Human Peripheral Lymphocyte Cultures Research Articles

In vitro study of the effect of resveratrol purified from the skin of Iraqi black grape (Vitis vinifera) on lymphocyte cultures isolated from the blood of patients with lymphoma.

The natural stilbene compound resveratrol (RSV) was extracted and purified locally from the black grape skin (Vitis vinifera) cultivated in Iraq. Cultures of human peripheral lymphocytes were obtained from the blood samples of patients with and without lymphoma to be treated with RSV at different concentrations. Three RSV concentration levels were subjected to isolated lymphocytes from blood samples of Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), and without lymphoma to estimate the change in TNF-α and IL-10. Resveratrol seemed to differently affect cytokines level in normal and lymphoma lymphocytes in relation to its concentration. The lowest resveratrol concentration (50 µg/ml) decreased TNF-α levels for patients without lymphoma and all NHL patients in contrast to the HL sample. Treating normal lymphocytes with a higher dose (1000 µg/ml) might elevate the levels of TNF-α in almost all samples. There was an inverse relationship between both cytokines in most treatments; with the increase in TNF-α level, there was a decrease in IL-10 level except in HL and normal lymphocytes treatment. The locally purified resveratrol could serve as a multi-target drug that modulates the immune system to improve body defense in patients suffering from lymphoma and in patients without lymphoma by altering cytokine levels in response to different resveratrol concentrations in a different manner.

Antimutagenic Effects of the Composition from Green Tea Leaves Extracts and Caucasian Persimmon Fruits.

On a culture of human peripheral blood lymphocytes, antimutagenic activity of a composition from extracts of green tea leaves and Caucasian persimmon fruits was established with a modification of the mutation process induced by chemical compounds producing an alkylating effect (nitrosomethylurea and sodium fluoride). A concentration dependence of the antimutagenic efficiency of the studied phytocomposite was shown. The highest antimutagenic efficiency was observed when a combination of green tea extract at a concentration of 0.01 μg/ml and persimmon fruit extract at a concentration of 0.001 μg/ml were used. Moreover, this combination was most effective against mutations induced by both nitrosomethylurea and sodium fluoride: the antimutagen efficiency factor was 0.53 and 0.55, respectively.

Cytomic analysis: a modern universal tool for biomedical and ecological and hygienic research (literature review). Part 2

In the second part of the review, examples of cytome analysis on both peripheral blood lymphocytes and human buccal epiteliocytes are given in the study of occupational risks and investigation of genome instabilities induced by alcohol and tobacco use. Much attention is paid to the cytome analysis application in environmental and hygienic research and the studies directed to evaluating natural and anthropogenic load to the human population, especially industrial, factors. The method of cytome analysis on primary culture of human peripheral blood lymphocytes cultivated with a cytokinetic block (CBMN-cyt test) in its current state is described in detail, and the history of its development is briefly reflected. The method of cytome analysis on human buccal epiteliocytes (BMN-cyt) is also analyzed. The review contains photomicrographs of genetic cell damages that are markers in micronuclei (MN) tests. We used PubMed, Web of Science, ResearchGate, Scopus, eLibrary databases as the sources of literature.

Cytogenetic and Oxidative Status of Cultured Lymphocytes after Exposure to Extracts from Rhizopogon Mushrooms (Agaricomycetes) In Vitro.

In this study, acetone and water extracts obtained from edible mushrooms, Rhizopogon luteolus Fr. and Rh. roseolus (Corda) Th. Fr., containing important bioactive components were used. Total antioxidant capacity (TAC) and total oxidative stress (TOS) levels were examined on human peripheral lymphocyte culture treated with the respective extracts. Levels of genetic damage and cytotoxic effects of the respective extracts on lymphocytes were also tested. In general, when TAC levels of the extracts on cells were examined, a concentration-dependent increase was observed; a negative correlation was found between TOS data and concentration. Genotoxicity tests (chromosome aberration and micronucleus analysis) revealed that the concentrations of the extract applications did not significantly (P > 0.05) change genotoxicity on human peripheral lymphocyte culture compared to the negative control. Considering all of the results obtained, it was determined that applications of Rh. luteolus and Rh. roseolus extracts, especially at concentrations of 50 and 100 mg/L, increased the TAC value of lymphocytes, which play an important role in the human immune system, without causing genetic or oxidative stress.

