Reactivity of Langerhans cells after application of different chemicals--an ultrastructural analysis.

The goal of this study was to investigate changes in epidermal Langerhans cells after application of different chemicals (acetone, 60% alcohol, 5% nickel sulphate, iodisole, and 0.1% 2,4-dinitrochlorobenzene) on the skin of volunteers. The skin of eight volunteers was treated with acetone, 60% alcohol, iodisol, 5% nickel sulphate, and 0.1% 2,4-dinitrochlorobenzene (DNCB). After application of DNCB, Langerhans cells (LCs) showed increased accumulation of Birbeck granules (Bgs). Alcohol and nickel sulphate caused alternative changes, mainly cytoplasmic vacuolation, in LCs. Nickel sulphate was even responsible for the disappearance of dendrites. Both chemicals have cytotoxic effects on LCs: cytoplasmic organelles and Bgs disappear and subsequently, the antigen-presenting activity of epidermal LCs is inhibited. We did not found any morphological changes in LCs after application of acetone.

The role of electron microscopy in the rapid diagnosis of viral infections--review.

Electron microscopy (EM) allows fast visualization of viruses in a wide range of clinical specimens. Viruses are grouped into families based on their morphology. Viruses from various families look distinctly and these morphological variances are the basis for identification of viruses by EM. The identification to the family level is often sufficient for the clinician or recognition of an unknown infectious agent. Diagnostic EM has two advantages over enzyme-linked immunosorbent assay and nucleic acid amplification tests. After a simple and fast negative staining, EM allows fast morphological identification and differential diagnosis of infectious agents contained in the specimen without the need for special considerations and/or reagents. Nevertheless, EM has the disadvantage of being unsuitable as a screening method.

Interaction of Borrelia burgdorferi sensu lato with Epstein-Barr virus in lymphoblastoid cells.

Since the possibility of interruption of latent EBV infection has been suggested by the induction of the lytic virus cycle with chemical substances, other viruses, and by immunosuppression, we hypothesized that the same effect might happen in B. burgdorferi sensu lato infection as happens in Lyme disease patients with positive serology for both agents. We have observed EBV replication in lymphoblastoid cells after superinfection with B. garinii and B. afzelii strains after 1 and 4 h of their interaction. We found that viral and borrelial antigens persisted in the lymphoblasts for 3 and 4 days. Morphological and functional transformation of both agents facilitate their transfer to daughter cells. Association with lymphoblasts and internalization of B. garinii by tube phagocytosis increased replication of viruses more successfully than B. afzelii and chemical inductors. Demonstration of such findings must be interpreted cautiously, but may prove a mixed borrelial and viral cause of severe neurological disease.

9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP)--a potential drug against hematological malignancies--induces apoptosis.

Antitumor activity of the acyclic nucleotide analogs PMEDAP, PMEA, and PMEG was studied on a model of a spontaneous T-cell lymphoma in inbred SD/cub rats. Significant therapeutic effects were recorded after a treatment with 16 daily doses of PMEDAP at 5 mg/kg applied to the vicinity of the growing lymphoma. Identical administration of PMEA, or PMEG at a daily dose of 0.1 mg/kg did not affect the survival of lymphoma-bearing animals compared with untreated controls. A decrease in the lymphoma weight during PMEDAP administration was accompanied by the suppression of mitotic activity in neoplastic cells and increased chromatin condensation as witnessed by karyological examinations. Electron-microscopy showed the morphology of apoptotic cells (shrunken cells with condensed chromatin, apoptotic bodies) in lymphoma cell suspensions. An increase of nuclear DNA fragmentation was found during PMEDAP administration compared with spontaneous DNA fragmentation of untreated control lymphomas. These results indicate that PMEDAP application induces apoptosis in in vivo growing lymphomas. The antitumor effect of PMEDAP lasts only during the administration of the drug. After its cessation progression of neoplasia was reestablished.

Therapeutic effect of heat shock on T-cell lymphoma in inbred Sprague-Dawley rat.

