In vitro proliferation of macrophage depleted human peripheral blood lymphocytes.

The incorporation of thymidine by normal human peripheral blood lymphocytes was tested in vitro following various culture conditions. A significant increase of thymidine uptake was observed in cultures depleted of plastic adherent, nylon wook adherent, or phagocytic cells. This proliferative activity occurred in the presence of various sera but was not due to a blastogenic response to a foreign protein, since it was also observed when autologous plasma was the only source of protein in the culture medium. The similarities and differences between this 'spontaneous' proliferative phenomenon and other blastogenic responses which are regulated by macrophages are discussed.     

In vitro effects of 3-acetyl-deoxynivalenol on the immune response of human peripheral blood lymphocytes

The effects of 3-acetyl-deoxynivalenol (3-AcDON) on the in vitro mitogen responses and the antibody producing ability of human peripheral blood lymphocytes were evaluated. 3-AcDON inhibited the proliferative response to pokeweed mitogen and concanavalin A at a lower concentration (100 ng/ml) as compared to phytohemagglutinin (200 ng/ml). The antibody producing ability was inhibited by 3-AcDON concentrations of greater than or equal to 200 ng/ml. Higher concentrations of 3-AcDON (greater than or equal to 300 ng/ml) produced severe suppression of plaque forming cell response in vitro and reduced the total yield of lymphocytes without altering cell viability. The results of this study indicate that 3-AcDON produces immunosuppressive effects in a dose dependent manner in vitro.     

In vitro synthesis of IgG by peripheral blood lymphocytes in chronic liver disease.

In vitro IgG synthesis by peripheral blood mononuclear cells (PBM) from patients with chronic liver disease (CLD) was studied. In addition, the effect of pokeweed mitogen (PWM), polyadenylic-polyuridylic acid complexes (poly A:U) and thymosin fraction 5 on IgG synthesis was determined. Unstimulated cultures of PBM from patients with chronic active hepatitis (CAH) and alcoholic cirrhosis (AC) synthesized significantly higher quantities of IgG than the controls. Moreover, there was a direct correlation between serum IgG concentrations and the quantity of newly synthesized IgG in these unstimulated cultures. PWM, poly A:U and thymosin each stimulated increased IgG synthesis in the controls. While neither poly A:U nor thymosin enhanced IgG synthesis in patients with CLD, PWM increased IgG synthesis in CAH but not AC. These results indicate that spontaneous in vitro B cell synthesis of IgG is enhanced in CLD and may reflect antigenic stimulation in vivo.     

In vitro evaluation of the genotoxicity of acetamiprid in human peripheral blood lymphocytes

Acetamiprid, a neonicotinoid insecticide, is commonly used both in agriculture and domestic areas against a wide range of insects. The potential genotoxicity of a commercial formulation of acetamiprid (Mosetam 20 SP, containing 20% acetamiprid as the active ingredient) on human peripheral blood lymphocytes was examined in vitro by sister chromatid exchange (SCE), chromosomal aberrations (CAs), and micronucleus tests. Cells were treated with 25, 30, 35, and 40 mug/ml of acetamiprid for 24 and 48 hr. Acetamiprid induced SCEs and CAs significantly at all concentrations and treatment times and micronucleus formation was significantly induced at 30, 35, and 40 mug/ml of acetamiprid as compared with both the control and solvent control. Acetamiprid decreased the proliferation index (PI) at the two highest concentrations (35 and 40 mug/ml) for the 24-hr treatment period and only at the highest concentration (40 mug/ml) for the 48-hr treatment period when compared with the control and solvent control. Peripheral lymphocytes exposed to all concentrations of acetamiprid showed significant decreases in mitotic index (MI) and nuclear division index (NDI) for both treatment periods when compared with both the control and solvent control. Furthermore, acetamiprid decreased the MI in both treatment periods, and the NDI only in the 24-hr treatment period to the same extent as the positive control, mitomycin C (MMC). This study presents the first in vitro evidence for the genotoxicity of a commercial formulation of acetamiprid in human peripheral lymphocytes.     

