[Applications of fluorescent semiconductor nanocrystals in microscopy and cytometry].

Quantum dots (QD) nanocrystals consisting of CdSe core with ZnS shell are a novel class of fluorophores with tremendous potential in microscopy and cytometry techniques. The unique optical features of Qdots, namely, high photostability and extinction coefficient, wide absorption and narrow emission spectra, and large Stokes shift make them desirable fluorescent tags for diverse biomedical applications. Applications of this novel technology in microscopy and cytometry produce reliable multicolor specimens due to increased photostability, ability for multiplexing and narrow emission spectra of nanocrystals. QD conjugates are available on the market and could be prepared in the laboratory. This paper describes the application of QD-conjugates for immunophenotyping and FISH assessment of cells and tissues, and the requirements for microscope and flow cytometer reengineering for successful use of QD in multiplex fluorescent format. Despite the considerable progress, important methodological issues still need to be solved in terms of QD nanocrystals' size, heterogeneity, functionalization and stability of their conjugates. We discuss practical approaches and challenges that need to be addressed to make QD immunostaining a standard method in biology.

Differential stimulation of monocytic cells results in distinct populations of microparticles.

BACKGROUND: Microparticles (MPs), small vesicles shed from stimulated cells, permit cross-talk between cells within a particular environment. Their composition is thought to reflect their cell of origin, and differs according to whether they are produced by stimulation or by apoptosis. Whether MP properties vary according to stimulus is not yet known. METHODS: We studied the characteristics of MPs produced from monocytic THP-1 cells upon stimulation with lipopolysaccharide or a soluble P-selectin chimera, using proteomics, flow cytometry, western blotting, and electron microscopy. RESULTS: Utilizing a novel criterion of calcein-AM staining to define MPs, we found that MP populations were similar with respect to size, presence and organization of cytoskeleton, and expression of certain antigens. The MPs shared the same level of procoagulant activity. We found that MPs also have distinct characteristics, depending on stimuli. These include differences in phosphatidylserine expression and expression of proteins from specific subcellular locations such as the mitochondria, and of unique antigens such as leukocyte-associated immunoglobin-like-receptor (LAIR)-1, which was found only upon stimulation with the soluble P-selectin chimera. CONCLUSION: We found that the properties of MPs depend on the stimulus that produced them. This supports the concept that monocytic MPs differentially modulate thrombosis, inflammation and immune regulation according to stimulus.

Possible role of natural killer cells in pemphigus vulgaris - preliminary observations.

Pemphigus vulgaris (PV) is an autoimmune blistering disease that affects the skin and multiple mucous membranes, and is caused by antibodies to desmoglein (Dsg) 1 and 3. Natural killer (NK) cells have a role in autoimmunity, but their role in PV is not known. NK cells in the peripheral blood leucocytes (PBL) of 15 untreated Caucasian patients with active PV were studied and compared with healthy controls for the expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules. CD56+ CD16- CD3- NK or CD56+ CD16+ CD3- NK cells from the PBL of PV patients co-express MHC class II and co-stimulatory molecule B7-H3 without exogenous stimulation. CD4+ T cells from the PBL and perilesional skin of PV patients were co-cultured with CD56+ CD3- NK cells from the PBL of the same patients; in the presence of Dsg3 peptides underwent statistically significant proliferation, indicating that NK cells functioned as antigen-presenting cells. Supernatants from these co-cultures and serum of the same patients with active PV had statistically significantly elevated levels of interleukin (IL)-6, IL-8 and interferon-gamma, compared with controls indicating that the NK cells stimulated CD4+ T cells to produce proinflammatory cytokines. In these experiments, we present preliminary evidence that NK cells may play a role in the pathobiology of PV.

Role of nitric oxide in the inhibition of cytochrome P450 in the liver of mice infected with Chlamydia trachomatis.