Антимутагенные эффекты композиции из экстрактов листьев зелёного чая и плодов кавказской хурмы

On a culture of human peripheral blood lymphocytes, antimutagenic activity of a composition from extracts of green tea leaves and Caucasian persimmon fruits was established with a modification of the mutation process induced by chemical compounds producing an alkylating effect (nitrosomethylurea and sodium fluoride). A concentration dependence of the antimutagenic efficiency of the studied phytocomposite was shown. The highest antimutagenic efficiency was observed when a combination of green tea extract at a concentration of 0.01 μg/ml and persimmon fruit extract at a concentration of 0.001 μg/ml were used. Moreover, this combination was most effective against mutations induced by both nitrosomethylurea and sodium fluoride: the antimutagen efficiency factor was 0.53 and 0.55, respectively.

Studying the mutagenic activity of drinking water and soil samples selected from Kentau and adjacent territories

In the course of carrying out a comprehensive assessment of the state of the environment and health of the population of the city of Kentau and adjacent territories, a study of the mutagenic activity of drinking water and soil samples taken in the territory of Kentau and adjacent settlements, the background territory and the comparison territory was carried out. In the course of the study, it was revealed that individual samples of drinking water and water extracts of soils cause a significant increase in the frequency of chromosomal abnormalities in the cultures of human peripheral blood lymphocytes and in the Allium-test. Analysis of the total mutagenic activity of the samples under study indicates the absence of a fundamentally contaminated and/or clean territory in the studied region, which indicates their general contamination with a tendency to increase contamination in some places.

Cytogenetic and oxidative effects of three lichen extracts on human peripheral lymphocytes.

In the present study, we investigated cytogenetic and oxidative [total antioxidant capacity (TAC), totaloxidant status (TOS)] effects of methanol and water extracts of Cladonia chlorophaea (Flörke ex Sommerf.) Sprengel, Dermatocarpon miniatum (L.) W.Mann and Parmelia saxatilis (L.) Ach. on cultured human lymphocytes. In addition, different phenolic compounds in the extracts were quantified by high performance liquid chromatography (HPLC) analysis. As a result of HPLC analysis, methanol extracts of all lichen species tested had higher phenolic compounds. Likewise, methanol extracts of eachlichen increased TAC levels in lymphocytes more thanwater extracts. The TOS levels of the cells treated with different concentrations (1-100mg/L) of the extracts decreased due to the increasing concentration of the extracts. Genotoxicity experiments revealed that the tested lichen extracts did not significantly increase (p>0.05) thelevel of genotoxicity on human peripheral lymphocyte culture compared to the negative control group. The results showed that C.chlorophaea, D.miniatum and P.saxatilis lichens, which were found to be a rich source of phenolic compounds, might be of interest in the pharmaceutical and food industries.

Antigenotoxic and Antimutagenic Effects of Andrographis paniculata , a Traditional Medicinal Herb against Genotoxicity of Cyclophosphamide: An In Vitro Study on Human Peripheral Lymphocytes

