little skate were clearly different from the mammalian and avian ones; they had a different process system - extra large forms were frequently seen, and the meningeal and perivascular cells were spread along the surface instead of forming endfeet by processes. The differences between Squalus and Raia astroglia were much like those found between reptiles versus mammals and birds. It suggests independent and parallel glial evolutionary processes in amniotes and chondrichthyans, seemingly correlated with the thickening of the brain wall, and the growing complexity of the brain. There is no strict correlation, however, between the replacement of radial ependymoglia with astrocytes, and the local thickness of the brain wall.

Glial fibrillary acidic protein-immunopositive structures in the brain of a Crocodilian, Caiman crocodilus, and its bearing on the evolution of astroglia.

Caiman crocodilus, as a representative of the order Crocodilia, was used in immunohistochemical studies. Immunohistochemical procedures were performed on free-floating sections using a monoclonal antibody against porcine glial fibrillary acidic protein (GFAP) and employing standard avidin-biotin complex methodology. The astroglia of Caiman exhibited robust immunoreactivity to the antibodies raised against mammalian GFAP. In Caiman, the predominant GFAP-immunopositive elements are the radial ependymoglia, similar to other reptiles. The regional variability of glial architecture in Caiman, however, seems greater than in other reptiles so far examined, although it is less compared with chickens. We suggest that this finding corresponds to a more advanced "regional adaptation" of the glial structure in Caiman compared with other reptiles. The main feature that distinguishes the astroglia of Caiman from those of other reptiles is the widespread occurrence of GFAP-immunopositive astrocytes. These cells are limited in lizards and snakes, are not present in turtles, but are found in every major brain area in Caiman. However, even in Caiman, astrocytes are only intermingled with radial glia and are not the predominant glial element of any brain area. The occurrence of astrocytes does not correlate with brain wall thickness. Despite their origin from different ancestral groups of stem reptiles (synapsid or diapsid), mammals and birds exhibit some common general features in their glial architecture and GFAP distribution: 1) predominance of astrocytes and 2) absent or limited GFAP immunopositivity of several brain areas. The present study demonstrates that, even in Caiman, a representative of the reptilian group most closely related to birds, these features are present only in part, suggesting that, in mammals and birds, they have evolved independently.

Integrated RT-PCR/nested PCR diagnosis for differentiating between subgroups of plum pox virus.

An RT-PCR/nested PCR technique was developed for the simultaneous detection and typing of plum pox virus (PPV) and its major types--Dideron (D), Marcus (M), El-Amar (EA) and Cherry (C). Degenerated oligonucleotides were synthesized for the general detection of PPV, flanking the coding sequence for the N-terminal portion of the coat protein (CP), within which strain-specific differences were identified. On the basis of these characteristic differences, degenerated primer pairs were designed to differentiate between the four major subgroups of the virus in nested PCR reactions. The validity of the technique was tested on viral strains and cloned cDNAs overlapping the CP region. High specificity was observed with no detectable cross-reactions. The results of general PPV detection with the new primers and those of the PCR-based detection of the 3' non-coding region of the viral genome correlated with complete coincidence. The PCR typing results correlated well with those of the RsaI-RFLP and serological typing and revealed a surprisingly high incidence of PPV-D in Hungary.

A comparison of intermediate filament markers for presumptive astroglia in the developing rat neocortex: immunostaining against nestin reveals more detail, than GFAP or vimentin.

The present study compares the immunopositive elements in the developing rat cortex between the day of birth (P0) and the 18th postnatal day (P18), after immunostaining against nestin, vimentin and glial fibrillary acidic protein (GFAP). Nestin immunostaining revealed more structural details than either vimentin or GFAP, or they together. While vimentin immunostaining preferred radial glia and GFAP preferred astrocytes, nestin immunostaining detected both. Stellate-shaped astrocyte-like cells were already seen at P0 and cells of typical astrocytic morphology were numerous at P3, and were predominating elements from P7, whereas GFAP-immunopositive astrocytes were very scarce even at P7, and became numerous only by P11, when nestin immunopositivity started to disappear. Nestin immunostaining revealed such structures which were not seen in GFAP- or vimentin immunostained sections: cell body-like structures 'hanging' at the end the radial fibers, seeming to divide with their fibers, or having astrocyte-like processes. Nestin immunostaining is therefore highly recommended for studies of the glial architecture in the early post-natal brain development.

[First detection of human calicivirus infection in Hungary]. / Humán calicivírus-fertózés elsó magyarországi igazolása.

Human caliciviruses (HuCVs) are important pathogens all over the world. They cause acute non-bacterial gastroenteritis in humans in all age-groups. The aim of the study was to detect HuCV infection in Hungary. Stool samples examined were received from a food-borne outbreak in nursery and elementary school in Szeged and Algyó areas November 1998. Reverse transcription-polymerase chain reaction (RT-PCR) method was successfully used for the detection of HuCVs in all stool samples received. This is the first description of HuCVs in Hungary and the verification of them as causative agents in a food-borne outbreak.

Milkborne campylobacter infection in Hungary.

