CC BY
16УДК 57.086.3:616-091.5:612.82: 616-002.828
electron microscopy
of autopsy material
from the human brain cryptococcosis and aids
1stepanova A.A. (head of the laboratory)*, 1vasilyeva N.v. (director of the institute, head of the chair), 2Yamaguchi M. (associate professor), 2shimizu K. (assistant professor), 2Kawamoto s. (professor)
1 North-West State Medical University named after I.I. Mechnikov: Kashkin Research Institute of Medical Mycology and Chair of Medical Microbiology, St. Petersburg, Russia; 2 Medical Mycology Research Center, Chiba University, Chiba, Japan
© Collective of authors, 2015
The cells of Cryptococcus neoformans var. neoformans and human brain macrophages in autopsy material of cryptococcosis AIDS patient has been studied by light and transmission electron microscopy. Use of paraffin blocks for electron microscopic investigation of human brain cryptococcosis was proposed.
Key words: autopsy archives material, brain, cryptococcus, cryptococcosis, light and electron microscopy, macrophages, polysaccharide capsule
электронная микроскопия аутопсийного материала головного мозга пациента больного криптококкозом и спидом
1 степанова А.А. (зав. лаб.), 1 Васильева н.В. (директор института, зав. кафедрой),
2 ямагучи м. (адъюнкт-профессор), 2 Шимицу K. (ассистент профессора), 2 Кавамото с. (профессор)
1 Северо-Западный государственный медицинский университет им. И.И. Мечникова: НИИ медицинской микологии им. П.Н. Кашкина и кафедра медицинской микробиологии, Санкт-Петербург, Россия; 2 Центр исследований по медицинской микологии, Университет, Чиба, Япония
Контактное лицо: Степанова Амалия Аркадьевна, тел.: (812) 303-51-40
© Коллектив авторов, 2015
С помощью методов световой и электронной микроскопии клетки Cryptococcus neoformans var. neoformans и макрофаги были изучены в головном мозге ВИЧ-инфицированного пациента на примере аутопсийного материала. Предложен метод использования парафиновых блоков для электронно-микроскопического исследования криптококкоза головного мозга человека.
Ключевые слова: аутопсийный архивный материал, головной мозг, криптококк, криптококкоз, макрофаги, полисахаридная капсула, световая и электронная микроскопия
introduction
The ultrastructure of C. neoformans var. neoformans cells were in general investigated in vitro [1-2 and etc.] and in host tissues of experimental cryptococcosis in murine [3, 4; Feldmesser M., et al. // Infect. Immun. -
2000. - Vol. 68; Feldmesser M., et al.// Microbiology. -
2001. - Vol. 147]. There is little information in scientific literature about the ultrastructure of cells of cryptococcus in human tissues with cryptococcosis. However, many issues of their pathogenesis remain unclear. Until now, the authors of the special publications tried to explore the relationships in the complex the cells of immune system of the host^the cell pathogenic fungus, whereas ultrastructural «portrait» of pathogenic C. neoformans var. neoformans has not been the subject of special study.
The primary fixation of human tissues for electron microscopy through the very small sizes of sample often complicate the detection of small and a few in numbers of the required objects. In this regard, in the present work attempt was made to use the paraffin blocks of autopsy material for subsequent purposeful electron microscopic study.
materials and methods
The dead women, which long time suffered from mixed infection: HIV, cryptococcosis and tuberculosis, during the mycological study of cerebrospinal fluid were sown cultures of C. neoformans var. neoformans. The patient has been medical treated with amphotericin B and fluconazole. During the posthumous histological research was also confirmed diagnosis of generalized cryptococcosis with a primary affection of the brain.