Anticancer effect of heat shock, either alone or in combination with the drug PMEDAP, and cold water immersion stress were studied in an in vivo model of s.c. transplanted rat T-cell lymphomas in an inbred Sprague-Dawley rat line (SD/cub). Significant anticancer effect was induced by repeated sessions of heat shock; decrease of s.c. lymphoma weight and prolongation of survival time of treated rats was found to be dependent on the number of HS sessions. Much stronger therapeutic effect was observed after repeated heat shock in combination with PMEDAP administration. Light and electron microscopy studies were performed to characterize the alterations within the lymphomas. Morphologically, cellular alterations corresponding with apoptosis were observed in lymphoma cells after repeated heat shock. Indirect immunoperoxidase technique was used to detect HSP 72/73 protein(s), p53 and Bcl2 proteins in lymphomas heated directly or indirectly. The induction of HSP 72/73 protein(s) was found in the lymphoma tissues from autopsied animals exposed to heat shock; the intensity of its expression was dependent on the experimental design. The expression of p53 and BcL2 proteins was not changed in lymphoma cells of HS treated animals as compared to that of untreated lymphoma bearing controls; the Bcl2 protein was present in both treated and untreated lymphomas, and the p53 protein remained undetectable in all samples. Contrary to the heat shock, the cold stress did not suppress growth of lymphomas and, furthermore, accelerated the infiltration of parenchymatous organs with lymphoma cells.

Polymerase chain reaction for detection of guinea pig adenovirus.

Lack of in vitro cultivation methods has inhibited the development of rapid, reliable diagnostic procedures for adenovirus-associated necrotizing bronchopneumonia in guinea pigs. Because polymerase chain reaction (PCR) techniques are well established for human adenoviruses, primers for the amplification of guinea pig adenovirus DNA were evaluated. The DNA for PCR was purified from the lung tissue of spontaneously infected and healthy guinea pigs. Adenovirus DNA could only be detected in the lungs of the infected animals. Subsequent sequence analysis of PCR products revealed that the guinea pig adenovirus is a distinct adenovirus.

Treatment of transplanted spontaneous rat T-cell leukaemia with local administration of recombinant murine interleukin-2.

Spontaneous rat CD4+CD8-T-cell leukaemia transplanted in syngeneic recipients served as an experimental model system for IL-2 therapy. As a source of IL-2, supernatants from in vitro cultured plasmacytoma cell line X63-m-IL2 secreting constitutively recombinant murine IL-2 were utilized. Administration of IL-2, s.c. to the vicinity of the tumour inoculum, suppressed tumour growth. The tumour-inhibitory IL-2 effects were time- and dose-dependent. When the treatment has started 10 days after the challenge with 10(4) leukaemia cells, IL-2 inhibitory effects on the lymphoma growth in situ were demonstrated by lower tumour weight combined with necrotic changes. No histological signs of lymphoma generalization were found in parenchymatous organs of IL-2-treated rats in contrast to the untreated controls. No histological or functional injuries to the kidneys due to IL-2 administration were found. The results of effector cell phenotyping demonstrated the kinetics of the CD4+/CD8+ ratio characterized by CD4+ T-cell depletion and resulting increase in the percentage of CD8+ PBL.

Electron microscopic demonstration of the interaction of liposomes and cells in vitro.

We have investigated the interaction of liposomes with the continuous cell lines P388D1 and L-132 and mouse peritoneal macrophages. To distinguish the liposomes from other vesicular structures, we have used liposomes with incorporated protein G and gold. A heterogeneous mixture of multilamellar liposomes 30 nm up to 1000 nm in size has been employed. Samples were examined at different time intervals. We found differences in the uptake of liposomes with regard to size and rate. Cells of a P388D1 monolayer took up liposomes by endocytosis very early after addition of liposomes and the number of lysosomes in their cytoplasm increased. In L-132 cells, first a fusion occurred between liposomes and the cell cytoplasmic protrusions, in the cytoplasm of which the mitochondria had multiplied. Peritoneal macrophages phagocytosed mainly large multilamellar liposomes and the membranous system of Golgi apparatus was the most prominent structure in the cytoplasm. Phagocytosis in P388D1 and L-132 cells was noted sporadically as late as 24 h after addition of liposomes to the cells.

Electron microscopic demonstration of the penetration of liposomes through skin.

To verify the penetration of liposomes through skin, we have used liposomes with encapsulated protein G-gold conjugate in a gel vehicle. Skin samples were examined 2, 4, 24, 48, and 72 h after liposome application. Our findings show that the penetration of liposomes through skin depends mainly on their size. Liposomes up to 600 nm in diameter penetrate through skin rather easily, whereas liposomes 1000 nm and more in diameter remain interiorized in the stratum corneum. The main penetration of liposomes proceeds along the hair sheaths as indicated by larger amounts of free liposomes in the corium of guinea pigs.