Andrographis paniculata (family: Acanthaceae) is a medicinal herb—used in Indian system of medicine (Ayurveda, Siddha, and Unani), traditional and folk systems to treat various illnesses. This study examined the phytochemical constituents of ethanol extract from A. paniculata and its protective effect against genotoxicity caused by cyclophosphamide (CPA). Phytochemical screening and estimation of total phenolic content were analyzed using standard methods. The bioactive components from the ethanol extract of A. paniculata (EAP) were analyzed using gas chromatography-mass spectrometry. To investigate the protective effect of EAP against CPA-induced genotoxicity, human peripheral lymphocyte cultures were used. To test the antigenotoxic and antimutagenic effects of EAP, lymphocytes were treated with different concentrations of extract (50∼250 mg/mL) alone and co-treated along with CPA+EAP for 48 h. The cells were analyzed for structural chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) in control, CPA treated, and CPA+ EAP co-treated lymphocytes. Results of the study revealed that the lymphocyte cultures which had 48 h continuous exposure to EAP (50∼250 mg/mL) did not show any significant changes in CAs and SCE frequencies. These results substanti-ate the antimutagenic nature of the extract. Furthermore, the lymphocytes co-treated with CPA along with extract showed a significant reduction in CAs (reduced from 26.50±2.50% to 11.00±1.00%) and SCEs (reduced from 9.92±0.63 per cell to 4.56±0.18 per cell). These results suggest that A. paniculata is protective against CPA induced genotoxicity and put forward its possible use as a supplement with chemotherapeutic drugs.

Cytokinesis-Block Micronucleus Assay Using Human Lymphocytes as a Sensitive Tool for Cytotoxicity/Genotoxicity Evaluation of AgNPs

Silver nanoparticles (AgNPs) are the most used nanomaterials worldwide due to their excellent antibacterial, antiviral, and antitumor activities, among others. However, there is scarce information regarding their genotoxic potential measured using human peripheral blood lymphocytes. In this work, we present the cytotoxic and genotoxic behavior of two commercially available poly(vinylpyrrolidone)-coated silver nanoparticle (PVP–AgNPs) formulations that can be identified as noncytotoxic and nongenotoxic by just evaluating micronuclei (MNi) induction and the mitotic index, but present enormous differences when other parameters such as cytostasis, apoptosis, necrosis, and nuclear damage (nuclear buds (NBUDs) and nucleoplasmic bridges (NPBs)) are analyzed. The results show that Argovit (35 nm PVP–AgNPs) and nanoComposix (50 nm PVP–AgNPs), at concentrations from 0.012 to 12 μg/mL, produce no changes in the nuclear division index (NDI) or micronuclei (MNi) frequency compared with the values found on control cultures of human blood peripheral lymphocytes from a healthy donor. Still, 50 nm PVP–AgNPs significantly decrease the replication index and significantly increase cytostasis, apoptosis, necrosis, and the frequencies of nuclear buds (NBUDs) and nucleoplasmic bridges (NPBs). These results provide evidence that the cytokinesis-block micronucleus (CBMN) assay using human lymphocytes and evaluating the eight parameters provided by the technique is a sensitive, fast, accurate, and inexpensive detection tool to support or discard AgNPs or other nanomaterials, which is worthwhile for continued testing of their effectiveness and toxicity for biomedical applications. In addition, it provides very important information about the role played by the [coating agent]/[metal] ratio in the design of nanomaterials that could reduce adverse effects as much as possible while retaining their therapeutic capabilities.

The Determination of the Effects of Wild Thyme (Thymbra spicata L.) and Cumin (Cuminum cyminum L.) Extracts on SCE (Sister Chromatid Exchange) in Human Lymphocyte Chromosomes

The purpose of this work is to determine of T. spicata L. and C. cyminum L. plant extracts on sister chromatid exchange rate in human peripheral lymphocyte culture as in vitro . Human blood lymphocyte cells, T. spicata L. and C. cyminum L. plant extracts were allowed to interact with doses of 0.05 µL/mL, 0.10 µL/mL, 0.15 µL/mL and 0.20 µL/mL for 24 hours. When trial groups where plant extracts were applied were compared with negative control and mitomycin-C (MMC) which was used as positive control, it was determined that the extract doses applied led to an increase in sister chromatid exchange rate. Also, it was found that increasing concentrations of plant extracts caused cell replication index to decrease. Between C. cyminum L. doses and RI, a negative correlation (r = -0.95) was observed and between T. spicata L . doses and RI a negative correlation was observed (r = -0.94) respectively. As a result of this study; T. spicata L. and C. cyminum L. plant extracts were found to have genotoxic and clastogenic effects on human peripheral lymphocytes.