In vitro antibody response of primed rabbit peripheral blood lymphocytes to group A variant streptococcal polysaccharide

Peripheral blood lymphocytes obtained from rabbits at various times after a primary course of immunization with streptococcal Group A-variant vaccines were successfully stimulated in vitro with this vaccine. Day 5 cultures contained about 81 % IgM and only 19 % IgG plaque-forming cells, day 10 cultures contained only 38 % IgM and 62 % IgG plaques, and day 15 cultures contained predominantly IgG plaques (83 %) and far fewer IgM plaques (17 %). Concomitantly, IgM and IgG secretion into the media was shown. Analysis of the secreted IgG antibodies by isoelectric focusing showed in principle that the clones stimulated in vitro were those that had determined the in vivo response. These data demonstrate the circulation of clonal progenitor cells in the rabbit peripheral blood.     

Enumeration of human peripheral blood lymphocytes secreting immunoglobulins of major classes and subclasses in healthy children and adults

The reverse enzyme-linked immunospot assay was modified to enumerate peripheral blood mononuclear cells (PBMC) secreting IgG1–4, IgA1–2, and IgM. Anti-human IgG F(ab')₂ and mouse monoclonal antibodies specific to each of the isotypes were used as solid-phase capture antibodies and developing antibodies, respectively. Although attempts were also made to detect IgD- and IgE-secreting cells (SC), their numbers in the peripheral blood were too few to be reliably estimated. The assay was applied to study healthy subjects including 21 neonates within 3 days of birth, 44 1- to 48-month-old children, and 32 adults. Sixty percent of these neonates had IgM SC in small numbers (<20 per 10⁶ PBMC), but only three neonates had IgSC of other isotypes. In contrast, by 1–2 months of age children had IgSC of all isotypes, including IgA2 and IgG4, often in higher numbers than adults. The relative frequencies of IgSC were IgA1 > IgG1 > IgM > IgG2 > IgG3 > IgG4 > IgA2 in the children and IgA1 > IgG1 > IgA2 > IgM > IgG4 > IgG2 > IgG3 in the adults. The order of the serum concentrations in the adults was IgG1 > IgG2 > IgA > IgM > IgG4 > IgG3. No correlation was found between the serum level and the IgSC number of individual isotypes (except for serum IgA and IgA1-SC). This new methodology should facilitate investigating the current status of immunoglobulin synthesis and the Ig repertoire in adults and children, in health and in disease.Dedicated to the memory of Dr. Charles Reimer.     

In vitro X-ray irradiation of human peripheral blood T lymphocytes enhances suppressor function

We studied the effect of in vitro X-ray irradiation on human peripheral blood T lymphocytes, with regard to their suppressor activity related to the concanavalin A (Con A)-induced suppressor system. To generate suppressor T lymphocytes, purified human T lymphocytes were incubated for 3 days in the first culture, with or without Con A. These lymphocytes were irradiated with various doses of X-ray before, mid or after the culture. After doing a second culture for 6 days, we measured the suppressive influence of these cells on T lymphocyte proliferation rates stimulated with allogeneic mononuclear cells, and B lymphocyte proliferation rates stimulated with pokeweed mitogen. Irradiation (1,000 rad) of cultures to which Con A had not been added induced much the same level of suppressor activity as seen in the cultures with Con A. The suppressor activity gradually increased with lapse of time from the irradiation to the suppressor cell assay. Suppressor T lymphocytes were resistant to X-ray irradiation and independent of DNA synthesis. On the other hand, irradiation-induced enhancement was minimal in cultures incubated with Con A, regardless of the irradiation time. As irradiation of human peripheral blood T lymphocytes was found to induce a suppressor function in vitro, clinical and experimental applications of irradiation in cases of a suppressed T lymphocyte function may be feasible.     

