Natural killer-like activity induced in spleen by IP injection of BCG

Summary Cytotoxic cells comparable to NK cells were induced in the spleens of C57B1/6 mice after IP injection of BCG. This activity was directed against target cells that were not spontaneously lysed by C57B1/6 splenocytes. Various procedures for identification of the effectors indicated that two different types of cytotoxic activity were involved.     

Lysis of murine macrophages by lymphokine-activated killer-like cells induced by BCG in vitro.

The lymphokine-activated killer (LAK)-like activity was found to be induced in mouse splenocytes cultured together with Bacillus Calmette-Guérin (BCG). The killer cells induced by BCG were capable of killing both NK-sensitive (YAC-1, P388D1) and NK-resistant (P815) tumor cells. As an important finding, they also lysed syngeneic macrophages (M phi). The anti-M phi killer activity appeared on day 2, and reached a peak on day 5 of culture. Phenotype analysis of the killer cells by depletion techniques using monoclonal antibody (mAb) and complement indicated that the majority of these anti-M phi killer cells were Thy-1+ and asialo GM1+. This M phi cytolysis could be inhibited by the addition of cold M phi, YAC-1 tumor cells, and P815 tumor cells, suggesting that the same population of the effector cells recognize M phi and tumor cells. The addition of anti-MHC class I, anti-MHC class II, anti-L3T4, or anti-Ly-2 mAb directly to assay cultures did not affect anti-M phi cytolysis, suggesting that the MHC molecules are not involved in the cytolysis of M phi by the BCG-induced killer cells. The addition of anti-LFA-1 mAb partially inhibited the cytotoxicity, suggesting importance of the contact between targets and effectors in the cytolysis. Our present data suggest that activation of murine lymphocytes with BCG induces LAK-like cells capable of killing a wide variety of tumor cells as well as M phi and this anti-M phi cytolysis is mediated by nonspecific killer cells.     

In vitro augmentation of natural killer activity of peripheral blood cells from cancer patients by a DNA fraction from Mycobacterium bovis BCG

Effects of a DNA-rich fraction from Mycobacterium bovis BCG (MY-1) on the natural killer (NK) activity of peripheral blood lymphocytes (PBL) from healthy donors and cancer patients were studied in vitro. The NK activity of PBL was assessed after incubating PBL for 24 hr in the presence or absence of MY-1 or that digested preliminarily with RNase or DNase. One microgram per ml of MY-1 or that digested with RNase augmented the NK activity of PBL from healthy donors. The activity of MY-1 was abolished by the digestion with DNase. Similarly, the NK activity in all of six patients with gastric cancer, 12 patients with colonic cancer, and six patients with uterine cancer was augmented by incubation with MY-1 (1 microgram/ml and 10 micrograms/ml), although the degree of augmentation varied depending upon the origin of PBL.     

Purification of natural killer-like Kurloff cells and arachidonic acid metabolism.

Pulmonary and splenic Kurloff cells have been purified from estrogen-treated guinea pig. Enzymatic digestion of lung tissue and mechanical dispersion of cells yielded about 650 x 10(6) viable cells. After centrifugal elutriation and centrifugation on continuous Percoll gradient, a population of high-density (1,100 g/ml) pulmonary Kurloff cells were obtained with high viability (approximately 99%) and purity (approximately 99%). Splenic Kurloff cells have been isolated by disruption of spleen tissue and centrifugation on continuous Percoll gradient. High-density splenic Kurloff cells (150 x 10(6) cells per spleen) were also obtained with high purity (approximately 99%) and viability (approximately 99%). Pulmonary and splenic Kurloff cells were incubated with various concentrations of arachidonic acid (10, 30 and 100 microM) in the absence or presence of 2 microM ionophore A23187. With 10 microM arachidonic acid the relative production of cyclooxygenase products was the following: TxB2 greater than PGE2 approximately PGI2. For an arachidonic acid concentration superior to 10 microM, the profile of release was PGE2 much greater than TxB2 greater than PGI2. Arachidonic acid metabolism through the 5-lipoxygenase pathway was also studied by incubating pulmonary or splenic Kurloff cells with 10 microM arachidonic acid in the absence or presence of 2 microM ionophore A23187, or in some experiments, with 2.5 microM leukotriene A4. Reverse phase HPLC profiles clearly indicated that high-density Kurloff cells did not express 5-lipoxygenase activity. However, these cells showed the ability to convert exogenous leukotriene A4 into leukotriene B4 suggesting the presence of LTA4 hydrolase activity. These data have been confirmed by a sensitive RIA method. This study constitutes the first report on the purification of pulmonary Kurloff cells and on arachidonic acid metabolism by these cells. The possible implications of Kurloff cells in various biological events are discussed.     

