Human Leukocyte Cultures Research Articles

Cellular source of interferon induced in human peripheral blood mononuclear leukocytes by mumps virus or by tumour cells persistently infected with mumps virus.

Human leukocyte interferon (IFN-a) was induced in cultures of human peripheral blood mononuclear leukocytes (PBML) from seropositive healthy donor infected by mumps virus, or by a human lung carcinoma cell line (Pc-10) persistently infected with mumps virus (Pc-10/M-V). Adherent cells (M0), non-T, non-B cells and B cells all produced IFN in response to mumps virus or Pc-10/MpV cells. The non-T, non-B cells co-cultured with Pc-10/MpV cells produced high-titred IFN. However, T cells did not produced any antiviral factors even in the presence of M0. Furthermore, T cells activated with phytohaemagglutinin (PHA-P), concanavalin A or treated with u.v.-irradiated mumps virus, or Pc-10/MpV cells pretreated with mitomycin C did not produce any antiviral factors in response to either mumps virus or Pc-10/MpV cells.

Generation in vitro of EBV-induced specific cytotoxic T cells in autologous serum avoids complications due to self-preferred foetal calf serum-specific T-cell cytotoxicity.

A previous report has established that in cultures of human mononuclear leukocytes, foetal calf serum (FCS) is capable of generating high levels of T cells preferentially cytotoxic for the autologous lymphoblastoid cell line (LCL). The present study compared the capacity of Epstein-Barr virus (EBV) to generate cytotoxic T cells in cultures of mononuclear cells grown in FCS in this system. Five EBV-seropositive and three seronegative donors were used and cultures were harvested at 14 days. With cultures from seropositive donors, whether grown in FCS or in autologous serum, EBV infection generated T cells cytotoxic for the autologous LCL; the response in uninfected control cultures was markedly lower. With seronegative donor cultures grown in FCS, there was virtually no difference in the capacity of T cells generated in infected or uninfected cultures to lyse the autologous LCL. Moreover, cells from seronegative donors cultured in human serum gave no detectable lysis of autologous LCL in either infected or uninfected cultures, clearly showing the absence of a response to EBV. This evidence shows that it is possible to distinguish the generation of specific cytotoxic T cells by FCS from generation by EBV, and with certain donors the apparently EBV-induced response may actually include a significant component induced by FCS in the medium. The cytotoxicity patterns of EBV-induced and FCS-induced T cells for autologous and allogeneic LCL targets showed a degree of parallelism, stressing the need for caution in interpretation of data obtained from cultures using FCS.

Antibody to staphylococcal enterotoxin A-induced human immune interferon (IFN gamma).

Antiserum to human gamma interferon (IFN gamma) was produced in rabbits immunized with partially purified (10(4.8) to 10(6.2) antiviral U/mg protein) staphylococcal enterotoxin A-induced IFN gamma. Staphylococcal enterotoxins, phytohemagglutinin M, concanavalin A, and pokeweed mitogen-induced antiviral activity in human leukocyte cultures was neutralized to undetectable levels by the antiserum. However, human leukocyte interferon (IFN alpha), human fibroblast interferon (IFN beta), and mouse interferons were not neutralized by the antiserum. After determining the antiserum was specific for IFN gamma and did not neutralize other known types of interferon, it was used with antibody to human IFN alpha to demonstrate the type(s) of interferon stimulated by some new inducers and antigens. Galactose oxidase- and calcium ionophore-induced interferons were neutralized to undetectable levels by the antiserum to IFN gamma. Interferon produced in leukocyte cultures from tuberculin-negative individuals stimulated with tuberculin-purified protein derivative or old tuberculin was IFN alpha, whereas interferon from tuberculin-positive individuals was a combination of alpha and gamma IFN. In addition, the antiserum neutralized the anticellular and natural killer cell enhancement activities of IFN gamma preparations. The specificity of this antiserum for IFN gamma indicates that it is an additional, powerful tool for identifying and classifying known and new interferons produced in vitro or in vivo and for investigating the role(s) of IFN gamma during the course of infectious, neoplastic, and autoimmune diseases.

