Macrophage Functional Heterogeneity in the <Emphasis Type="Italic">in vitro</Emphasis> Induced Immune Response

RNA from antigenically stimulated peritoneal macrophages is immunogenic in vitro^1–4. The studies of Fishman and Adler^5–6 suggest that the peritoneal macrophage population consists of at least three functionally distinct subpopulations. Although most peritoneal macrophages act as scavenger cells⁷, a second population—possibly less than one cell per 1,000—consists of cells that produce but do not necessarily secrete antibody^8,9 and respond to antigen by synthesizing informational RNA. On transfer to normal lymphoid cells, this RNA elicits IgM antibody with the allotypic specificity of the macrophage donor¹⁰. A third type of macrophage gives rise to the RNA-antigen complex responsible for the in vitro synthesis of IgG antibody with the allotypic specificity of the lymphocyte donor¹⁰.     

<Emphasis Type="Italic">In vitro</Emphasis> cytotoxicity of an endophytic fungus isolated from<Emphasis Type="Italic">Nothapodytes foetida</Emphasis>

An attempt has been made to assessin vitro cytotoxicity of an endophytic fungus fromNothapodytes foetida. Various human cancer cell lines (liver HEP-2, lung A-549, ovary OVAR-5, prostate PC-3, cervix Hela, colon HCT-15, oral cell line KB, CNS SNB-78, were used.In vitro cytotoxicity of camptothecin (CPT) isolated from the fungus was done where OVAR-5 cell line showed maximum inhibition and HEP-2 cell line was least sensitive with this compound.In vitro cytotoxicity of fractions/extracts from endophyte was carried out where ethyl acetate fraction showed sufficient growth inhibition against all the cell lines.     

Induction of Immunity and Tolerance to the Dinitrophenyl Determinant <Emphasis Type="Italic">in vitro</Emphasis>

THE immune response in dissociated lymphoid cell cultures offers an opportunity to investigate the interaction of antigen with the surface receptors of immunocompetent cells. Using polymerized flagellin of Salmonella adelaide (POL), evidence was obtained that in vitro processes as different as immunity and tolerance both depend on the direct interaction between antigen and antigen-sensitive cells^1–4. The use of chemically defined determinants in place of natural antigens could simplify the study of the molecular mechanisms underlying immunity and tolerance. Systems used in the past to induce immunity to defined determinants in vitro involved either a particulate antigen⁵ or spleen fragment cultures⁶ and were therefore unsuitable for the detailed study of the interactions occurring on the surface of lymphoid cells. A new system had to be devised. Here I describe the induction of a primary immune response to a hapten–protein conjugate in dissociated spleen cell cultures and the immune tolerance to a chemically defined determinant in vitro.     

Transfer of Immunity to Tumour Isografts by the Systemic Administration of Xenogeneic “Immune” RNA

SPECIFIC immunoreactivity can be conferred on lymphoid cells by incubation with RNA-rich extracts prepared from lymphoid tissues exposed to specific antigens in vivo¹ and in vitro^2,3. We have shown transfer of immunity to tumour specific antigens in vivo⁴ and in vitro⁵ by incubation of syngeneic spleen cells in vitro with RNA extracted from the lymphoid tissues of xenogeneic or syngeneic animals immunized with the tumour to be treated. Administration of these spleen cells to normal animals decreased the development and growth of isografts of the same tumour.     

Restoration of the Immune Response to Sheep Erythrocytes by a Serum Factor

THE immune response of CBA mice to sheep erythrocytes (SRBC) is known to be thymus-dependent because strain members thymectomized during the first few hours of life exhibit a marked inability to respond to this antigen^1,2. Experiments with isoantisera suggested that a cell to cell interaction is involved in this response. Thymus cells per se do not develop into haemolytic plaque-forming cells, but in some, so far obscure, way they cause cells of bone marrow origin to become producers of haemolytic plaques^2,3. A study of spleen cells from neonatally thymectomized (NNT) mice in a tissue culture system indicated that the decreased responsiveness to SRBC is also expressed in vitro. In that case 15×10⁶ NNT spleen cells produced only 500 haemolytic plaques when assayed on day 4 of culture. But when 15×10⁶ thymus cells were added to identical cultures of NNT spleen cells at inception, the number of haemolytic plaque forming cells increased to 2,300 (ref. 4). When an equivalent number of thymus cells alone were incubated with SRBC there was no response.     

