Autologous stimulation of human lymphocyte subpopulation.

The background stimulation universally seen when lymphocytes are cultured in vitro has been shown to be markedly lowered by reducing the proportion of B lymphocytes. B-rich fractions of lymphocytes had extremely high background stimulation. It is concluded that stimulation of T cells, probably by autologous B cells, provides the most probable explanation for the findings described.

Role of antibodies in the specificity of natural cell-mediated cytotoxicity.

Freshly isolated effector cells expressing natural cell-mediated cytotoxicity (NCMC) against cultured target cells possessed antibodies attached to Fc receptors on their cell surfaces. A wide variety of natural antibodies in the circulation combined with the receptors on effector cells to allow recognition of the many specificities on cultured target cells and resulted in reactions with an overall appearance of nonselectiveness. The role of antibodies in the specificity of NCMC was demonstrated by recovery of NCMC by trypsinized effector cells when incubated in serum. With absorbed serum, activity was selectively weaker against the absorbing target cell. When effector cells were reconstituted with antibodies eluted from the absorbing cells, selective cytotoxicity for that cell was detected. The specificity of effector cells reconstituted with eluted antibodies confirmed the results from previous studies on specificity by direct cytotoxicity and by competitive inhibition of cytotoxicity and supported the role of antibodies in NCMC.

Determination of specificity in natural cell-mediated cytotoxicity by natural antibodies.

Natural cell-mediated cytotoxicity (NCMC) and antibody-dependent cell-mediated cytotoxicity (ADCC) appeared to involve similar cytotoxic mechanisms; effector N-cells killed target cells with IgG antibodies attached to the Fc receptor determining the specificity of the reaction. In NCMC the wide range of specificities detected by natural antibodies provided an effector system capable of recognizing numerous antigens on cultured target cells. When several target cells were tested concurrently, an apparent nonselective cytotoxicity resulted. The specificity of individual reactions against each of the target cells could be demonstrated by selective inhibition with competitor cells. The inhibition of cytotoxicity by competition and the effect of proteases on the effector cell for NCMC, but not for ADCC, initially suggested an antibody on the surface of the natural cytotoxic effector cell. This suggestion was supported by the loss of activity with treatments that removed immunoglobulins on the effector cell and by the recovery of reactivity with incubation of the cells in normal human serum. Absorption of the reconstituting serum with target cells resulted in loss of activity against that target cell, substantiating the role of natural antibodies.

The nonselective cytotoxic cell (N cell).

When effector lymphocytes were reacted with cultured human tumors, the total cytotoxic reaction could be divided into selective and nonselective components. The nonselective part of the reaction was due to a cell type called N cells. Fractionation of effector suspension indicated that N cells were neither T nor B cells. Like B cells, N cells did not form rosettes with sheep erythrocytes; they were retained by columns coated with lg or antiserum to lg and died preferentially when stored at an ambient temperature. However, N cells were differentiated from B cells by their inability to form complement-receptor rosettes and by their survival when incubated at 30 degrees C. The effect of the nonselective cytotoxic cell must be differentiated from selective activity in studies of specificity for cell-mediated cytotoxicity.

Interaction analysis of selective and nonselective cell-mediated cytotoxicity.

The coexistence of selective and nonselective cytotoxic cells in effector suspensions required a method of separating the two effects before specificities in cell-mediated cytotoxicity could be investigated. A method of analysis was derived which used the average cytotoxicity for each effector and target to estimate selective and nonselective cytotoxic effects. The analysis clearly detected specificity in tests of cell-mediated lympholysis, and application to tests of cell-mediated cytotoxicity on cultured human tumor cells showed that selective reactions were found.

Search for specificity in natural cell-mediated cytotoxicity.