Herpes simplex virus infection in human monocyte cultures: dose-dependent inhibition of monocyte differentiation resulting in abortive infection.

Monocyte-enriched cultures of human blood leukocytes were exposed to herpes simplex virus (HSV) at different multiplicities of infection (m.o.i., from about 10 to 0.001 p.f.u./cell). Highest maximum progeny virus titres were invariably obtained with low initial m.o.i., i.e. those between 0.01 and 0.0001 p.f.u./cell, while little if any infectious progeny was produced in cultures inoculated with the highest virus concentrations. By the time of maximum virus production, i.e. 5 to 7 days after inoculation, monocytes in the uninfected cultures had mostly differentiated to macrophages. This differentiation was partially inhibited in cultures initially exposed to the higher concentrations of HSV. Synthesis of HSV antigens was detected by indirect immunofluorescence both in the high m.o.i. cultures and in the productively infected cultures. By this criterion, a maximum of 10 to 15% of all adherent cells became infected in both culture types. It is suggested that the higher doses of HSV, by inhibiting cellular maturation, also prevent the subsequent completion of its own infectious cycle.

In vitro virulence marker: growth of dengue-2 virus in human leukocyte suspension cultures.

We wished to find a simple, biologically relevant method to evaluate the virulence of dengue viruses for human beings. Since cells of mononuclear phagocyte lineage may be important sites of dengue infection in primates, we evaluated the permissiveness of these cells to dengue virus as a correlate of virus virulence. Two wild-type, large-plaque, monkey-virulent dengue-2 virus strains and two small-plaque, monkey-avirulent dengue-2 virus strains were evaluated for their ability to replicate in human peripheral blood leukocyte cultures supplemented with enhancing antibody. One of the small-plaque strains was demonstrated to have reduced virulence for man. Wild-type dengue-2 viruses replicated readily in peripheral blood leukocyte suspension cultures, whereas small-plaque dengue-2 strains did not. Differences between our data and results obtained by other workers employing adherent peripheral blood leukocytes are discussed. Antibody-enhanced growth of dengue virus in suspension cultures of human peripheral blood leukocytes gives promise of being a simple in vitro system for characterizing dengue virus virulence.

A factor with procoagulant activity produced by stimulated human leukocyte cultures

Human leukocyte cultures stimulated by phytohemagglutinin (PHA) or purified protein derivative (PPD) produce and release a procoagulant (PCF) that can correct the clotting defect in Factor VIII, but not Factor VII- or Factor X-deficient plasma. Since exogenous anticoagulants can suppress cellular immune responses, it is likely that certain patients with coagulation defects may have impaired reactions as well. The site of action of PCF, as elucidated by our in vitro studies, suggested that Factor VIII-deficient individuals should have normal delayed hypersensitivity responses, but Factor VII-deficient individuals should have diminished reactivity. In the limited number of patients available for study, this proved to be the case. These findings demonstrate the existence of a procoagulant that is either a lymphokine or lymphokine-induced monokine and provide indirect evidence for its role in in vivo reactions of cellular immunity.

Mechanism of Action of Human B Cell-Activating Factor

Abstract Human monocytes have been reported to release factors that can elicit distinct responses from a number of different target cells. Three of these activities are compared in this report. Supernatants of endotoxin-stimulated monocytes have been shown to stimulate the direct plaque response of T cell deficient splenocytes in vitro (BAF assay), to stimulate the thymidine incorporation by thymocytes (mitogen assay), and to synergize with phytohemagglutinin in the stimulation of thymocytes (PHA-enhancing assay). By gel filtration, plaque stimulating activity was recovered as a single peak with a m.w. of approximately 15,000 daltons when tested on nude splenocytes. When assayed on normal C57BL/10CR splenocytes, a second peak of plaque-stimulating activity was observed near 35,000 daltons. Mitogenic activity was observed both within the albumin peak and coincident with the 15,000 dalton plaque stimulator. Finally, PHA-enhancing activity was found to be broadly distributed between 8000 and 66,000 daltons, and included molecules that co-eluted with the 35,000 and 15,000 dalton plaque stimulators. Upon further purification of the 15,000 dalton material by isoelectric focusing, the plaque-enhancing, mitogenic, and PHA-enhancing activities co-eluted. This observation raised the possibility that all three activities of the 15,000 dalton material were due to the effects of a single factor on T cells. Some investigators favor this possibility based upon the study of the supernatants of human mixed leukocyte cultures (XRF). Here it is shown that spleen cell populations can be prepared that responded to BAF but not XRF, and vice versa. Therefore, studies on XRF cannot be used as evidence for the mechanism of the action of BAF. These results raise the possibility that this single factor may effect more than one target cell and may be responsible for a number of the sequellae of the stimulation of the reticuloendothelial system by bacterial products.