Inhibition of <Emphasis Type="Italic">E. coli</Emphasis> DNA Polymerase II by Ara-CTP

THE potent antileukaemic agent, 1-β-D-arabinofuranosylcytosine (ara-C), specifically inhibits DNA synthesis in bacterial and animal cells^1,2. Although the exact mechanism of inhibition has been in doubt, it seems likely that it occurs at the DNA polymerization reaction itself^2–5. We describe here the effects of ara-CTP, which is the most prominent form of ara-C in the cell, on in vitro replicative systems^6–11 of E. coli and on isolated DNA polymerases II and III^12–15.     

Dexamethasone treatment in vitro resulted in different responses of two fractions of phagocytes of the holothurian Eupentacta fraudatrix

Echinoderm phagocytes are considered to be analogues to vertebrate macrophages. Previously, the phagocytes of some echinoderm species were divided into two fractions with unclearly identified functional properties. This study aims at modeling the immune response of two phagocyte fractions (P1 and P2) of the holothurian Eupentacta fraudatrix to the synthetic glucocorticoid hormone dexamethasone (Dex) in vitro and at comparison of the effects of such pretreatment on humoral cooperation of each phagocyte fraction with another type of immunocytes, morula cells. During 48-h incubation, Dex (0.1–100 μM) induced apoptosis in a direct (in the P1 fraction) or reverse (in the P2 fraction) concentration-dependent manner. In addition, 100 μM Dex differently affected the cytokin-like substance level in the P1 and P2 phagocyte fractions. Moreover, the supernatants of the Dex(100 μM)-pretreated phagocytes induced opposite changes in the IL-1-like substance level in morula cells. These results indicate a striking functional difference between the two phagocyte fractions. The data obtained provide a new insight into the evolution of macrophage response and into the prospects of the use of in vitro holothurian phagocyte model.     

Simian Tumour Virus Proliferation in Inoculated <Emphasis Type="Italic">Macaca mulatta</Emphasis>

AN oncorna-type virus (M-PMV) was detected in a spontaneous breast tumour of a female rhesus monkey^1,2. This virus is morphologically and biochemically similar to oncogenic RNA viruses of other species^3–5. Using in vitro tissue culture assays and electron microscopy we have observed proliferation of infectious virus in hyperplastic lymph nodes of M-PMV inoculated infant rhesus monkeys.     

Effect of Rifampicin on the Development of Tumours induced by Adenovirus in Male Hamsters

INITIAL in vitro studies established that rifampicin, one of a group of rifamycin SV derivatives^1,2, prohibits bacterial growth and phage replication by binding to a polypeptide component of the microbial DNA-dependent RNA polymerase^3–7. The trachoma agent and related psittacosis-lymphogranuloma agents are also inhibited in vitro and in embryonated eggs by this drug⁸. Further studies have shown that rifampicin is active against a number of bacteria in vivo, both after parenteral and oral administration^1,9,10. It also inhibits malaria in mice¹¹ and trachoma in monkeys^12,13 and is of special value in the treatment of human tuberculosis^14–16. The low toxicity of the rifamycins in mammals¹⁷ has been attributed to the observed relative insensitivity of mammalian RNA polymerase to the rifamycins in vitro^3,18.     

Histone or Bacterial Basic Protein required for Replication of Bacteriophage RNA

IN spite of the apparent simplicity of RNA bacteriophage, several proteins, both phage and bacterial, are required for the synthesis of Qβ RNA in vitro. The polymerase complex alone contains one phage-coded and three host proteins^1,2. The specific role of these proteins in Qβ RNA replication is unknown, but because they demonstrate an associative interaction and are always found with active enzyme, it has been suggested that all four contribute to polymerase activity¹.     