In situ entry of oligonucleotides into brain cells can occur through a nucleic acid channel

Brain tissue has become a challenging therapeutic target, in part because of failure of conventional treatments of brain tumors and a gradually increasing number of neurodegenerative diseases. Because antisense oligonucleotides are readily internalized by neuronal cells in culture, these compounds could possibly serve as novel therapeutic agents to meet such a challenge. In previous in vitro work using cell culture systems, we have demonstrated that intracellular delivery requires a vector such as cationic liposomes since free oligonucleotides remain largely trapped in the endocytic pathway following cellular uptake. Here we studied the cellular uptake properties of oligonucleotides by explants of rat brain (brain slices), and by in vivo brain tissue after administration of oligonucleotides by bolus injection. In contrast to in vitro uptake, we show that in brain slices oligonucleotides were taken up by neuronal and nonneuronal cells, irrespective of their assembly with cationic liposomes. In either case, a diffuse distribution of oligonucleotides was seen in the cytosol and/or nucleus. Uptake of oligonucleotides by brain slices as a result of membrane damage, potentially arising from the isolation procedure, could be excluded. Interestingly, internalization was inhibited following treatment of the tissue with antibody GN-2640, directed against a nucleic acid channel, present in rat kidney cells. Our data support the view that an analogous channel is present in brain tissue, allowing entry of free oligonucleotides but not plasmids. Indeed, for delivery of the latter and accomplishment of effective transfection, cationic lipids were needed for gene translocation into both brain slices and brain tissue in vivo. These data imply that for antisense therapy to become effective in brain, cationic lipid-mediated delivery will only be needed for specific cell targeting but not necessarily for delivery per se to accomplish nuclear deposition of oligonucleotides into brain cells and subsequent down-regulation of disease-related targets.     

In vitro augmentation of macrophage-activating-factor release from peripheral blood cells of cancer patients by a DNA fraction from Mycobacterium bovis BCG

Effect of a DNA-rich fraction from Mycobacterium bovis BCG (MY-1) on the macrophage-activating-factor (MAF)-activity release from peripheral blood lymphocytes (PBL) of patients with benign diseases and patients with gastric cancer or colorectal cancer was studied in vitro. The MAF activity was augmented by the addition of 10 micrograms/ml of MY-1 in many cases. Augmentation of the MAF activity in benign disease, gastric cancer and colorectal cancer in stages I to III was observed in five of six cases, eight of 10 cases and five of eight cases, respectively. The activity of MY-1-induced MAF was partially abolished by the treatment with monoclonal anti-interferon-gamma (IFN-gamma) antibody. These results suggest that MY-1 augments MAF-activity release from PBL by inducing IFN-gamma and other MAF-like substances.     

In situ detection of autoanti-idiotype antibody-forming cells induced by influenza virus infection.

In situ immunocytochemical-staining methods combined with computer-aided image analysis were employed to examine autoanti-idiotype antibody-forming cell expansion in vivo. Autoanti-idiotype antibody-forming cells were demonstrated in the spleens of C57BL/6J (B6) strain mice intranasally infected with the influenza virus A/Hong Kong/168/(H3N2)[R] X-31. Autoanti-idiotype B cells were detected and elevated in spleen tissues after secondary influenza infections compared to normal B6 mice, and were specific for a dominant idiotype antibody called PY206 reactive with the influenza virus hemagglutinin. Influenza virus-infected BALB/c mice, which make little PY206 Id, did not have increased autoanti-Id against PY206 compared to normal mice. Results provide evidence for an idiotype network-mediated stimulation of autoanti-idiotype B cell production during influenza virus infection.     