Studies of the Producer Cell of Interferon in Human Lymphocyte Cultures

The producer cells of interferon were studied in human leucocyte cultures stimulated by a variety of stimulants, including phytohemagglutinin (PHA), pokeweed mitogen (PWM), Corynebacterium parvum (CP) and Herpes Simplex Virus (HSV). When the cells were fractionated by the use of neuraminidase-treated sheep red blood cells (SRBC), the T cell population responded with interferon production to PHA and PWM but not to CP or HSV. However, the non-T population showed a vigorous response to the latter two stimuli. In contrast, nylon column eluate cells enriched for T cells responded well to CP and HSV with production of interferon. To resolve these contradictory data, we have used combinations of techniques. Nylon column eluate cells were further separated by SRBC and it was found that the nylon non-adherent rosetting cell did not produce interferon in response to HSV or CP whereas the nylon-nonadherent non-rosetting cell did. In additional experiments more elaborate techniques were used. Leucocytes were treated by plastic adherence and iron filings, passed over a nylon column and subsequently over an Ig-anti-Ig column, then rosetted with SRBC. Again only the non-rosetting population produced interferon. In parallel experiments the capacity of the different cell populations to lyse three types of target cells in a chromium release assay as a test for natural killer (NK) cell activity was investigated. There was some correlation between interferon production and NK cell activity. Thus, our data indicate that interferon is produced by non-T, non-B cells, possibly by cells related to NK cells.

Partial purification of a bone-resorbing factor elaborated from human allogeneic cultures

Human mixed leukocyte culture supernatants contain a factor(s) capable of effecting resorption of fetal rat bones in vitro. We have investigated procedures for the large-scale generation and partial purification of this bone-resorbing factor(s). Human mononuclear leukocytes obtained by leukapheresis from two healthy donors (average of 1 × 10 10 cells/donor) were cultured together (2.5 × 10 6 cells/ml) for 40 hr, the supernatants harvested and assayed for bone-resorbing activity by measurement of 45Ca release from fetal rat bones in vitro. These supernatants consistently induced significant bone resorption when compared to supernatants from a single donor's cells. Neither the release of this bone-resorbing factor nor its biologic activity were abrogated by addition of indomethacin. The bone-resorbing factor(s) eluted from Sephadex G-75 at an apparent molecular weight of 11,000 to 18,000 and appeared only in the breakthrough when fractionated on DEAE-cellulose (0.005 M phosphate, 0.02 M NaCl, pH 7.5). A concentrated eluate containing bone-resorbing activity after two cycle Sephadex G-75 gel filtration and DEAE-cellulose chromatography was applied to anionic polyacrylamide disc gels. A single peak of bone-resorbing activity was eluted, although this activity could not be attributed to a single stainable protein band. The results indicate that human mixed leukocyte cultures elaborate a low-molecular-weight boneresorbing factor(s) resembling the previously-described osteoclast-activating factor. Methods presented for the large-scale generation and partial purification of this activity should facilitate clarification of the role of this mediator in tissue destruction.

Sister chromatid exchanges in human leukocyte chromosomes: spontaneous and induced frequencies in early- and late-proliferating cells in vitro.