Differences in antigenicity between the human osteosarcoma cell line TE 85/B and its feline sarcoma virus-infected subline NIH E1041 were detected by competitive inhibition of natural cell-mediated cytotoxicity (NCMC). Whether the differences could be attributed to the viral infection was investigated by absorption and elution studies of antibodies that determine the specificity of NCMC against the cell lines. Antibodies from the serum of healthy individuals were first absorbed onto target cells against which they were to be tested and then eluted to provide antibodies putatively specific for the target cells. Trypsin-treated effector cells were restored with the absorbed serum or eluted antibodies and tested against TE 85/B and its intentionally infected sublines. The differences observed previously between TE 85/B and NIH E1041 were extended to the detection of small differences in antigenicity among all sublines. Separately maintained sublines from the same culture became antigenically different with continuous passage. The causes for these specific changes were unknown, but a role for the control of these antigens by NCMC was suggested. Differences in antigenicity between virus-infected sublines cultured separately need not be related to the virus infection.

Specificities in human cell-mediated cytotoxicity.

Specificity of natural cell-mediated cytotoxicity was investigated through selective reactions detected by direct cell-mediated cytotoxicity and by inhibition of cytotoxicity through competition. Assuming that target cells reacting alike in direct cytotoxicity shared common antigens, we classified 10 target cells into three groups by target antigens: TA (target antigen) 1, 2, and 3. Partial confirmation of the three groups was achieved in the cross-competition assay. The distinction of TA 1 as a group was clear but some cross-reactivity existed between TA 2 and TA 3 cells.

Decline of natural nonselective cell-mediated cytotoxicity in patients with tumor progression.

Lymphocytes isolated from the blood of patients and healthy donors include a population of cells that destroy target cells in the direct cell-mediated cytotoxic assay with little indication of specificity. This natural reaction is the dominant feature of most cell-mediated cytotoxic tests and, although it appears to be mostly nonselective, it possesses some selective activity. The observed cytotoxicity from these reactions depends mostly on the reactivity of the effector cell; when several effector cells are tested on different target cells, the relative order of activity is usually maintained on the different target cells. When this natural cytotoxicity was analyzed without regard to the type of cancer of the patient or of the target cells, a weak decline in the average reactivity was observed with increasing tumor involvement.

Granulocytes as effectors in cell-mediated cytotoxicity of adherent target cells.

In the microassay for cell-mediated immunity, detachment of adherent target cells from the wells occurs to an even greater extent when tested with granulocytes than when tested with lymphocytes. Intact cells are not necessary since the sonic extract from granulocytes causes the same effect. The reaction by granulocytes appears to be mediated by enzymes, and is inhibited by the presence in the wells of hyaluronic acid. Moreover, it is limited to the detachment of adherent target cells, since neither intact nor sonically disrupted granulocytes exhibit cytotoxic activity in the chromium release assay. The detachment of target cells is also inhibited by heparin which may be used to specifically mullify the effect of granulocytes contaminating lymphocyte suspensions.

Natural and antibody-dependent cell-mediate cytotoxicity to cultured target cells superinfected with Epstein-Barr virus.

The relationship between natural cell-mediated cytotoxicity (NCMC) and antibody-dependent cell-mediated cytotoxicity (ADCC) was examined in an Epstein-Barr virus-infected target cell system. The total ADCC reactivity to Epstein-Barr virus-infected target cells varied considerably with different effector cells, indicating contributions to specificity by the effector cells as well as by antibodies in the sera. To investigate the role of each reactant, the effector cells, sera, and target cells were tested according to a three-dimensional experimental design and examined for selectivity with the two- and three-way interaction analysis. The two-way analysis was applied to different planes from the experiment to examine special interactions involving two of the three reactants. Selective ADCC was examined through the results from sera versus target cells, selective NCMC was examined by effector cells versus target cells, and the relationship between NCMC and ADCC was examined through the final plane of effector cells versus sera. A three-way interaction analysis applied to the same results supported the conclusions from the two-way analysis and allowed further inquiry into the concurrent role of three reactants. The design and analysis used in the study allowed detection of selective ADCC and NCMC for Epstein-Barr virus-infected target cells and variations in the efficiency of ADCC by different effector cells and in the modulation of NCMC by different sera.

Excitation energy transfer from tryptophan residues of peptides and intrinsic proteins to diphenylhexatriene in phospholipid vesicles and biological membranes.