Migration of natural suppressor cells from bone marrow to peritoneal cavity by live BCG

Delayed-type hypersensitivity (DTH) responses were suppressed in mice inoculated with bone marrow cells from mice that had been injected with 10(8) colony-forming units (CFU) of live BCG. Upon analysis of this DTH-suppression by the use of a macrophage migration inhibition (MI) assay, the in vitro correlate of DTH, suppressor macrophages in the peritoneal cavity were found to play an important role in DTH suppression. However, neither suppression of DTH nor production of suppressor macrophages was observed in mice inoculated with bone marrow cells from mice that had been injected with methotrexate (MTX), a folic acid antagonist, and 10(8) CFU of live BCG. Moreover, suppressor cells against the MI activity of peritoneal exudate cells from BCG cell wall-immunized mice existed in bone marrow cells from normal mice, natural suppressor (NS) cells, and they were sensitive to MTX. In addition, these NS cells phagocytized carbonyl iron particles, were adherent to Sephadex G-10, and had Fc receptors, but they had no B or T cell markers, suggesting that these cells belonged to a macrophage compartment. From this evidence, we hypothesized that the origin of suppressor macrophages in the peritoneal cavity induced by live BCG injection was MTX-sensitive NS cells in bone marrow, and that these NS cells were stimulated by a small dose of live BCG trapped in bone marrow after i.v. injection of a high dose of live BCG and migrated from bone marrow to the peritoneal cavity.     

Salvage of the nucleic acid base queuine from queuine-containing TRNA by animal cells

The incorporation of queuine into tRNA and its fate upon tRNA turnover has been studied in the Vero and L-M cell lines. An assay was developed using [³H]dihydroqueuine to detect the queuine acceptance and, thus, the queuine content of tRNA in intact cells. While L-M cells can use only queuine, Vero cells can use either queuine or its nucleoside, queuosine, to form queunine-containing tRNA. Since queuosine is not a substrate for the enzyme which incorporates queuine into tRNA, Vero cells must generate queuine from its nucleoside. When Vero cells are labelled with [³H]dihydroqueuine, the half life of acid insoluble radioactivity is 52 days in queuine-free medium and 3.1 days in queuine-containing medium, indicating that [³H]dihydroqueuine is salvaged from tRNA and reused by Vero cells, but that exogenous queuine can compete with the salvaged [³H]dihydroqueuine. When L-M cells are labelled with [³H]dihydroqueuine, the half life of the acid insoluble radioactivity is 1.2 days in the presence or absence of queuine, indicating the absence of queuine salvage in L-M cells.     

In situ studies on incorporation of nucleic acid precursors into Chlamydia trachomatis DNA.

Chlamydiae are obligate intracellular bacteria that are dependent on eukaryotic host cells for ribonucleoside triphosphates. The purpose of the present study was to determine whether Chlamydia trachomatis obtains deoxyribonucleotides from the host cell. The study was aided by the finding that host and parasite DNA synthesis activity could be distinguished by their differing sensitivities to aphidicolin and norfloxacin. Results from isotope incorporation experiments indicated that any nucleobase or ribonucleoside that could serve as a precursor for host DNA synthesis could also be utilized by C. trachomatis for DNA replication. C. trachomatis utilized only those precursors which the host cell converted to the nucleotide level. Pyrimidine deoxyribonucleotides were efficient precursors for host DNA synthesis; however, they were not used by C. trachomatis. On the other hand, purine deoxyribonucleosides are rapidly catabolized by host cells, it is necessary to regulate their metabolism to determine whether they serve as direct precursors for C. trachomatis DNA synthesis. This was partially achieved by using a hypoxanthine-guanine phosphoribosyltransferase-negative cell line and using deoxycoformycin and 8-aminoguanosine as inhibitors of (deoxy)adenosine deaminase and purine nucleoside phosphorylase, respectively. The results indicated that purine deoxyribonucleosides are efficiently utilized for host cell DNA synthesis even if degradation pathways are inhibited and salvage to ribonucleotides is minimized. In sharp contrast, the purine deoxyribonucleosides were utilized by C. trachomatis as precursors for DNA synthesis only when host catabolic pathways and salvage reactions were intact. High-pressure liquid chromatographic analysis of nucleotide pools extracted from host cells pulsed with radiolabeled precursors suggests that infected cells transport and phosphorylate all deoxynucleosides as effectively as mock-infected control cultures. In aggregate, these results show that chlamydiae do not take up deoxyribonucleotides from the host cells.     