Biological relevance of CuFeO2 nanoparticles: Antibacterial and anti-inflammatory activity, genotoxicity, DNA and protein interactions

Heterometal oxide nanoparticles of bioessential metals are shedding new light to nanoparticle-inspired bioapplications. Pairing bioreactive elements like copper and iron can affect the redox dynamic and biological profile of the nanomaterial. Given the complexity of physicochemical properties, biological activity and toxicity concerns, extensive exploration is demanded, especially when active and less active oxidation states participate as in case of cuprous-ferric delafossite CuFeO2 (copper(I)-iron(III)), a less widespread nanomaterial. In that vein, CuFeO2 nanoparticles were synthesized and biological profile was evaluated in comparison with cuprous oxide (Cu2O NPs) counterpart, an already established antimicrobial agent. Interactions with bacteria, proteins and DNA were examined. Cu2O NPs exhibited stronger antibacterial activity (IC50 < 25 μg/ml) than CuFeO2 NPs (IC50 > 100 μg/ml). In vitro exposure of nanoparticles on plasmid DNA unveiled toxicity in the form of DNA damage for Cu2O and enhanced biocompatibility for CuFeO2 NPs. Genotoxicity estimated by the frequency of sister chromatid exchanges, cytostaticity based on the proliferating rate indices and cytotoxicity based on the mitotic indices at human peripheral lymphocyte cultures were all significantly lower in the case of CuFeO2 NPs. Furthermore, through in vitro albumin denaturation assay, CuFeO2 NPs showed better performance in protein denaturation protection, correlating in superior anti-inflammatory activity than Cu2O and similar to acetylsalicylic acid. Synergy of copper(I)-iron(III) in nanoscale is apparent and gives rise to fruitful bioapplications and perspectives.

Study the impact of astaxanthin on developing of genomic instability in human peripheral blood lymphocytes irradiated in vitro on G2 phase of cell cycle

To identify the possibility of modification by astaxanthin the level of genome damages induced by gamma quanta in the culture of human peripheral blood lymphocytes exposed in vitro on postsynthetic (G2) phase of the first mitotic cycle. Peripheral blood lymphocytes from four apparently healthy volunteers 35-51 years old were cultivated using modified micromethod. To obtain genomic damages in G2 phase of the first mitotic cycle the part of cultures was irradiated by γ quanta in dose 1.0 Gy through 46 hours of cultivation. Astaxanthin in final con centration 20 μg/ml was exposed to lymphocytes' cultures before the irradiation. Cytogenetic analysis the uniform ly stained slides of metaphase chromosomes was carried out to determine the frequencies of chromosome and chro matid types of aberrations. Using the method of individual cells electrophoresis (Comet assay) the relative level of DNA damages (Tail Moment index) and the frequency of apoptotic cells with high level of DNA fragmentation were evaluated. Mean group frequencies of chromosome aberrations after gamma irradiation of lymphocytes in vitro exceed ed those without radiation exposure and were 72.35 ± 1.17 and 2.46 ± 0.30 per 100 metaphases, respectively (p < 0.001), mainly due to chromatid type of aberrations (58.32 ± 1.29 per 100 metaphases). Adding of astaxanthin into culture medium before the irradiation did not result in changes as in the frequency of chromosomal damages (71.54 ± 1.34 per 100 metaphases) as in the spectrum of aberrations - also prevailed chromatid type of aberrations (58.47 ± 1.47 per 100 metaphases). The increase of Tail Moment index after radiation exposure (from 3.84 ± 0.36 to 12.06 ± 1.88, respectively, p < 0.001) and lack of significant impact of astaxanthin on this index in the irradiated lym phocytes (8.96 ± 2.39, p > 0.05) was established, ie astaxanthin didn't change the relative level of radiation induced DNA damages. Also apoptogenic effect of astaxanthin was not found: frequency of apoptotic cells were (2.25 ± 1.49) % in cultures of intact lymphocytes, (2.08 ± 1.54) % in irradiated cultures and (1.78 ± 1.25) % under joint action of gamma radiation and astaxanthin (p > 0.05). Noimpactofastaxanthinongenomicinstabilityinducedbygammairradiation invitroinculturesof human peripheral blood lymphocytes on postsynthetic (G2) phase of first mitotic cycle had been established.