Activity and Properties of Macrophage Migration Inhibitory Factor produced by Mixed Lymphocyte Cultures

THE macrophage migration test is an in vitro demonstration of delayed hypersensitivity. Supernatant fluids of sensitive lymphocytes cultured for 24 h in the presence of specific antigen contain migration inhibitory factor (MIF) that arrests the migration of macrophages of unsensitized animals in vitro¹,². In vivo, it induces delayed skin reactions³. The use of the macrophage migration test, based on differences of transplantation antigens in donor and recipient, to show histocompatibility has been suggested⁴. The test was also recommended as an indicator of immunological reactivity after organ transplantation, to demonstrate impending rejection⁵. It can demonstrate homograft sensitivity, for migration of peritoneal exudate cells (containing lymphocytes and macrophages) of CBA mice previously sensitized by grafts from A/Jax donors was inhibited when they were mixed with peritoneal exudate cells of the donor strain. However, histocompatibility was not demonstrated, for mixtures of peritoneal exudate cells of ungrafted CBA mice and A/Jax mice migrated regularly during the 24 h test⁶.     

RNA Polymerase III of <Emphasis Type="Italic">Blastocladiella emersonii</Emphasis> is Mitochondrial

MULTIPLE RNA polymerases have been shown to exist in a wide variety of eukaryotic organisms^1–5. Two nuclear polymerases have been found in all the cells studied, each with a specific location and a specific function: the DEAE fraction I enzyme is located in the nucleolus and may be involved in the synthesis of ribosomal RNA^1,2,5,6; the DEAE fraction II enzyme is located in the non-nucleolar nucleoplasm and functions in the synthesis of DNA-like RNA^2–5,7. The DEAE fraction III enzyme was reported to exist in sea urchin¹, the aquatic fungus B. emersonii⁵ and to be present sometimes in rat liver preparations^1,8. Although there have been some reports that polymerase III is nuclear, Horgen and Griffin⁵ showed that the enzyme was sensitive to the prokaryotic RNA polymerase inhibitor rifampicin. They suggested that the fraction III enzyme may be mitochondrial, formed as the result of organelle contamination in their crude nuclear preparations. The results of this study show that the DEAE fraction III enzyme in B. emersonii is a mitochondrial enzyme, most likely functioning in the synthesis of mitochondrial RNA. The rifampicin sensitivity of the enzyme is further evidence of a prokaryotic origin of mitochondria^9,10.     

Effect of Anti-immunoglobulin Antisera on Homograft Rejection in Mice

HETEROLOGOUS antisera against immunoglobulins or their component protein chains have been shown to inhibit the immune response in a variety of systems. Antibodies against mouse immunoglobulins, for example, inhibit the response of mouse spleen cells cultured in vitro^1–3. Antibodies against the heavy chains of chicken IgM (anti-μ), administered during embryonation and again at hatching, have produced agamma-globulinaemia in bursectomized chickens⁴, apparently by plasma cell line elimination⁵. Graft-versus-host (GVH) and delayed hypersensitivity reactions have been suppressed in neonatal mice by in vitro pretreatment of injected lymphoid cells with antiserum against light chains⁶. Similar pretreatment with univalent fragments (Fab) of anti-immunoglobulin antibodies has diminished the GVH reaction in adult mice⁷.     

Effect of R Factors on Rifampicm Resistance in <Emphasis Type="Italic">E. coli</Emphasis>

THE bactericidal effect of rifampicin, a semi-synthetic rifamycin, is due to its action on DNA-dependent RNA polymerase¹ and all rifampicin-resistant mutants of Escherichia coli contain an altered RNA polymerase with an increased resistance to rifampicin in vitro^2–4. While studying a possible curing effect of rifampicin on E. coli R factors, we observed that R⁺ recombinants of some rif-r mutants are more sensitive to rifampicin (Table 1). Of the cells harbouring certain R factors, less than 1% are able to form colonies on rifampicin-supplemented agar, while with certain others there is no detectable effect.     