In April 1998, an annual 2-day animal farm sale was held in Hódmezóvásárhely, where 500 to 600 visitors consumed unpasteurized milk. The first signs of disease began 2 days after the end of the sale. Fifty-two people from a wide age range fell ill, primarily with inflammatory enteritis. These cases included 34 with Campylobacter positivity: 30 with Campylobacter jejuni and 4 with Campylobacter coli. Environmental samples (raw milk, udder swabs, and rectal swabs from 12 cows in the suspected herd) were tested 2 weeks after the first signs of the disease, and two rectal swabs were found to be positive for C. jejuni. Initially, the epidemic seemed to be sporadic and, accordingly, only 26 human and 2 animal Campylobacter isolates were reserved for randomly amplified polymorphic DNA analysis. This comparative analysis verified that fecally contaminated milk was the source of the outbreak. The DNA-banding patterns of 20 C. jejuni isolates (19 human and 1 animal) were identical. The antibiotic susceptibilities of the Campylobacter isolates were determined, and only six C. jejuni (human) isolates, one C. coli (human) isolate, and one C. jejuni (animal) isolate were resistant to tetracycline, both by disk diffusion and by E test (antimicrobial gradient strip for the quantitative determination of susceptibility or resistance of microorganisms). No plasmid was detected in these tetracycline-resistant isolates. The endotoxin production of Campylobacter isolates was examined via the cytopathogenic effect on the Vero cell line. This effect exhibited various degrees of positivity in 19 cases. Only two human C. jejuni isolates displayed + + + + positivity. Both isolates were from patients who had required antibiotic therapy and hospital care.

Lesions do not provoke GFAP-expression in the GFAP-immunonegative areas of the teleost brain.

In the mammalian and avian brains the predominant astroglial elements are astrocytes, and the distribution of GFAP-immunopositivity is rather uneven, some large brain areas being almost devoid of GFAP-immunopositivity. In these areas however, an intense GFAP-immunopositivity appears following injury. In the teleost brain most of the areas are GFAP-immunopositive and ependymoglia predominates. However, a large area, the layer of the sensory neurons in the vagal lobe, is devoid of GFAP. The question arises, whether the lack of GFAP-immunopositivity in the teleost brain is also due to the repression of the GFAP-production, as in birds and mammals, or due to the lack of cells capable of expressing GFAP. To answer this question, stab wounds were made in the vagal lobe of goldfish, as well as in the tectum, in which moderately dense but highly organized GFAP-immunopositive glia has been detected in intact animals. In the layer of the sensory neurons in the vagal lobe no GFAP-immunopositivity appeared even after lesions had been introduced. In the tectum, a rather slight increase of the intensity of the immunostaining was observed in the glial fibers near the lesions but no typical reactive glia similar to that found in mammals or birds, was observed. The results suggest that a lesion does not provoke GFAP-expression in GFAP-immunonegative brain areas in teleosts, in contrast to what is observed in mammals and birds.

Axon growth failure following corpus callosum lesions precedes glial reaction in perinatal rats.

The present study compares the glial reactivity and the axon growth following corpus callosum (CC) lesions, in perinatal rats. Lesions were performed on fetal (E17 to E20) and early postnatal (P0 and P2) rats. The reactive glia and the presence of neural fibers were detected by immunohistochemical staining of glial fibrillary acidic protein (GFAP) and neurofilament protein (NFP), respectively. The callosal axons failed (at least in part) to penetrate the lesioned area already after E18 lesions, and the lesioned area was always impenetrable for axons after E20 and P0 lesions. In these cases, the lesioned part of the CC was completely or nearly devoid of GFAP as well as NFP. The distributions of the immunopositivities to GFAP and NFP also coincided with each other, both in the intact part of the CC and along the alternative courses of the callosal axons. GFAP-immunopositive reactive glia accompanied to the deficiency of NFP-immunostaining only when animals were lesioned at P2. Nestin immunostaining revealed astrocytes or their precursors already at P0, but reactive glia were detected only after P2 lesions, as with immunostaining to GFAP. The results suggest that the age after which the lesioned area proves to be impenetrable for axons can precede that age after which lesions provoke glial reaction. In this case the inhibition of axon growth is to be attributed to factors other than to the reactive glia. The presence of nestin-positive cells suggests that the lack of reactive glia along the lesion track was not due to the absence of astrocytes, but rather due to the lack of their reaction to lesion. In this developmental stage astroglia, when activated, seem to promote the growth of axons.

Plectin immunopositivity appears in the astrocytes in the white matter but not in the gray matter after stab wounds.

Plectin is associated with intermediate filaments, such as glial fibrillary acidic protein (GFAP) and vimentin, which have an important role in the glial reactions after lesions. The present study investigates the plectin immunoreactivity following stab wounds in the cortex and the underlying white matter of adult rats. Without lesion, plectin immunoreactive astrocytes were not found in the cortex and only scarcely in the white matter. Following lesion, a number of astrocytes became immunopositive in the white matter, while no immunoreactivity was found in the overlying cortex. GFAP and vimentin showed intense immunopositivity in both areas. Therefore, the participation of the plectin seems to distinguish the glial reactions developed in the white or gray matters.

Recent advances in biohydrogen research.

A fundamental and principal difficulty of the future energy supply is that the formation of fossil fuels is much slower than the rate of their exploitation. Therefore the reserves which can be recovered in an energetically feasible manner are shrinking parallel with an increasing world-wide energy demand. Among the alternative energy carriers, hydrogen is preferred because it is easy to transport and store and it burns to environmentally friendly water vapour when utilized. Hydrogen can be produced in biological systems, however, our understanding of the molecular details is just emerging.