The effect of an antiphlogistic incorporated in liposomes on experimentally induced inflammation.

The antiphlogistic Ibuprofen incorporated in liposomes caused a decrease of the inflammatory edema induced by Carrageenan in the distal part of the rat's hind leg after both the intramuscular and percutaneous administration. The antiphlogistic effect of free Ibuprofen in the cream was weaker. Intramuscular administration of empty liposomes slowed down in the initial stages the development of inflammation and slightly diminished the size of edema.

Interaction of virulent and non-virulent Yersinia enterocolitica strains and an epithelial and a phagocytic cell lines.

Morphological alterations of HEp-2 and P338D1 cells were detected as result of Yersinia virulent strains action only. A non-virulent strain caused none of these alterations even 24 h post infection (p.i.). The internalization of the bacteria was demonstrated by double fluorescence staining. Adherence and beginning of cell invasion of virulent strains was detected 30 min p.i. already. Two hours p.i. these bacteria were in great numbers inside the cells of both lines. The non-virulent Yersinia strain was found only in the P338D1 macrophages at 2 and 24 h p.i. but in smaller numbers than virulent strains. Electron microscopy confirmed that internalization of virulent strains Y526 and Y527 was done by the phagocytosis of both cell lines. Even intracellular replication of these virulent strains was observed in both cell lines at 2 and 24 h p.i. Both bacterial strains were disintegrated as well as they multiplied inside the cells. In strain Y526 disintegration of bacteria prevailed, whereas their replication predominated in the strain Y527. At 24 h p.i. cells infected with strain Y527 were sac-like, with rudiments of the cytoplasm and organelles, packed with bacteria that were released after cell membrane rupture. Cells infected with strain Y526 were metabolically active and even at 24 h p.i. predominately contained disintegrated bacteria, but even in this case replication and release of bacteria was observed.

[Construction of recombinant vaccinia viruses]. / Konstrukce rekombinant viru vakcínie.

Expression system based on vaccinia virus (VV) is used both for recombinant protein production in vitro and as an alive vaccine. The article summarizes various strategies for recombinant VV construction, and describes preparation of recombinants expressing various forms of S and C genes of hepatitis B virus (HBV). It is shown, that the CV-1 cells infected with these recombinants synthesized the MS (middle) and the LS (large) polypeptides of the surface antigen (HBsAg) and the nucleocapsid antigen (HBcAg) polypeptide and the polypeptide HBeAg. Posttranslation modification of all expressed proteins was the same as at HBV infection.

Electron-microscopic demonstration of virus particles in acute lymphoblastic leukaemia in Sprague-Dawley rats.

Retrovirus-like particles were detected by the negative staining method in supernatants of lymph node and spleen cell suspensions prepared from Sprague-Dawley rats with spontaneous acute lymphoblastic leukaemia (ALL). Similar particles were found in supernatants of cell suspensions from a lymphoma that developed after inoculation of lymph node and spleen cell suspension prepared from animals with spontaneous ALL into the subcutis of Sprague-Dawley recipients. On ultrathin sections, budding forms of the virus particles were seen as a dot at the periphery of neoplastically transformed cells.

Electron microscopy of Langerhans cells and Borrelia burgdorferi in Lyme disease patients.

To investigate dermal and epidermal involvement in the presence of Borrelia burgdorferi and to analyze the role of Langerhans cells and keratinocytes, 14 cases of erythema chronicum migrans and two controls were studied by means of electron microscopy, using negative staining and sectioning techniques. Using immunoelectron microscopy and histochemistry, positive results for B. burgdorferi were disclosed in 5 cases of erythema chronicum migrans and 3 cases of neuroborreliosis which were confirmed by cultivation. We cultured 4 stains of B. burgdorferi from the skin, 1 from blood and 2 from cerebrospinal fluid in BSK medium. Near to the centre of erythema chronicum migrans with focal necrosis were both a dissolved basal membrane and keratinocyte desmosomes surrounding damaged B. burgdorferi cells in the epidermis. Markedly oedematous keratinocytes and Langerhans cells with B. burgdorferi were released into lymphocyte infiltrates. At the periphery of all erythema chronicum migrans lesions, keratinocytes were well preserved while all dendritic cells seemed to be vacuolated. Above foci of B. burgdorferi located perivascular or among collagen fibers, Langerhans cells were frequent and more granulated. The possible role of Langerhans cells in the identification and elimination of B. burgdorferi is discussed.