The mitochondrial apoptotic pathway is induced by Cu(II) antineoplastic compounds (Casiopeínas®) in SK-N-SH neuroblastoma cells after short exposure times.

The family of Copper(II) coordination compounds Casiopeínas® (Cas) has shown antiproliferative activity in several tumour lines by oxidative cellular damage and mitochondrial dysfunction that lead to cell death through apoptotic pathways. The goal of this work is looking for the functional mechanism of CasIIgly, CasIIIia and CasIIIEa in neuroblastoma metastatic cell line SK-N-SH, a paediatric extra-cranial tumour which is refractory to several anti-carcinogenic agents. All Cas have shown higher antiproliferative activity than cisplatin (IC50=123μM) with IC50 values of 18, 22 and 63µM for CasIIgly, CasIIIEa and CasIIIia, respectively. At low concentrations and early times (4h), these compounds cause a disruption of the mitochondrial transmembrane potential (Δψm). Concomitantly, an important depletion of intracellular glutathione and an increase of reactive oxygen species (ROS) hydrogen peroxide and radical superoxide were observed. On the other side, the lower cytotoxic effect of Casiopeínas on cultures of human peripheral blood lymphocytes (IC50CasIIgly =1720µM, IC50 CasIIIEa =3860µM and IC50 CasIIIia =4700µM) show the selectivity of these compounds over the tumour cells compared with the non-transformed cells. Chemically, glutathione (GSH) interacts with Casiopeínas® through the coordination of sulphur atom to the metal centre, process which facilitates the electron transfer to get Cu(I), GSSG and the posterior production of ROS. Additionally, the molecular structure of CasIIIia as nitrate is reported. These results have shown that the anticarcinogenic activity of Casiopeínas® on neuroblastoma SK-N-SH is through mitochondrial apoptosis due to the enhanced pro-oxidant environment promoted by the presence of the coordination copper compounds.

The impact of astaxanthin on radiation-induced chromosome aberrations in human peripheral blood lymphocytes in vitro

Aim. Research objective is to establish the possibility of modifying the astaxanthin (a carotenoid from a xanthophyll group) radiation-induced cytogenetic effects in human peripheral blood lymphocytes (PBLs) in vitro. Methods. The cultivation of PBLs from four conventionally healthy volunteers, the preparation and analysis of uniformly stained slides of metaphase chromosomes. Astaxanthin in final concentrations of 2, 10 and 20 µg/ml was added into the culture of PBL prior to the incubation before irradiation with γ-quanta in a dose of 1 Gy. Results. Astaxanthin did not affect the level and spectrum of chromosome damage in non-irradiated PBLs both in individual persons, and along the group on average (P&gt;0.05), indicating a lack of mutagenic activity. The effect of astaxanthin at a concentration of 20 μg/ml on irradiated PBLs resulted in a significant reduction of radioinduced cytogenetic effect in all donors. Medium-group level of chromosome aberrations decreased almost 3 times and was characterized by statistically significant (P&lt;0.001) decrease in frequency of chromosomal type aberrations due to the classical unstable cytogenetic markers of radiation effect, dicentrics and ring chromosomes. Conclusions. Astaxanthin at a concentration of 20 µg/ml was found to reduce the mutagenic effect of ionizing radiation, thus suggesting its powerful radioprotective potential. Keywords: astaxanthin, culture of human peripheral blood lymphocytes, radiation mutagenesis, chromosome aberrations, radioprotective effect.