We present in this article only one ofthe most successful example from the large number of approved variants of possible use of paraffin blocks for subsequent electron microscopic studies. The pieces of brain tissue were fixed for the light microscopy in 10% formalin solution. The paraffin sections were stained with hematoxylin-eosin. After studying of section in the light microscope, from the part of paraffin block, in which we observed the greatest concentration of cells of C. neoformans var. neoformans, we cut out pieces (2x2x10 mm) and treated according to the following scheme: dewaxing in xylol (3 hours in the thermostat at 60 °C for softening and partial mechanical removal of paraffin, twice for 6 hours at room temperature), conducting through a series of ethanol solution (96 °C - 60 minutes, 70 °C - 30 minutes, 50 °C - 30 minutes, 30 °C - 30 minutes), washed twice for 15 minutes in cacodylate buffer (pH 7,2), then fixed
•■k
for 3 hours at room temperature in a mixture of 3% solution of glutaraldehyde and paraformaldehyde, then it was post-fixed in 1% osmium tetroxide, prepared on the same buffer. Then the samples were treated with increasing ethanol concentration from 30% to 90% (in 70%-hydrated ethanol was added 2% uranylacetate), absolute ethanol for 30 minutes, a mixture of absolute ethanol and acetone (1:1), absolute acetone for 30 minutes and fractional mixtures of absolute acetone and epoxy resin «epon-araldite» (9:1 - 30 minutes; 8:2 - 30 minutes; 7:3 - 45 minutes; 6:4 - 45 minutes; 5:5 - during night at room temperature).
Then the samples of tissue were placed in a plastic capsule and embedded in the epoxy resin. For better impregnation of the epoxy resin in the pieces of brain, we put the epoxy resin with samples for 3 days in a desiccator with exsiccant, after which they were placed in the thermostat for three days at 60° C for polymerization.
Then we preparated semi-thin sections (2-5 |m) from the epoxy blocks on the Pyramitome 11800 (LKB) after what , then they glue with 50% acetone on glass slides, stained with a toluidine blue and then examined in a light microscope. The blocks on Pyramitome were trimmed for subsequent obtaining the ultrathin sections on Ultratome V (2088, LKB) with glass knives. Ultrathin sections were collected on a grid without film then were stained for 10 minutes with 2% solution of uranyl acetate and 5 minutes by lead citrate according to standard methods. Light-microscopic research and shooting of the sections was performed on a microscope Leica DMR and electron microscopy - transmission electron microscope Jem-100SX (Tokyo, Japan).
results and discussion
In the semi-thin epoxy sections of the zone of brain pia mater, which we stained with toluidine blue (Fig. 1 a), we revealed the fungal yeast cells lying free in brain tissue and inside of the macrophages (Fig. 1c). In the brain tissue occasionally we observed a single (Fig. 1 b, arrows) or a few yeast cells of cryptococcus.
Fig. 1. General view (a) of the human brain tissue and the cells of cryptococcus (b-v) in light (a-e) and transmission electron microscope (h-v). Explanation for this and another figures:
AFC - aggregation of fungal cells; C - capsule; CW - cell wall, DC - daughter cell, DCW - debris of the cell wall, DPC - debris of the capsule, FB - fibrous bodies; FC - fungal cell, M - macrophage, Ma - macrovacuole, MC - mother cell, Mc -microvacuole, Mf - microfibrils, N - nucleus, Nu - nucleolus, S - scar, Sl - slime. Numeration on Fig. 1 demonstrated the layers in composition of the capsule. Scale: a = 1 mm; b = 10 |m; c - f = 0.5 |m; g, r = 2 |m; h = 7 |m; i, m = 1 |m; j-n, q, u, v = 3 |m; o, p = 0.8 |m; s = 1.5 |m
The cells of C. neoformans var. neoformans
We identified fungal cells inside (Fig. 1 c) and outside of macrophages (Fig. 1 b, d, f) during the investigation of the semi-thin sections. All investigated under electron microscope free localized cells of cryptococcus were in different stages of dying. We can distinguish among them four main 6 types of cells. Cells of cryptococcus of 1st type (« 25%) had no nucleus and organelles, they were rich with storage substances, contain moderate electron density cytosol, at least - fragments of membranes of different lengths and clusters of particles with similar morphology with human immune-deficiency virus (Fig. 1 n, arrows). On semi-thin sections its content and the lower layer of the capsules were stained in light blue color, while the cell walls - in dark blue (Fig. 1d).
The final stages of death were typical for the fungal cells of 2nd type (« 28%), they had also no nucleus and organelles. In the yeast cells between the plasmalemma and the cell wall was observed varying degrees of deposition of fine-fibrillar material, apparently, slime.
The yeast cells of 3rd type (« 35%) were completely dead; they usually retain the form of the once-living
yeast cells and contain material whose morphology was similar with slime. Contents and cell walls of the fungi of the 2nd and 3rd types after staining with toluidine blue differ with its dark blue color, whereas the polysaccharide capsule was colorless (Fig. 1 e) or pale blue.