Vaccinia virus recombinants co-expressing hepatitis B virus surface and core antigens.

Using the Praha strain of vaccinia virus (VV) two double recombinant VVs expressing the surface and capsid HBV proteins (HBsAg and HBcAg) under the control of the P7.5 promoter were constructed. In the first construct the gene coding for HBsAg was inserted into the HindIII J fragment (TK gene) and the gene coding for HBcAg was inserted into the HindIII M fragment (host range, K1L gene) of the VV genome. To test whether the expression of the foreign genes was influenced by the insertion site, in the second construct their locations were inversely changed. When compared with single VV-HBV recombinants expressing either HBsAg or HBcAg, the double recombinants expressed in vitro approximately the same amounts of the respective antigens. The particles formed by either HBsAg or HBcAg expressed by recombinant viruses, were isolated and examined by electron microscopy. Particles composed of both HBsAg and HBcAg were not detected in cultures infected with one of the double recombinants. The residual virulence in 3-week-old mice of the single recombinants was not markedly altered by the insertion of the second gene. The immunogenicity in mice of both the single and double recombinants was comparable and was not influenced by the location of the HBV genes in VV genome, as revealed by antibodies developed against the respective antigens.

Cytostatic effect of 9-(2-phosphonomethoxyethyl) adenine (PMEA). II. Lymphoblastic leukemia in Sprague-Dawley rats.

Two cases of spontaneous acute lymphoblastic leukemia in the inbred strain of Sprague-Dawley (Prague) rats have been observed. Since its reliable transplantability in syngeneic recipients was established, leukemic animals were used to test the cytostatic effect of PMEA. The treatment resulted in a significant prolongation of survival time of the treated animals. At the same time histological examination of PMEA-treated and untreated animals indicated that the drug effectively slows down the growth of lymphoma at the site of inoculation and inhibits the subsequent progression of tumor cells in the lung, liver, spleen and lymph nodes.

Some aspects of the presence of surface--associated acid mucosubstances on larval Taenia saginata infecting suitable and unsuitable hosts.

The coat of acid mucosubstances occurring on the bladder surface was studied in Cysticercus bovis (larvae of Taenia saginata) developing in specific (cattle) and nonspecific (some other ruminants) intermediate hosts. In cysticerci developing in nonspecific intermediate hosts, this coat is absent or poorly developed even at the time when it is completely formed on the bladder surface of C. bovis from cattle. If the cysticercus develops up to the infective stage in a nonspecific intermediate host, it is usually localized outside the muscles: in the brain, lungs or liver. Its bladder is then also covered by a coat of acid mucosubstances. The absence or insufficient development of the protective coat on larvae in nonspecific intermediate hosts is explained by their early and strong tissue reaction.

Cysticercus bovis: pinocytosis in the Cysticercus tegument.

The authors have found that pinocytosis occurs in the tegument of C. bovis from the fourth week after infection. Electron-lucid bladders surrounded by plasma membrane were encountered in the distal cytoplasm. The pinocytosis occurred in form of micropinocytotic bladders only in the bladder tegument but not in the scolex. The bladders appeared first in the superficial part of the distal cytoplasm. During the following periods of development of the larva they were dispersed in the whole distal cytoplasm and were found even in the processes of subtegumental cells and inside these cells near the heterolysosomes.

Pathology of experimental Cysticercus bovis infection in the reindeer (Rangifer tarandus Linné, 1758).

In the present study, the tissue reaction of the brain, skeletal muscles and heart in experimental C. bovis infection of the reindeer is described. There is non-purulent cysticercal leptomeningites with formation of multinucleate symplasms in the cerebral meninges, and lymphocytic encephalitis in the superficial layers of the cerebral cortex. The tissue reaction around the morphologically differentiated cysticercus in the meningeal location is similar to that in muscle cysticercosis of cattle. In muscles and heart, larvae die very soon after infection and they are resorbed in the course of formation of fibroplastic granulation tissue around them. C. bovis reaches the infective stage in cerebral localization only. The authors suppose that this phenomenon is due to a certain degree of immunological tolerance in the brain.