In this study, we attempted to determine the effect of a systemic infection with Chlamydia trachomatis on cytochrome P450(CYP)-dependent metabolism in mice. Furthermore, we wanted to assess if these effects were mediated through NO. BALB/c(H-2d) female mice were inoculated intraperitoneally with the C. trachomatis mouse pneumonitis (MoPn) biovar, and induction of NO synthase (NOS) was detected by measuring [NOx] levels and inducible NOS protein content in peritoneal macrophages by Western blotting. Recovery of C. trachomatis from liver, lung, and spleen peaked at 4 days postinfection. Following cotreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, there was a significant increase in the intensity and the length of the infection. Six days after inoculation with C. trachomatis, CYP1A- and CYP2B-mediated metabolism in the liver of the mice was diminished up to 49% of control levels. However, when animals were treated with N(G)-nitro-L-arginine methyl ester at days 4 and 6 postinfection, the decrease in the metabolism of CYP1A and CYP2B was largely blocked. These results suggest that C. trachomatis infection can depress cytochrome P450 in a manner similar to other types of infections and that NO is likely to be a mediator of this depression. This finding may be of significance to patients taking drugs that are metabolized by phase I enzymes during infections with some bacteria such as C. trachomatis.

Role of neutrophils in controlling early stages of a Chlamydia trachomatis infection.

Inoculation of mice with the granulocyte-depleting monoclonal antibody RB6-8C5 showed that neutrophils are critical protective effector cells during a Chlamydia trachomatis infection. In addition, administration of monoclonal antibody 2E6 demonstrated that extravasation of neutrophils into the peritoneal cavity in response to inoculation with the C. trachomatis MoPn biovar is dependent on the surface beta-2 integrin molecule CD18.

A modified PCR-based method for rapid non-radioactive detection of clinically important pathogens.

We have devised a sensitive and rapid method for the detection of several bacterial pathogens in clinical specimens using PCR. This method has been named Direct Labeling and Detection Procedure (DLDP) and is based on the direct incorporation of a nonradioactive digoxigenin label (DIG-11-dUTP) into a microbial species-specific gene fragment during amplification. Following amplification, the resulting PCR products are cleansed of nonincorporated DIG-11-dUTP, spotted onto a nylon membrane, fixed by UV-crosslinking and the labeled DNA is visualized by digoxigenin detection reagents. Using cultivated reference bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa) we were able to demonstrate a rapid and sensitive detection of < 20 CFU of bacteria in human secretions (sputum, urine, mucous). The present study suggests that DLDP can be used as a reliable method for indication of bacteria in clinical or environmental specimens with the proviso that the selected corresponding oligonucleotide primers provide amplification of strong species-specific genes.

Overexpression of Gs alpha subunit in thyroid tumors bearing a mutated Gs alpha gene.

Point mutations occurring at codon 201 of the gene coding for the alpha subunit of the stimulatory G protein impair intrinsic GTPase activity and lead to a constitutive activation of adenylate cyclase. We have examined thyroid follicular and papillary carcinomas and follicular adenomas and found samples that bear this mutation at codon 201 of the Gs alpha gene. Both mutation-positive and mutation-negative tissue samples were investigated for the level of Gs alpha expression relative to a pool of normal thyroid tissue, using immunoblotting against two (mid-region-specific and C-end-specific) antipeptide antibodies. Using 8000 g and 100,000 g membrane fractions of homogenized tissues we have demonstrated that the Gs alpha proteins in normal ad neoplastic thyroid tissues are represented by three isoforms: 43 kDa, 45 kDa and 52 kDa. We have quantified and compared the amount of Gs alpha protein and find it is overexpressed in mutation-bearing tissue samples.

[Effect of the lymphocytosis-stimulating factor from Bordetella pertussis on murine lymphoid and hemopoietic cells]. / Vliianie limfotsitozstimuliruiushchego faktora Bordetella pertussis na limfoidnye i krovetvornye kletki myshei.

Effect of B. pertussis lymphocytosis-promoting factor (LPF) on the lympho-hematopoietic system of mice was studied. The injection of LPF was shown to sharply enhance endogenous colony formation and to induce a severe depletion of thymus cells, reaching its maximum of day 4. Thymocytes obtained on day 2 or 3 after the injection of LPF produced a suppressive effect on endogenous colony formation. The proliferative activity of hematopoietic stem cells sharply increased under the influence of LPF, though it had no radioprotective action. On the following day after the injection of LPF a steep rise in the number of hematopoietic stem cells was observed in the blood of mice: their content increased 20-fold in comparison with the control level. These data may be important for the evaluation of the side effects of pertussis vaccine on the lympho-hematopoietic system.