An efficient excitation energy transfer from tryptophan residues of intrinsic membrane proteins to an extrinsic fluorescent probe (diphenylhexatriene) has been demonstrated in rat erythrocyte ghosts. To correlate this transfer with the localization of the probe, a model system has been investigated. It consists of peptides containing lysine and tryptophan residues bound to negatively charged phosphatidylserine vesicles. Absorption and fluorescence spectroscopies were used to follow peptide binding and diphenylhexatriene incorporation. Peptide binding is accompanied by a blue shift of the tryptophan fluorescence together with an increase of the quantum yield and of the fluorescence decay time. An experimental Föster critical distance value of 4.0 nm was found for energy transfer from tryptophan residues of peptides to diphenylhexatriene which approaches the range of calculated values (3.1-3.7 nm) using a two-dimensional model. These results demonstrate that efficient energy transfer can occur from tryptophan residues of intrinsic proteins to diphenylhexatriene without any interaction between diphenylhexatriene and proteins in biological membranes.

Triple helix-directed psoralen crosslinks are recognized by Uvr(A)BC excinuclease.

Pyrimidine oligonucleotides bind to the major groove of an oligopyrimidine-oligopurine DNA sequence by triple helix formation. A 14-mer oligopyrimidine 3'-psoralen-conjugate (P) and a doubly modified 5'-acridine/3'-psoralen-oligonucleotide (PA) were photo-crosslinked to their target site. The crosslinked complexes were tested regarding their sensitivity to Uvr(A)BC excinuclease/DNA complex formation and excision, and compared to free psoralen crosslinked to the same site (M). An electrophoretic mobility-shift assay showed that the crosslinked triple-helix did not hamper formation of the (A)2B complex under conditions where the third strand was bound to its target. In vitro excision experiments performed on damaged DNA fragments containing crosslinked 5-methoxypsoralen (M-target) confirmed that the psoralen photoadduct was recognized by Uvr(A)BC and that excision occurred at the crosslinked site. The major cleavage reaction took place on the 5'-side of oligopurine strand. The excision was less efficient on the 5'-side of the pyrimidine strand. The 3'-side incision either on the purine or pyrimidine strand was even weaker. With optimal Uvr(A) concentrations, it was observed that the incision reaction on (P)- and (PA)-modified targets was clearly inhibited compared to the (M)-modified target, reflecting an effect of the oligonucleotide on the recognition/excision process. These results demonstrate that a triple helix is efficient in promoting inhibition of Uvr(A)BC excision nuclease activity. These results could account for divergent findings concerning the effects of triple helix-forming oligonucleotides on repair systems and open new perspectives to study DNA repair processes through the use of bi-substituted triple helix-forming oligonucleotides.

Double helices with parallel strands are formed by nuclease-resistant oligo-[alpha]-deoxynucleotides and oligo-[alpha]-deoxynucleotides covalently linked to an intercalating agent with complementary oligo-[beta]-deoxynucleotides.

Oligo-[alpha]-thymidylates have been synthesized and covalently linked to an intercalating agent (an acridine derivative) and/or to a p-azidophenacyl group. These molecules bind to a complementary oligo-[beta]-deoxynucleotide. A strong stabilization is obtained by covalent attachment of the acridine derivative at the 5' end of the oligo-[alpha]-deoxynucleotide. Upon excitation of the p-azidophenacyl group with ultraviolet light, the oligo-[alpha]-thymidylate is crosslinked to its target sequence. These crosslinks are converted to chain breaks under alkaline conditions. This allows an unambiguous assignment of the orientation of the two oligonucleotide chains. As expected, beta-beta hybrids have an antiparallel orientation, whereas the two chains of alpha-beta hybrids are parallel independently of whether an intercalating agent is covalently linked to the alpha-oligo-nucleotide. Oligo-[alpha]-thymidylates covalently linked to an acridine derivative are highly resistant to endo- and exonucleases. Therefore, they could be used as anti-messengers to block mRNA translation in vivo under conditions where oligo-[beta]-deoxynucleotides are usually hydrolysed.

Triple-helix specific ligands stabilize H-DNA conformation.