Human leukocyte cultures were pulse-treated with the trifunctional alkylating mutagen trenimon in a final concentration of 10(-7) M for 15--20 h after culture start, i.e., in the G1 phase of the cell cycle. At 24 h after culture start bromodeoxyuridine (BUdR) was added to the trenimon-treated cultures and to several untreated cultures running in parallel. The series treated with BUdR only and the series treated with BUdR+trenimon were each used to prepare two cultures at different culture times. Mitoses were collected during consecutive intervals of 12 h from 30 h up to 102 h after culture initiation by colcemid. For all preparation times (42 h, 54 h, 66 h, 78 h, 90 h, and 102 h) the frequencies of first, second, and third and further mitoses were determined in the BUdR- and in the BUdR+trenimon-treated series. In the trenimon-treated series a clear cell cycle delay was detected as compared with the normal distribution of different types of mitoses found in series treated with BUdR only. Spontaneous and trenimon-induced sister chromatid exchange (SCE) frequencies were determined in second mitoses occurring at 66 h, 78 h, 90 h, and 102 h after culture start. For all these preparation times about six SCE per metaphase were consistently found in BUdR-treated, and about 19 SCE per metaphase in BUdR+-trenimon-treated series, indicating a homogeneous sensitivity of early- and late-proliferating cells with respect to the induction of SCE.

Improved technique for human leukocyte cultures

An improved technique for cultivation of peripheral human lymphocytes is presented. The setting of the blood cells is replaced by a wash in Ficoll to remove the polymorphonucleocytes.

Recent data on the examination of the mutagenic effect of a dinitro- o-cresol-containing pesticide by different test methods

The mutagenic effect of the 50% dinitro-o-cresol-containing pesticide Krezonit E was assessed by the following test methods: (1) human leukocyte cultures in vitro; (2) mouse bone-marrow chromosome preparations in vivo; and (3) examination of gametic mutations (a) in mouse test preparations, (b) with dominant lethality, and (c) by the examination of F1 embryonic chromosomes. According to the results the dinitro-o-cresol-containing pesticide is mutagenic. In all test systems aberrations both in the somatic and in the gametic cells could be detected. There was a significant increase in the rate of breaks and deletions and of the univalents in the diakinesis phase of meiosis found to be pathognomic by Schleiermacher [Humangenetik 3, 127–133 (1966)]. The chromosome aberrations of the F1 embryos were significantly higher in the first, second, and third week after the treatment of the male parent than in the control group. According to these findings the conclusion can be drawn that damage in the more mature forms of spermiogenesis can be better detected by examining the F1 embryonic chromosomes, while the aberrations of the early forms can be shown much better by dominant lethality.

Comparative studies of chromosomal aberration and mutagenicity of trivalent and hexavalent chromium

The comparative cytogenetic and mutagenic effects between trivalent and hexavalent chromium were investigated. Five chromium compounds, K 2Cr 2O 7 and K 2CrO 4 containing Cr 6+, and Cr(CH 3COO) 3, Cr(NO 3) 3 and CrCl 3 containing Cr 3+, were examined for their ability to induce chromosomal damage in cultures of human leukocytes, for their reactivity with DNA by a rec-assay system and for mutagenicity in the E. coli Hs30R test system. Chromosome-breaking activity was significantly higher for the compounds with hexavalent than trivalent chromium, the efficiency being in the decreasing order K 2Cr 2O 7 > K 2CrO 4 >> Cr(CHCOO) 3 > Cr(NO 3) 3, CrCl 3. In the rec-assay and mutation assay, hexavalent (K 2Cr 2O 7 and K 2CrO 4) and trivalent Cr(CH 3COO) 3) compounds gave positive results, their mutagenic potential being higher in the same order of clastogenic magnitude.