The 4th type of cryptococcus cells (» 5%) were distinguished by its light content and absence of the polysaccharide capsule. They are characterized by thin, friable, light, often deformed cell walls, which had caused a large variety of their forms. Toluidine blue staining was clearly revealed only the cell walls which had the light blue color, while the contents of the fungal cells and polysaccharide capsule were not stained (Fig. 1 f).
The 5th types of fungal cells (2%) were in early stages of monocaryotic hypha formation (1 g, arrow). And finally, the 6th type of fungal cells (5%) was in different stages of budding (Fig. 1 h). As a rule, budding yeast cells were dead and contained the fine-fibrillar material, which was similar with slime.
The sizes of the cryptococcus cells, on average was about 7.20 |m. Specific ultrastructural features of the fungal cells of 1st type were the presence of storage substances in the form of fibrosinous bodies (from 0.05 x 0.11 to 6.0 |m) and small (0.20 |m) rosettes (Fig. 1 k, m) of moderate electron density glycogen. Mostly in the cy-tosol we observed only one type of storage substances -fibrosinous bodies. Only in small number of cells we note presence of fibrous bodies with rosettes of glycogen, the number of last one varied from one cell to another (Fig. 1 k, m). It should be noted, that in small number of yeast cells the number of fibrous bodies varied from 3 to 7 (Fig. 1 i), whereas in the dominant its number we observed only one large fibrosinous body, which occupied the all the cell (Fig. 1 j-m). Sometimes in the cytosol of this cells possible revealed the profiles of «incipient» conceived fibrosinous bodies (Fig. 1 i, arrows). The deposition of matrix of this bodies occur between the two (on the section) dark thin (5-6 nm) lamella with high electron density. There are astonished the morphological variety of the fibrosinous bodies, among which possible recognized rod-shaped, lenticular, conical, polygonal (Fig. 1 i), bowl-shaped (Fig. 1 k), irregular (Fig. 1 k), rounded (Fig. 1 l) or V-shaped (Fig. 1 m). Fibrosinous bodies had a homogeneous matrix with the moderate electron density. The outside they delimited with thin (5-6 nm), dark, often discontinuous thin lamella, what was described for fibrosinous bodies of Melampsora lini [Hassan Z.M., Littlefield L.J., 1979]. Previously fibrous bodies were described in the conidia of Podoshaera oxyacanthe (= Erysiphe clandestina) [Zopf W., 1887], in conidia of the species from genus Blumeria and Microsphaera [Niger F.W., 1901; Yarwood C.E., 1973], paraphysis-like cells of Puccinia coronata f. sp. avenae [Harder D.S., 1976], mature teliospores of Gymnosporium juniper-verginianae [Mims C.W., 1977], in the pedicel cells of the uredium of Melampsora lini [1979], in the cell of the uredospore stipes of Physopella zeae [Hearth M.C., Bonde M.R., 1983], in the conidia and sometimes in the conidiog-enous cells of 5 species of the powdery mildew from 3
genus - Erysiphe communis, Microshaera alphitoides, Sphaerotheca pannosa, S. fulgineae, S. mors-uvae [Vasi-lyev А.Е., Kamaletdinova F.I., Jitnikova I.P., 1988]. Note, that the fibrosinous bodies were the rare type of storage substances of the fungal cell. This is the first report about the presence of fibrosinous bodies in the cells of cryptococcus, which infected the human brain. Recently this type of reserve substances has been described in cells of one of the 16 studied in culture conditions strains of C. neoformans var. neoformans [2] and the vegetative mycelium of Aspergillus versicolor [5] also growing in vitro. It is interesting to note, that this type of storage substances we detected in mature cells of strong virulent strain 1175, growing in vitro.
The cytosol of the described type cells of C. neoformans var. neoformans was characterized by moderate electron density with rarely distributed free ribosomes. Sometimes it was possible to observe the aggregations (from 20 to 25 on the cell section) ofparticles, in which size (30-40 nm) and morphology (three-layer membrane vesicles, surrounded by a crown of ribosome-like structures sitting on a short stalk), were similar with the human immunodeficiency virus (Fig. 1 n). It may be possible to suggest the two scenarios for interpretation of this observation: 1) the virus-like particles may penetrate in fungal cell content from the human brain tissue; 2) the virus-like particles may immigrate from content of fungal cell in human brain tissue. In first case, cells of cryptococcus may participate in distribution of HIV. In second case, cells of cryptococcus may be source of HIV and distribute its in human body. But it may be possible to suggest the presence of two this scenario.