Under superhelical stress, oligopurine-oligopyrimidine mirror-repeat sequences are able to adopt H-DNA conformations where a triple-helical and a single-stranded structure co-exist. We have previously shown that a benzo[e]pyridoindole derivative (BePI), an antitumor drug interacting more tightly with triplex than with duplex DNA, strongly stabilizes intermolecular triple helices formed upon binding of homopyrimidine oligonucleotides to the major groove of double-stranded DNA at oligopurine-oligopyrimidine sequences. Here we show that an intramolecular triple helix is also strongly stabilized by this ligand. In vitro elongation performed by different DNA polymerases (bacteriophage T7, Escherichia coli or Taq polymerase) could be irreversibly inhibited by the H-DNA structure in the presence of BePI. A mirror-repeat polypurine-polypyrimidine sequence inserted between the E. coli beta-lactamase gene (conferring ampicillin resistance) and its bla promoter strongly inhibited transcription of the beta-lactamase gene in vivo. In the absence of supercoiling, transition to the H-conformation did not occur, but BePI stabilized the H-DNA structure induced by supercoiling as shown by chemical probes (chloroacetaldehyde). The results presented here open a new field of investigation for antitumor agents targeted to a novel class of genetic structures able to regulate gene expression.

Site-specific cleavage of single-stranded and double-stranded DNA sequences by oligodeoxyribonucleotides covalently linked to an intercalating agent and an EDTA-Fe chelate.

An oligodeoxythymidylate, oligo [d(T8)], was covalently linked to an acridine derivative via its 3' end and to EDTA via its 5' end. The octathymidylate was targeted to a single-stranded DNA fragment 27 nucleotides in length containing an octadeoxyadenylate sequence. In the presence of Fe(II) and a reducing agent (dithiothreitol) cleavage reactions were induced in the nucleotide sequence. The extent of the reaction was dependent on oligo concentration, salt concentration and temperature. Dissociation of the complexes at high temperature or low salt concentration abolished the site-specific cleavage reactions. Treatment of the reacted DNA with piperidine or piperidine-formiate strongly enhanced the yield of cleavage reactions demonstrating that damages were induced on nucleic acid bases by the EDTA-Fe complex covalently linked to the octathymidylate. At high salt concentration (1 M NaCl) or in the presence of spermine and ethylene-glycol a triple helix was formed involving the 27-mer DNA fragment and two oligo[d(T8)]. One of the oligo[d(T8)] was bound parallel and the other antiparallel to the oligo[d(A8)] complementary sequence. Cleavage reactions were induced on both sides of this oligo[d(A8)] target sequence. When a 27-mer duplex was used as a target the oligo[d(T8)] was bound in a parallel orientation with respect to the oligo[d(A8)]-containing strand in the major groove of the double helix. Cleavage reactions were induced on the oligo[d(A8)]-containing strand by the EDTA-Fe chelate attached to the 5' end of the oligo[d(T8)].

Sensitive quantitation of nitric oxide by EPR spectroscopy.

A recent method for NO detection is electron paramagnetic resonance (EPR) with ferrous and mononitrosyl dithiocarbamate (Fe2+ (DETC)2) for spin trapping [Menon, N.K., et al., J.Mol. Cell Cardiol., 23:389; 1991]. However, by this technique, we failed to detect the spectrum of the NOFe2+ (DETC)2 complex in biological systems because of the low solubility of Fe2+ (DETC)2 and rapid oxidation of NOFe2+ (DETC)2 complex. To overcome these problems, we modified this method by adding albumin to solubilize Fe2+ (DETC)2 and Na2S2O4 as a strong reductant to increase the sensitivity and stability of the EPR spectrum of the NOFe2+ (DETC)2 complex. The optimal concentrations of these reagents were 3.3 mM of Fe2+ and DETC, 33 mg/ml albumin and 2 M Na2S2O4. The detection limit was less than 10 pmol/ml under these conditions. By this modified method, we succeeded in quantifying NO production from porcine aorta induced by forskolin.

Oligodeoxynucleotides covalently linked to intercalating agents: a new class of gene regulatory substances.