In vitro effect of statins on cytokine production and mitogen response of human peripheral blood mononuclear cells

The in vitro effect of the hydrophilic statin - pravastatin - and three hydrophobic statins - atorvastatin, lovastatin and simvastatin - on the production of IL-1beta, IL-1ra, IL-2, IL-6 and IFN-gamma by human peripheral blood mononuclear cells (PBMC) and their response to mitogens was examined. Lovastatin and simvastatin increased the production of IL-1beta in a dose dependent manner and reduced secretion of IL-1ra at high concentration. These two statins exerted a dose dependent inhibitory effect on IL-2 production and reduced the secretion of IFNgamma at high dose. Atorvastatin did not affect IL-1beta, but suppressed IL-1ra, IL-2 and IFNgamma production. Atorvastatin, lovastatin and simvastatin caused a dose dependent inhibition of mitogen-induced proliferation of PBMC. IL-6 production was not affected by any one of the statins. While pravastatin caused a slight reduction in the proliferative response of PBMC to PHA, it did not affect either their response to Con A and PWM, or the secretion of cytokines tested.     

In vitro response of peripheral blood mononuclear cells to phytohemagglutinin and interleukin-2

The serological determination of class II antigens is still a mandatory test prior to allotransplantation. It is known that these antigens are normally expressed on B lymphocytes and monocytes. The B lymphocytes that constitute 10% to 15% of total blood lymphocytes are the cells currently used for HLA-DR typing. To avoid HLA-DR typing difficulties, or even impossibilities that are frequently encountered among some patient groups, we studied the response of peripheral blood mononuclear cells--as an alternative source of cells for class II antigen typing--to in vitro mitogen and interleukin-2 activation and propagation. Although the patients included in this study were selected having previously known HLA-DR typing difficulties, all could be adequately typed by this method.     

Early protein synthesis in activated human peripheral blood mononuclear cells

Though B-cell division and Ig synthesis in response to pokeweed mitogen (PWM) require interaction with T-cells and monocytes, it is not clear which earlier events in B-cell activation share this requirement, and which are the result of direct interaction of mitogen with the B-cell. Having previously shown that the acceleration of lecithin synthesis in human B-cells at 16-20 hr requires both T-cells and monocytes, we now examine whether B-cells require similar interactions to increase their protein synthetic rate, another important activation event. At 21-24 hr of PWM stimulation, the stimulation index (SI) for incorporation of [35S]methionine into protein was 2.1 +/- 0.4 for unfractionated cells, 1.7 +/- 0.1 for B-cells, 2.5 +/- 0.1 for T-cells, and 3.4 +/- 0.5 for monocytes. Thus monocytes contributed substantially to early mitogen-induced protein synthesis by human peripheral blood mononuclear cells. When the monocyte/B-cell fraction (MB) and T-cell fraction (T) were mixed at various ratios in PWM-stimulated cultures, synergy was apparent at MB:T ratios of 1:1 and 1:2, indicating cell interactions augmented early mitogen-driven protein synthesis in at least one of these cell types. However, much or all of this synergy could be attributed to T-cells, whose protein synthetic response was augmented by B-cells and monocytes. In contrast, the early increase in B-cell protein synthesis appeared to be independent of cell interactions, since their SI of 1.7 was not influenced by varying the proportion of M- or T-cells over a 50-fold range. These contrasting results between two contemporary events fits the hypothesis that one (accelerated phospholipid synthesis) requires a first signal plus one or more cell interaction signals, whereas the other (accelerated protein synthesis) requires only the first signal.     

Induction of T cell growth factor synthesis in human peripheral blood lymphocytes by staphylococcal protein A

Staphylococcal Protein A (SpA) induces T cell growth factor (TCGF) production in cultures of human peripheral blood mononuclear cells (PBMC) with titers equivalent to those induced by PHA. The optimal time and cell concentration for TCGF production were found to be the same for SpA and PHA. TCGF induction by SpA was blocked by addition of human AB-serum or human IgG, as was the mitogenic effect of SpA. An easy and inexpensive procedure is described for the quantitative removal of SpA from TCGF without loss of TCGF activity.     

In vitro studies of suppressor cell function in human peripheral blood mononuclear cells.

Peripheral blood mononuclear cells (PBMC) from normal donors, pre-cultured at 37 degrees C for 24 hr before the addition of mitogen, demonstrated an enhanced proliferative response. This may be due to the loss of a subpopulation of suppressor cells during the incubation period. Still further enhancement was observed when pre-culturing was prolonged for 48 hr, while cells pre-incubated at 4 degrees C showed no increased responsiveness. Concanavalin A (Con A) pre-activated PBMC supressed the mitogen response of responder cells. More marked suppression was observed when the concentration of Con A used to induce the suppressor cells was increased. It was not possible to activate suppressor function in cells which had been kept in vitro for longer than 48 hr. These findings support the concept of the existence and function of suppressor cells, and that the suppressive influence is short-lived in vitro culture.     