Modulation of phytohemagglutinin-mediated lymphocyte stimulation by egg lecithin

Egg lecithin at 200 μg/ml or greater was found to abrogate blastogenic responses of human leukocyte cultures stimulated with phytohemagglutinin (PHA), concanavalin A (ConA), purified protein derivatives of tuberculin (PPD), candidin or streptokinase and streptodornase (SKSD) while leukocyte aggregation mediated by PHA, however, occurred irrespective of the presence of the lipid. Inhibition of PHA-mediated responses occurred when the lecithin was added simultaneously with the mitogen but the extent of inhibition decreased with delay in the addition of the lipid. When added 48 h after their exposure to PHA, the presence of lecithin did not change the pattern of [ 3H]TdR incorporation. Prior sonic treatment of an aqueous suspension of lecithin was found to render the lipid more effective in arresting lymphocyte responses than the unsonicated control. In entity, these results indicated that the lipid did not affect the viability of the leukocyte cultures nor did it seem that the lipid may interfere with the binding of the mitogen per se. It was concluded therefore, that inhibition by lecithin may most likely occur early at a time following binding of the mitogen but before the cells are committed to cellular division and it seems that the plasma membrane might be a likely site of action of the lipid.

Testing in vitro of an indirect mutagen (cyclophosphamide) with human leukocyte cultures Activation by liver perfusion and by incubation with crude liver homogenate

The indirect mutagen cyclophosphamide was tested in human whole blood cultures with respect to chromosome-breaking activities and suppression of mitotic indices after activation by liver perfusion and crude liver homogenates with and without cofactors. Both methods produced nearly the same effects, with the exception of liver homogenate without cofactors which had only weak metabolic activities.

Production of migration inhibitory factor (MIF) by human leukocytes following exposure to Epstein-Barr virus

The ability of Epstein-Barr virus (EBV) to induce a cell-mediated immune response (CMI) in cultures of human leukocytes was investigated. Partially purified EBV, obtained from culture fluids of AV-1 cells, was inactivated by uv-irradiation. Inactivated virus was mixed with peripheral leukocytes from Hodgkin's disease (HD), infectious mononucleosis (IM) and malignant lymphoma patients as well as from normal individuals in an in vitro culture system. Production of migration inhibitory factor (MIF), as measured by guinea pig macrophage migration inhibition (MMI), was utilized as an indicator of CMI response. Significant differences in MIF response were observed subsequent to exposure of the cells to EBV. Leukocytes from patients in each of the disease categories tested exhibited greater MIF production than did those from the normal controls. There were significant differences in MIF production by leukocytes from the malignant and non-malignant disease categories. Serum from each subject was examined for immunoglobulin specific for EBV capsid antigen (anti-VCA). Although the majority of individuals within the disease categories tested had elevated anti-EBV serum titers, no correlation could be made between elevated anti-VCA titer and levels of MIF production.

In vitro testing of an indirect mutagen (cyclophosphamide) with human leukocyte cultures. Activation by a non-enzymatic hydroxylation system (undenfriend system)

In vitro testing of an indirect mutagen (cyclophosphamide) with human leukocyte cultures. Activation by a non-enzymatic hydroxylation system (undenfriend system)

Chromosome-damaging effect of betel leaf

The chewing of betel leaf with other ingredients is a widespread addiction in India. The chromosome damaging effect was studied in human leukocyte cultures. There was an increase in the frequency of chromatid aberrations when the leaf extract was added to cultures.

In vitro testing of an indirect mutagen (cyclophosphamide) with human leukocyte cultures: Activation with liver microsomes and use of a dialysis bag

In vitro testing of an indirect mutagen (cyclophosphamide) with human leukocyte cultures: Activation with liver microsomes and use of a dialysis bag

Human sister chromatid exchange caused by methylazoxymethanol acetate

The incidence of sister chromatid exchange was determined in human leucocyte cultures treated with methylazoxymethanol acetate. In all individuals examined, treated cultures manifested a significantly higher frequency of sister chromatid exchanges than controls. Two concentrations of MAM AC were tested in blood cultures from nine individuals. The concentrations varied from individual to individual since they were determined by means of individual dose-response curves, which involved [ 3H]-thymidine incorporation in PHA-stimulated short-term lymphocyte cultures versus MAM AC concentration. The lower concentration was less than the TD 50 dose. Compared to control cultures, the lower concentration caused a higher incidence of sister chromatid exchange in eight of nine individuals. The cumulative mean value for all control cultures was 5.32 exchanges per cell while that for cultures treated with the higher concentration was 10.73.