[Immunologic analysis of the pathology developing in mice as a results of intrauterine infection with the influenza virus]. / Immunologicheskii analiz patologii, razvivaiushcheisia u myshei v rezul'tate vnutriutrobongo zarazheniia virusom grippa.

The progency of C57BL/6 mice consisting of three groups: with signs of slow influenza infection ("dwarf"), "nude-like" resembling nude mice, and "nude-like" with spontaneous fur growth, was examined. The slow influenza infection in "dwarf" mice was found to be characterized by marked immunosuppression manifested by a sharp reduction of the number of antibody- and rosette-forming cells and blasttransformation of spleen lymphocytes into T- and B-mitogens. The most marked immuno-suppression was found in the "dwarfs" born to the females infected with the virus enriched with standard virions. "Nude-like" animals also had marked immunosuppression (particularly with regard to the rosette-formation), however, the "dwarfs" appeared to have more marked affection of B-cells as compared with "nude-like" mice. Gradual restoration of fur in a portion of "nude-like" animals (spontaneous growth) was due to sharp stimulation of immune responsiveness in them as manifested by a two-fold (as compared with the controls) increase in the number of antibody- and rosette-forming cells and normalization of spleen cell response to T- and B-mitogens. Differences between nude and "nude-like" mice consisting in the latter in the affection of not only T- but also B-link of immunity are discussed.

[Stimulation of postradiation regeneration of erythropoiesis in mice with Bordetells pertussis vaccine]. / Stimuliatsiia postradiatsionnoi regeneratsii éritropoeza u myshei pod deistviem vaktsiny Bordetella pertussis.

Inoculation into mice of killed B. pertussis vaccine (10(10) microbial cells) one day before their sublethal irradiation (6.0 Gy) was accompanied by accelerated regeneration of erythropoiesis in the bone marrow, particularly in the spleen as was judged by the 59Fe incorporation. B. pertussis also induced an increase in endocolonization when inoculated 4-5 days after irradiation. The latter suggests a possible effect of vaccine on the hematopoietic cells, less differentiated than erythropoietin-sensitive cells (ESC), inasmuch the sensitivity of the ESC to erythropoietin commonly appeared at the later stages. When B. pertussis was inoculated into BALB/c mice one day before their infection by Rauscher's leukemia virus noticeable activation of leukemogenicity was observed. It is believed that the reason for this is the stimulation of erythroid target cells for the virus after B. pertussis vaccination. The data obtained indicate that B. pertussis vaccine activates erythropoiesis in both normal and irradiated mice.

[Proliferation of spleen cells in response to Bordetella pertussis]. / Proliferatsiia kletok selezenki v otvet na Bordetella pertussis.

Vaccine strain 305 of B. pertussis in a dose of 10(8)-10(11) cells was shown to be mitogenic for splenocytes of BALB/c mice and nude mice. When added in a dose of 10(10) B. pertussis exerted a more pronounced mitogenic effect than phytohemagglutinin P, which was less powerful, however, than that of Con A, B. pertussis caused a greater stimulation of DNA synthesis in lymphocytes than B mitogens whose action depended on the differentiation stages of B lymphocytes. This is likely to hint towards a possible action of B. pertussis on immature B cells and/or their precursors. The cells of T lineage (T1 cells and/or T precursors) can also be involved.

[Action of Bordetella pertussis vaccine on mouse hematopoietic stem cells]. / Deistvie vaktsiny Bordetella pertussis na stvolovye krovetvornye kletki myshei.

Intravenous inoculation of killed Bordetella pertussis vaccine into BALB/c, CBA, C57Bl/6 mice or (CBA x C57Bl/6) F2 hybrids 1 day or 3 days before sublethal irradiation (5.5 Gy) was shown to sharply increase the endogenous colony formation in the spleen 9 days after irradiation. Moreover, the CFUs content of the spleen and bone marrow was also enhanced 1 and 3 days after vaccination of the mice with 10(10) cells B. pertussis as revealed by the exocolonization technique (Till and McCulloch). Thus, a single injection of B. pertussis vaccine hastened the hematopoietic recovery after irradiation.