Initial Nucleotide Frequencies of Bacterial RNA synthesized during Amino-acid Starvation or Changes of Carbon Source

THERE is growing evidence to indicate that RNA synthesis in bacteria is regulated through adjustment of the frequency of initiation of new RNA molecules. In this framework an understanding of the process of initiation of RNA synthesis takes on a special importance and for this reason we have investigated in varying conditions the composition of the 5′ terminal or first-inserted nucleotide. It has been previously shown that such initiations do not occur randomly, either in vivo¹ or in the proper conditions in vitro^2,3, but that RNA chains are exclusively initiated with the purines, adenosine and guanosine. Additionally, there was a recent suggestion based on in vitro studies that the nucleotide guanosine-3′-diphosphate-5′-diphosphate (MS1), proposed to be a regulatory agent in RNA synthesis, functioned by specifically depressing the frequency of initiation of a large fraction of RNA molecules beginning with guanosine⁴. Here we report, however, that in vivo, in conditions in which regulation of RNA synthesis is manifest, the ratio of molecules initiated with adenosine and guanosine is not changed.     

<Emphasis Type="Italic">In vitro</Emphasis> Detection of Enhancing Antibody by the Macrophage Migration Test

THE immunological basis of enhanced allograft survival in inbred strains of mice is well established: humoral antibody, by some mechanism which is not yet clear, interferes with the cell-mediated host response to alloantigens¹. Various in vitro correlates of transplantation immunity in the mouse have been developed^2–9 and we have adapted one, the technique of macrophage migration from capillary tubes⁹, as an in vitro method to detect enhancing antibody.     

Autocrine Survival Factors of a Cytotoxic CTLL-2 Cell Line

Cell survival in multicellular organisms is controlled by numerous cytokines, growth factors, and autocrine survival factors. Autocrine survival factors remain the least studied. The aim of this work was to study the autocrine factors which control survival of a CTLL-2 cytotoxic cell line: isolation and characterization of biologic activity along with physicochemical features of the active molecules have been performed. The conditioned medium of CTLL-2 cells containing autocrine factors was separated by gel filtration into four fractions: A, B, C, and D (according to the order of their efflux from the column). The biological activity of the fractions was tested by the MTT assay with the low density 5 days culture as a cell survival model. The testing of the ability of the fractions to support the cell survival in culture has shown that fractions A and B were active, whereas fractions C and D were not. The presence of a peptide of the molecular mass of 1157 Da in active fractions A and B has been detected by MALDI-TOF-mass-spectrometry. Considerable amount of lactate in fractions A and B, which flowed out from the column along with the peptide, has been detected with an enzymatic lactic acid assay. The lactate concentration in fraction A was 3.72 ± 0.11 mM and it was 0.83 ± 0.06 mM in fraction B. The obtained data suggest that fractions A and B contain supramolecular complexes of the peptide (M 1157 Da) with different lactate content. The peptide in a free form has not been found in the CTLL-2 cell conditioned medium.     

Availability of Label in <Superscript>3</Superscript>H-Thymidine for DNA Synthesis in Cultured Macrophages

DURING a study of the developmental potentialities of promacrophages from bone marrow, blood, peritoneal fluid and spleen of rat and rabbit, it was found that a pool of long-lived labelled DNA precursors was formed both in vivo and in vitro while ³H-thymidine was used as label.     

Intracellular localization of anti-tumor immune RNA.

Syngeneic spleen cells from normal, non-immune Fischer 344/N rats and allogeneic spleen cells from normal Wistar-Furth rats became cytotoxic, in vitro, to chemically induced Fischer rat sarcoma (MC3-R) target cells following incubation with xenogeneic Immune RNA (I-RNA) extracted from spleens of guinea pigs immunized with MC3-R tumor cells. I-RNA extracted from intact spleen cells or from the cytoplasmic fraction of spleen cells were equally active. RNA extracted from isolated spleen cell nuclei was inactive, as were all RNA fractions from spleen cells of nonspecifically immunized guinea pigs. Syngeneic I-RNA extracted from intact spleen cells or the cytoplasmic fraction of cells from spleens of Fischer rats bearing growing MC3-R transplants mediated cytotoxic reactions against MC3-R target cells when incubated with normal Fischer rat spleen cells. RNA from the nuclei of spleen cells of rats bearing MC3-R tumors was considerably less active. All RNA fractions from spleen cells of normal non-immune Fischer rats were inactive. The immunologically active component of xenogeneic and Syngeneic I-RNA, therefore, were found to be localized in the cytoplasm of specifically sensitized lymphoid cells.