Analysis of Chromosome Aberrations Contained in Vitro Human Peripheral Blood Lymphocytes After Treatment with Ceftriaxone

The invention and use of antibiotics in treating infections is one of the greatest achievements of the twentieth century medicine. Antibiotics are one of the categories of pharmaceuticals with a broad and increasing application. The goal of this paper is to analyze the influence of different set of test concentrations of ceftriaxone antibiotics on the occurrence of chromosome aberrations after in vitro treatment with concentrations: 0.15 mg/ml, 0.25 mg/ml and 0.50 mg/ml. In the study was used the blood of healthy donors in vitro, treated with different concentrations of ceftriaxone. Ceftriaxone is a semisynthetic cephalosporine third generation antibiotic, with broad spectrum of activity and with specific characteristics (Nerlove et al. 1996). As a biomarker of genetic damage are used the methods of cultivation of peripheral blood lymphocytes and analysis of chromosome aberrations. Cytogenetic analysis of ceftriaxone genotoxicity was performed in 48-hour culture of human peripheral blood lymphocytes by test of standard chromosome aberration analysis according to Moorhead, with certain modifications. Insight into the frequency and type of chromosome aberrations is obtained by analyzing 100 metaphases per sample. By the analysis of 100 metaphases per sample was determined that the relative frequency of metaphases with chromosome aberrations is increased with increasing concentrations of ceftriaxone in lymphocyte culture. The increase in the frequency of structural aberrations was also positively correlated with the applied ceftriaxone concentrations. Metaphases with numerical and structural aberrations are registered in lymphocyte cultures treated with ceftriaxone concentration of 0.25 mg/ml and 0.50 mg/ml, but this increase was not significant compared to the control cells cultures. Significantly increased frequency of metaphases with structural chromosome aberrations in cultured human peripheral blood lymphocytes treated with concentrations of 0.25 mg/ml and 0.5 mg/ml compared to the control confirming clastogenic potential of this drug. Ceftriaxone also expressed aneugenic activity at the highest test concentration (0.50 mg/ml), confirming a statistically significant difference in the frequency of numerical aberrations in cultures treated with doses of 0.5 mg/ml.

338 Interferon alpha 21 levels are elevated in peripheral blood lymphocytes and aortic smooth muscle cells with the 9p21.3 coronary artery disease risk genotype

The 9p21.3 locus confers risk for coronary artery disease (CAD) by an unknown mechanism. A recent report identified several long-range enhancers, at CDKN2B, CDKN2A, MTAP and between the interferon omega-1 (IFNW1) and interferon alpha-21 (IFNA21) genes, whose chromatin structure is influenced by single nucleotide polymorphisms within the 9p21.3 risk locus as far as 1 million base pairs away. However, whether protein expression from the genes in the vicinity of these long-range enhancers is correlated with the 9p21.3 risk genotype was not known. Here, we applied immunoblot analysis using antibodies specific to the proteins encoded by the genes near the long-range enhancers to test for their association with the 9p21.3 risk genotype. Using protein extracts from primary cultures of human aortic smooth muscle cells (n = 6) and peripheral blood lymphocytes from patients with CAD (n = 18) genotyped for the 9p21.3 risk locus using SNP microarrays, we found a clear positive correlation (P < 10-4) between IFNA21 levels and 9p21.3 risk genotype in both cell types. No association between MTAP expression and 9p21.3 genotype was seen in either cell type. In contrast, CDKN2A protein expression in both cell types, and CDKN2B protein expression in aortic smooth muscle cells was negatively associated with 9p21.3 risk genotype. Thus, the presence of long-range enhancers influenced by the 9p21.3 risk locus does not predict the direction of associated gene expression. Elevated IFNA21 and reduced CDKN2A protein expression is linked to and likely accounts for the risk of CAD associated with the 9p21.3 locus.