In vitro production of IgE and IgG by sensitized human peripheral lymphocytes.

Antigenic stimulation of peripheral lymphocytes was made in two different groups of atopic patients, testing their capabilities to produce IgE and IgG in vitro. For this purpose, Marbrook's technique was used in the cultures. The immunoglobulins produced were measured in the supernatants by a sensitive double-antibody method. Results demonstrate that it is possible to produce enough IgE and IgG to be detected. Some important parameters had to be considered: appropriate selection of the patients and the antigens used in the antigenic stimulation, as well as the number of cells and the duration of the culture.     

Free DNA induces modification on the protein synthesis profile of human peripheral blood mononuclear cells of healthy donors

Understanding how free DNA might act as a signal between cells is important for knowing how DNA orchestrates immune responses and for optimizing the therapeutic of cancer, infection and immunologic diseases. This communication demonstrates that DNAs from different origins (bacteria, T. cruzi, HeLa cells) and synthetic oligonucleotide containing an unmethylated CpG motif are capable of inducing alterations in the protein profile of normal human leukocytes. As far as we know there have been no similar studies regarding the comparative effects of different free DNAs on early protein synthesis of human peripheral blood mononuclear cells.     

In vitro synthesis of IgE by human peripheral blood leucocytes. III. Release of pre-formed antibody

The appearance of IgE in supernatant fluids from cultures of human peripheral blood leucocytes (PBL) is employed in many laboratories as an index of antibody 'synthesis'. This study demonstrates that, unlike IgG, the majority of cell associated IgE is sequestered by PBL in a tightly bound form, which resists extraction via the freeze-thawing (F/T) techniques in current use. A method is presented for the quantitative extraction of cell associated IgE, involving brief acid treatment of whole cells, and is shown to yield up to 100% more IgE than the F/T procedure. The use of this extraction process on PBL before and after culture permits discrimination between release of pre-formed IgE and de novo synthesis, both of which are demonstrated to occur to widely differing degrees in PBL cultures from different donors.     

Antibodies to acetylcholine receptors in myasthenia gravis. In vitro synthesis by peripheral blood lymphocytes before and after thymectomy.

Pokeweed mitogen (PWM)-driven in vitro synthesis of antibodies to the acetylcholine receptor (PSA) was studied in non-thymoma patients with myasthenia gravis. In a group of 46 patients, the occurrence of PSA was related to the presence of the thymus or, in operated patients, the absence of a clinical effect of thymectomy. Sixteen patients were followed before and soon after thymectomy. PSA disappeared in all patients, at least temporarily, between 6 weeks and 1 year afterwards, independent of the clinical course and eventual clinical effect of the operation. A recurrence was found only in one of the five patients who derived no benefit from the operation. These findings support the hypothesis that the therapeutic effect of thymectomy can be explained by removal of a source of autoreactive lymphocytes. There was no correlation between the changes in serum levels of a-AChR and clinical improvement, suggesting a minor role of circulating peripheral blood lymphocytes (PBL) and the thymus in the total production of a-AChR.     

Inhibition of phytohaemagglutinin-induced autorosette formation of human peripheral blood lymphocytes by RNA or protein synthesis inhibitor.

The induction of autorosette forming cells (ARFC) with phytohaemagglutinin (PHA) in human peripheral blood lymphocytes (PBL) was examined. After 24 h of incubation with PHA, the level of ARFC in PBL was markedly increased but subsequently decreased to about one-third of the peak level at 96 h of culture. When PBL were pre-treated with actinomycin D, or cultured with puromycin, the induction of ARFC was completely blocked. Pre-treatment of PBL with mitomycin C (MMC) had no effect on induction of ARFC, whereas DNA synthesis was completely blocked. These data indicate that the generation of autologous red blood cell receptors is a relatively early event in PHA activated PBL, and that it is independent of DNA synthesis but dependent on RNA and protein synthesis.