Oligodeoxynucleotides have been covalently linked to a 9-aminoacridine derivative via their 3'-phosphate group. Specific complexes are formed with the complementary sequence of the oligonucleotide. The stability is strongly increased due to intercalation of the acridine derivative. Absorption, fluorescence, nuclear magnetic resonance and circular dichroism have been used to characterize complex formation. The stability of the complexes depends on the length of the linker between the acridine derivative and the 3'-phosphate group of the oligonucleotide. Oligonucleotides covalently linked to an intercalating agent can be used to selectively control gene expression. Transcription initiation can be blocked when such an oligonucleotide binds to the transcribed strand in the open complex formed by E. coli RNA polymerase with the bla promoter. With some oligonucleotides, non-specific effects on transcription can be detected, most probably due to binding of the modified oligonucleotide to RNA polymerase. Translation of the messenger RNA from gene 32 of phage T4 can be prevented by using an oligonucleotide complementary to the sequence upstream from the Shine-Dalgarno sequence. Inhibition of translation does not occur in the absence of the intercalating agent covalently linked to the oligonucleotide nor with oligonucleotides which do not have a target sequence on the mRNA.

Selective reactions in antibody-dependent cell-mediated cytotoxicity.

Selective reactions associated with HLA specificity were sought in antibody-dependent cell-mediated cytotoxic tests against HLA-typed lymphoblastoid lines using operationally monospecific HLA sera and effector cells from healthy individuals. Precise detection of HLA specificities was disturbed by the presence of natural antibodies in HLA antiserum and the effect of the serum and cells on target cell viability. Detection of HLA specificity was improved by absorption of the serum to remove natural antibodies and correction of the results for extraneous cell and serum effects.

Programmed filtration, a new method for removing large molecules and regulating albumin leakage during hemodiafiltration treatment.

During hemodiafiltration (HDF) treatment for chronic renal failure patients, replacing large volumes using high-flux membranes with relatively large pores is preferred from the standpoint of enhancing the elimination of large molecules (10 to 50 kd). Aggressive protein-permeable treatment often results in massive leakage of essential albumin, however, which may cause fatigue, hypotension, and a decrease in the plasma albumin concentration in some patients. During 5-hour conventional HDF treatment with the filtration rate or pressure set at constant values, fractional albumin loss in the dialysate was assayed, which revealed that the albumin concentration in the dialysate showed a maximum value in the beginning with a steep decline within 1 hour. Approximately 40% to 50% of the total amount of albumin leakage occurred during the first 30 minutes. Concomitantly the large molecules transferred into the pores by aggressive filtration during the beginning partially plugged the pores, resulting in a decrease in the permeability for beta(2)-microglobulin. From the standpoint of achieving the highest clearance for large molecules, while suppressing albumin leakage below the acceptable range, the optimal profiles for filtration conditions in HDF have been proposed, in which either the transmembrane pressure is regulated according to the sigmoid curve in the pressure control manner or the flow rate is set along the concave in the flow control manner. The profiles of pressure or flow as a function of time have been programmed and installed in a HDF machine to perform an optimal HDF treatment automatically. The new filtration methods gave significantly higher beta(2)-microglobulin removal and lower albumin leakage than conventional HDF methods with constant filtration.

Immune function in aging atomic bomb survivors residing in the United States.

Immunologic parameters were studied among survivors of the 1945 atomic bombs who now reside in the United States. Of all known survivors living in the U.S., about 40% (n = 189) participated in this study. Of those survivors on whom radiation exposure information was available (n = 168), 96% were exposed to less than 50 rad at the time of the bomb (ATB). Survivors were divided into two groups; those exposed to varying low doses of radiation (S+ group, exposed at less than or equal to 2500 m from the hypocenter) were compared with those exposed to "O rad" (S0 group, exposed at greater than 2500 m from the hypocenter). Of the former group, 92% were exposed to less than 100 rad and 89% to less than 50 rad ATB. Cellular immune responses, including natural cell-mediated cytotoxicity (NCMC), interferon production, and the mitogenic response to PHA, tended to be higher among S+ individuals, although only the difference for NCMC was statistically significant. This was suggestive of a trend which was consistent with the higher serum interferon levels and lower frequencies of detectable immune complexes and antimitochondrial antibodies among the S+ group, although these differences were not statistically significant. Other immunologic parameters which showed no trend included frequency of antinuclear antibodies, rheumatoid factor, levels of serum immunoglobulins, levels of isoantibodies and heteroantibodies, and the magnitude of the mixed lymphocyte reaction.