The evaluation of genotoxic potential of ornidazole, nitroimidazole, in lymphocyte culture of patients with amebiasis

The genotoxicity study of ornidazole (ONZ) was carried out on human lymphocyte chromosomes, using sister chromatid exchange (SCE) and micronucleus (MN). Thirty-two patients with Entemoeba histolitica infection who received 1000 mg/day for 10 days were included in this study. SCE and MN were measured before and after therapy. A statistically significant increase was observed in the SCE (P < 0.001) and MN frequencies (P < 0.001) after ornidazole therapy. It was concluded that ONZ has a potential geno- and cytotoxic effect in human peripheral lymphocyte cultures. For this reason, further, detailed studies are needed to elucidate the ONZ mechanism of genotoxicity and its carcinogenic potential.

Chromosomal damage induced by vanadium oxides in human peripheral lymphocytes

Fly ash, the inorganic residue resulting from the combustion of some fuels, may almost exclusively contain vanadium oxides, compounds which exert potential toxic effects on a wide variety of in vitro and in vivo biological systems. Because information related to the oxidation state responsible for inducing genotoxic effects is controversial, the aim of the present study was to evaluate the effects of three vanadium salts in vitro. Human peripheral lymphocyte cultures were exposed to 1, 2, 4, or 8 μg/mL of vanadium(III) trioxide, vanadium(IV) tetraoxide, or vanadium(V) pentoxide (V2O3, V2O4, or V2O5, respectively). These cultures were then screened for structural chromosomal aberrations, and mitotic index (MI) measurements were made. Cytogenetic evaluations showed that only V2O4 increased the percentage of aberrant cells (without gaps) and chromosome damage (including and excluding gaps), while all compounds led to a decrease in the MI. These results demonstrate that vanadium(III), vanadium(IV), and vanadium(V) are all capable of inducing cytotoxicity, but only oxidation state IV induces clastogenic effects.

Production of IgE antibody to Japanese cedar pollen allergen Cry j1 by short term culture of human peripheral blood lymphocytes

Peripheral blood lymphocytes (PBL) from a patient allergic to Japanese cedar pollens, were stimulated with IL-4, IL-13, CD40-Ligand and/or hydrocortisone in the presence of Epstein-Barr virus in 96-well round bottomed culture plates, and the secretion of IgE-class antibody against a Japanese cedar pollen allergen Cry j1 in the supernatants were examined. PBL cultured with IL-4, and IL-4 + CD40-Ligand showed the highest IgE secretion and the cultures were maintained for 30 days. However, we failed to expand the culture with high IgE secretion. It was suggested that patient's PBL stimulated with IL-4 were useful for short term IgE production to Cry j1.

Human T cells in hu-PBL-SCID mice proliferate in response to Daudi lymphoma and confer anti-tumour immunity.

In vitro culture of human peripheral blood lymphocytes (PBL) with Daudi (Burkitt lymphoma) cells results in selective proliferation of V gamma 9/V delta 2 T cells with high cytotoxicity against Daudi cells. After adoptive transfer into severe combined immunodeficient (SCID) mice, these cells exert specific anti-tumour activity against Daudi lymphoma. To test whether cytotoxic V gamma 9/V delta 2 T cells are induced in SCID mice, human PBL injected intraperitoneally were stimulated with irradiated Daudi cells (PBL/Daudi-SCID). After 7-14 days, PBL/Daudi-SCID had a significantly higher percentage of human gamma delta T cells in their peritoneal cavity, lymph nodes and blood than controls (PBL-SCID). DNA content analysis of T cell subsets from PBL/Daudi-SCID showed a significantly higher percentage of cells in S + G2 + M phases of the cell cycle in the TCR-gamma delta-1+ than in CD3+ cell population. Human cells recovered from PBL/Daudi-SCID showed specific cytotoxicity against Daudi cells. PBL/Daudi-SCID inoculated with a lethal dose of Daudi lymphoma survived significantly longer than controls. This protection was specific for Daudi cells and was not mediated by murine natural killer (NK) cells. Thus human peripheral blood T cells grafted in SCID mice proliferate in response to antigen and confer specific immunity.