The all investigated cells of cryptococcus outside were surrounded by a cell wall, the integral part of which was the so-called «scar» (Fig. 1 q), formed after the separation of the daughter cell from the mother one. The wall thickness of the fungal cells, which were not ingested by macrophages, in average was about 0.80 |m. We have revealed in the cell wall with varied thickness (from 0.3 to 0.7 |m) microfibriles with moderate electron density, forming several layers of different electron density (Fig. 1 p, s). Some yeast cells had a wall with thickness more than 0.7 |m, for which was typical high electron density and the presence of poorly distinguishable microfibrils (Fig. 1 i, k, l, n, o). The reason for the increase of electron density in the cell walls could be the deposition of melanin in them, as it was shown in several works [Nosanchuk J. D., Casadevall A. // Microbiology. - 2003. - Vol. 149; Luchnikof A.V., Abrosimov Ä.Y. - Moscow, 2001, etc.].
Outside the walls in all studied fungal cells were covered with a well-formed polysaccharide capsule, which was easy to identify under light (Fig. 1 d) and electron microscope (Fig. 1 p, s, t, v) considering its large thickness and low electron density. The thickness of the capsule in free lying yeast cells, on average was 9.50 |m, which was in 11.8 times higher than the cell wall and 1.3 times - the average diameter of mature cells.
In general, the analyzed cells developed on its surface a well-formed polysaccharide capsule. The
best preservation of morphological elements of the polysaccharide capsules could be observed in cells of fungal cells of the 1st type. In the composition of the capsules of this cells visible the three distinct layers (Fig. 1 p, s). In light matrix of the first layer revealed a clear network of radially oriented dark microfibriles. The upper boundary of the layer had a slightly wavy contour and the lower contour of the cell wall. The thickness of this layer was on average of 0.30 ± 0.02 mm. The second layer of capsules was dark and homogeneous, its average thickness was about 0.07 |m. In light matrix of 3rd layer, the final layer of polysaccharide capsules, were observed to moderately developed network of microfibrils, which have in the lower one third part -the large vesicular morphology and in the rest one - of the radial. Microfibrils of this layer had rather the large extent: they penetrated the all thickness of the last layer of the capsule. Its thickness from one cell to another varied within wide limits from 0.2 to 0.9|m.
In the work of Vasilyeva N.V. with co-author [4] on the example of mature cells 16 strains of C. neoformans var. neoformans were revealed the changes in the ultrastructure of polysaccharide capsules during in vitro^in vivo transition. Thus, typical for the mature cells of the cultures of the 4 types of polysaccharide capsules in vivo was changed in also 4, but principally different pattern of structure. Moreover, the authors did not find relationship between the structural type of the capsule and virulence of investigated strains.
Described in this paper the structural type of the polysaccharide capsules can be determinates as a variant of the 5th type (Fig. 2 a, b, see text) according to the classification of Vasilyeva N.V. with co-author [64], which was typical for cells of some strains of the fungus, which infects the lungs and brain of murine. A distinctive feature of its structure was the absence in the composition of the polysaccharide capsule of large cells of cryptococ-cus tapered macrofibriles adjacent with the electron-dense homogeneous second layer (Fig. 2 a, arrow).
Fig. 2. Diagram, illustrating the ultrastructure of the polysaccharide capsule of C. neoformans var. neoformans: a - the structure of the polysaccharide capsule of the 5th type in brain of murine (according the Vasilyeva N. V. et al., 2006); b - subtype of capsules of 5-th, which was described in the present work for the cells of C. neoformans var. neoformans infecting the human brain tissue
We revealed the consistent stages of deposition of slime-like material in the periplasmatic space - between the cell wall and the plasmalemma in the 2nd type of
yeast cells (Fig. 1 r), resulting that the plasmalemma force out in the center, and later completely undergo lysis. In our case, slime, which perhaps, synthesize and secreted by the plasmalemma of fungal cells, consisted of densely packed and strongly sinuous fibrils, which gave them a «vesicular» appearance.