In situ hybridization for the detection of human parvovirus B19 nucleic acid sequences in paraffin-embedded specimens

Parvovirus infection of pregnant women leading to a transplacentar infection of the fetus may result in hydrops fetalis, and ultimately in intrauterine death of the fetus. In situ hybridization with a biotinylated as well as with a 35S-labeled probe for human parvovirus B19 was performed on formalin-fixed paraffin-embedded (FFPE) tissue from a fetus suffering from non-immunologic hydrops fetalis. Histology was suggestive of viral infection probably with human parvovirus. Parvovirus DNA could be detected and precisely localized mainly in the nuclei of erythroid precursors cells within fetal blood vessels of all organs examined. There was no detection of B19 nucleic acid in parenchymal cells of the placenta or the fetal organs, nor within maternal blood cells. These findings are in agreement with the well-known properties of animal parvoviruses to replicate exclusively in proliferating cells. Taking into consideration the problems in diagnosing human parvovirus infection by light microscopy, we conclude that in situ hybridization with an appropriate non-radioactive probe is a valuable, rapid and safe complementary detection method for the diagnosis and study of human parvovirus infections. The 35S-labeled probe is more sensitive than the biotinylated probe, but has the disadvantages of lower resolution of the signal, longer duration of the assay, the hazard of radioactivity and the shorter shelf-life of the probe.     

In situ hybridization for the detection of human parvovirus B19 nucleic acid sequences in paraffin-embedded specimens

Parvovirus infection of pregnant women leading to a transplacentar infection of the fetus may result in hydrops fetalis, and ultimately in intrauterine death of the fetus. In situ hybridization with a biotinylated as well as with a³⁵S-labeled probe for human parvovirus B19 was performed on formalin-fixed paraffin-embedded (FFPE) tissue from a fetus suffering from non-immunologic hydrops fetalis. Histology was suggestive of viral infection probably with human parvovirus. Parvovirus DNA could be detected and precisely localized mainly in the nuclei of erythroid precursors cells within fetal blood vessels of all organs examined. There was no detection of B19 nucleic acid in parenchymal cells of the placenta or the fetal organs, nor within maternal blood cells. These findings are in agreement with the well-known properties of animal parvoviruses to replicate exclusively in proliferating cells. Taking into consideration the problems in diagnosing human parvovirus infection by light microscopy, we conclude that in situ hybridization with an appropriate non-radioactive probe is a valuable, rapid and safe complementary detection method for the diagnosis and study of human parvovirus infections. The³⁵S-labeled probe is more sensitive than the biotinylated probe, but has the disadvantages of lower resolution of the signal, longer duration of the assay, the hazard of radioactivity and the shorter shelflife of the probe.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

紫杉醇产生菌的细胞总核酸提取

树状多节孢（Nodulisporium sylviforme)为中国新记录属种,首次发现它可产生特效抗癌药物紫杉醇,国内外尚未见其DNA提取等方面的研究报道。为了获得高质量的DNA,利用单因素试验对其细胞总核酸的提取方法进行了探索。找到了一套适合紫杉醇产生菌DNA的提取较为简便易行可靠的方法。用此法直接从紫杉醇产生菌中提取DNA,并将所提取DNA进行随机引物扩增（RAPD）,得到了较清晰的扩增图谱。     

Delivery of nucleic acid into mammalian cells by anthrax toxin

Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells.     

Localization of nucleic acid sequences by EM in situ hybridization using colloidal gold labels

It is possible to combine hybridization to specimens on electron-microscope grids of nucleic-acid probes labelled nonisotopically with immunogold detection of hybrid sites to map the position of target sequences rapidly and precisely. The basic technique is described, and examples are provided to illustrate the types of questions which can be approached in the general area of higher-order chromosome organization and function. A combination of two differentially labelled probes and two different-sized gold particles permits the simultaneous detection of closely linked or interspersed sequences.     

Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH)

Bacteriophage–host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.