The 3rd, most common type ofyeast cells, represented the dead cells, the content of which was filled with fine-fibrillar material like slime (Fig. 1 q), which, in our opinion, allowed them to retain the rounded shape typical for the once-living cells. The cell walls and the contents of the fungal cells of this type after staining with toluidine blue had a dark blue color, whereas the polysaccharide capsule was colorless, which was associated with the lysis of the lower part of its layers. In our opinion, for the cells of this type typical the presence of slime-like material in its content, what in our opinion increased the adsorption of them toluidine blue.
In the fungal cells ofthe 2nd and 3rd types we observed at successive stages of degeneration of polysaccharide capsules, which occur in the following way. Initially, the note changes in the orientation of microfibrils of the 3rd layer with the changes of radial orientation on irregular (Fig. 1 s). Simultaneously occur the thinning of the second dark layer of capsule (Fig. 1 t) until to complete disappearance (Fig. 1 u). Then we noted the decrease of the density of microfibrils distribution in the first layer of the capsule, and a small part of which were extended. At the end we note a complete lysis of microfibrils. Simultaneously with lysis of polysaccharide capsules we reported a decrease of thickness and reduction of the electron density and the gradual lysis and microfibril of the cell wall.
The cells of 4th type were the rarest, as noted above; they did not contain fine-fibrillar material and posses light content and often deformed cell walls. The last peculiarity was caused a large variety of their morphological forms. The cells of this type were usually lose a polysaccharide capsule or have only its small remnants (Fig. 1 v).
Intact cells of cryptococcus in autopsy material of the brain in HIV-infected patient was absent, about what argue the absence in their cytosol of nuclei and often - plasmalemma. Besides, the presence of pictures of budding with involving of the fungal cells without live content, both outside and inside macrophages and the formation of fibrous bodies, was indicate that the population of fungal cells in the moment of antimycotics action was active. Absence in the fungal cells nuclei, mitochondria and often plasmalemma, as well, as other components of the cells, indicate that these processes occurred much earlier than the death of the patient.
Macrophages had dimensions - 5.0-12.0x15.2-25.7 | m, were rounded, ellipsoidal or iregular; with smooth, slightly sinuous or incorrectly folded part of cell periphery. They contained, as a rule, one nucleus (Fig. 3 a, f), which was characterized by variable topography. The most frequently occur macrophages with ellipsoidal nucleus (2.5 x 9.8 |m, Fig. 3 a), more rare pear shaped,
dumbbell or slightly irregular in shape (Fig. 3 f, h). Macrophages with 2 or more nucleous were also rarely observed (Fig. 3 c).
Fig. 3. Ultrastructure of macrophages (a, c, f) and its fragments (b, d, e, g, h) in the human brain. Scale: a = 3 |m; b, c, f = 6 |m; d = 4 |m; g, h = 5 |m
The degree of nucleus chromatization is rather, high. Condensed chromatin dominated, had the appearance of large electron-dense homogeneous blocks of various shapes, mainly localized near the inner membrane of the nucleus envelope. The described chromatin structures, as a rule, developed during apoptosis [Luchnikof A.V., Abrosimov A.Y. - Moscow, 2001]. The nucleolus was one, excentric, spherical (0.60 mm), with high-density, associated with the nuclear envelope through a block of condensed chromatin (Fig. 3 d). Nucleoplasm was electron-light. The envelope of the nucleus consists of two high contrast membranes. In small number of the macrophages, which were in different stages of apoptosis, the inner membrane of the nucleus envelope was formed the hemispherical shape (Fig. 3 a, arrows) invagination.
Inside the macrophages, as a rule, were present from 1 to 8 cells of yeast (Fig. 3 a, c, f, h). From the 52 analyzed macrophages, 31% contained one, 15% - two or three fungal cells, 10% - four or five, 6% - six and seven. And, finally, 7% of the macrophages contained from 7 to 9 cells of cryptococcus. The diameter ingested with macrophages fungal cells varied from 3.5 to 10.0 |m (average 6.7 |m). According to our observations, macrophages ingest only dead fungal cells (Fig. 3 b), about what demonstrated the absence of content, or intact organelles. This fact can be explained by the absence of living cells and among the same, but which
free lying in the pia mater. As exception, we observed the budding cells of cryptococcus, which mainly occur on sections of macrophages with greater its number (up to 7-9 on the median section). However, as in the case of budding free lying cells, they were not intact.
According to our observations, the destructive processes, which we rare observed in the fungal cells inside macrophages, at the first type, mainly concerned the lysis of the reserve substances (with the possible utilization of their decay products by macrophages), as well as free ribosomes, cytosole and residues of membranes of the once intact organelles. We also observe the successive stages lysis of the polysaccharide capsules and destructive changes in the fungal cell wall (Fig. 3 a, g, f). For polysaccharide capsules it were: the reduction of thickness, density and length and localization of microfibrils, the disappearance of layers, fragmentation (Fig. 3 g) and complete they lysis. For the cell wall it was progressive reduction of thickness and the electron density, demela-nization (for cells with dark cell walls), the appearance of local gaps (Fig. 3 h) and strong deformation (Fig. 3 g). Sometimes we observed the extrusion of «debris» of once ingested by macrophages of cryptococcal cells of in the form of fragmented walls with lower contrast (Fig. 3 h, arrow). This last fact also was described during light-microscopic investigation of the cells of C. neoformans, ingested by murine macrophages in vitro condition [6; Tucker S.C., Casadevall A. // Proc. Natl. Acad. Sci. (USA). - 2002. - Vol. 99. and etc].
The cytosol of macrophages with high electron density, rich with macro- (in the average of 1.20 |m) and microvacuoles (in the average of 0.50 mm), which probably may be analogues of primary and secondary lysosomes, as well as numerous, small (0.20 | m), light secretory vesicles (Fig. 3 a, f, arrow), a large part of which could be the result of fragmentation of the cisterns of the endoplasmic reticulum and dictyosome of the Golgi apparatus. In the content of macro- and microvacuoles was observed the accumulations of fine-fibrillar material, the density of localization of which was non-uniform. We often observed the pictures of the contact (Fig. 3 e, arrow) fusion of microvacuoles with a polysaccharide capsule of ingested cells of C. neoformans var. neoformans. In all investigated macrophages we note the absence of limiting their plasmalemma, that according to our opinion, as the fact of clarification of nucleoplasm in interphase nuclei, mitochondrial swelling and disappearance of their crists, fragmentation of elements of the endoplasmic reticulum and dictyosome of the Golgi apparatus, as well as the plasmalemmal lysis may be associated with the beginning of the destructive processes [Luchnikof A.V., Abrosimov À.Y. - Moscow, 2001].
resume
1. The use of paraffin blocks of the autopsy material for subsequent electron microscopic studies were acceptable, in particular, to study the pathogenesis of cryptococcosis of the human brain and revealing the structural peculiarity of fungal cells. They can be used
also for studies of virus-like particles in human tissues of macrophages in the brain tissue of dead patient. We
1. Yamaguchi M., Biswas S.K., Ohkusu M., Takeo K. Dynamics of the spindle pole body of the pathogenic yeast Cryptococcus neoformans examined by freeze-substitution electron microscopy //FEMS Microbiol. Lett. - 2009. - Vol. 296. - P. 257-265.
2. Vasilyeva N.V., Stepanova A.A., Sinitskaya I.A., Semenov VV. Comparative ultrastructural investigations of Cryptococcus neoformans strains with different virulence //Problems of Medical Mycology. - 2005. - Vol. 7, №2. - P. 99.
3. Vasilyeva N.V., Stepanova A.A., Sinitskaya I.A. Electron-microscopic investigations of developmental biology in the cells of lower and higher virulent strains of the Cryptococcus neoformans in vitro and in vivo // Problems of Medical Mycology. - 2007. - Vol. 9, №2. - P. 47-48.
4. Vasilyeva N.V., Stepanova A.A., Sinitskaya I.A. Ultrastructure of capsules of the mature cells of the Cryptococcus neoformans in vitro and in vivo // Problems of Medical Mycology. - 2006. - Vol. 8, №2. - P. 25.
5. Stepanova A.A., Sinitskaya I.A. Cytology of the cells of vegetative mycelium of the Aspergillus versicolor, growing in vitro // Problems of Medical Mycology. - 2006. - Vol. 8, №3. - P. 22-28.
6. Alvarez M., Casadevall A. Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages // Curr. Biol. - 2006. - Vol. 16, №2. - P. 2161-2165.
Поступила в редакцию журнала
Рецензент: С.М. Игнатьева
