Growth Of Human Lymphocytes Research Articles

The use of Bacterial Cellulose Produced from the Local Isolate Komagataeibacter xylinus TELE8 as an Antibacterial for Wounds

The present study aimed to use the bacterial cellulose pre-produced from local isolate Komagataeibacter xylinus TELE8 as antimicrobial dressings for wounds and compared their activity with specific artificial antibacterial. First, the toxicity of bacterial cellulose was examined in-vitro and in-vivo toward the shape and growth of human lymphocytes and then toward skin irritation, respectively. The results showed that there was no effect of bacterial cellulose dressings on the shape and numbers of human lymphocytes compared to the studied positive and negative control samples, also, the results of the study of the effect of bacterial cellulose dressings on the skin surface characteristics indicated that there was no erythema, edema, redness and dryness on the surfaces of the skin exposed to bacterial cellulose in test animals. Thereafter, the effect of BC, anti, and BC + anti dressings were studied using 12 male rabbit of 6 months old and were randomLy distributed into four groups included: a control group (without treatment), BC group (treated with bacterial cellulose), BC + anti group (treated with bacterial cellulose and sodium fusidate), and anti group (treated with sodium fusidate), all wounds in animals were treated for 15 days according to their group and the observations were recorded. The results showed clear and rapid wound healing in two groups, including the BC group and BC +Anti group compared with the anti-group and control group, also, the scores of skin irritation in the two previous groups were significantly lower than the anti-group and the control group for all days of the experiment; therefore bacterial cellulose may provide a promising effect in enhancing wound healing and preventing wound infections that act in delaying wound recovery.

Differential action of polyunsaturated fatty acids and eicosanoids on bleomycin-induced cytotoxicity to neuroblastoma cells and lymphocytes.

IntroductionThis study was conducted to examine whether bleomycin-induced growth inhibitory action on human neuroblastoma cells (IMR-32) is influenced by anti-inflammatory metabolites of polyunsaturated fatty acids (PUFAs): lipoxin A4 (LXA4), resolvin D1 and protectin D1 in vitro.Material and methodsThe in vitro study was conducted using monolayer cultures of exponentially growing IMR-32 cells. The effects of various PUFAs and eicosanoids and anti-inflammatory metabolites of PUFAs such as lipoxin A4 (LXA4), resolvin D1 and protectin D1 on the growth of IMR-32 cells and human lymphocytes in vitro were investigated. The potential of PUFAs, eicosanoids and LXA4, resolvin D1 and protectin D1 to modify the growth inhibitory effects of bleomycin was also studied in IMR-32 cells and human lymphocytes.ResultsPUFAs inhibited the growth of IMR-32 cells (EPA > DHA = AA > GLA = ALA > DGLA = LA) significantly (p < 0.001) while prostaglandins were found to be not effective. Bleomycin-induced growth inhibitory action on IMR-32 cells was augmented by PUFAs and its metabolites (p < 0.05). PUFAs and LXA4 did not inhibit the growth of human lymphocytes and bleomycin-induced growth inhibitory action was also not enhanced by these bioactive lipids.ConclusionsBioactive lipids have differential action on normal human lymphocytes and tumor cells in vitro. The apparent lack of effect of PUFAs in combination with bleomycin on the growth of human lymphocytes in comparison to their growth inhibitory action on IMR-32 cells suggests that PUFAs can be used in combination with bleomycin to target tumor cells with little concern over this combination’s effect on the growth of human lymphocytes. Further studies are warranted to evaluate these differential effects under in vivo conditions.

Tax peptide vaccine suppressed the leukemia in humanized mouse model of ATL

Cytotoxic T-lymphocyte (CTL) against HTLV-1 Tax has been demonstrated to play a vital role in controlling HTLV-1 infected cells in the infected carrier people. It was also shown that DNA immunization of mice with Tax expression vector suppressed the tumor growth of EL-4 lymphoma cells expressing Tax (EL4/Gax) through the activation of anti-Tax CTL [1]. Recently we established an HTLV-1-infected humanized mouse model (hu-NOG) to recapitulate ATL development and HTLV-1-specific immunity [2]. Here the efficacy of Tax-peptide vaccination was examined in the hu-NOG mouse model. Mixture of twelve overlapping peptides of 40-42 amino acids long encompassing whole Tax protein was inoculated subcutaneously three times weekly to hu-NOG mouse and then -irradiated HTLV-1 producing Jurkat cells were intraperitoneally injected to infect HTLV-1. While the control mice without immunization died of leukemia within two months of infection, Tax-immunization retarded the out growth of human lymphocytes after HTLV-1 infection and two out offive mice survived with alimited number of infected T-cells. IL-12 induction and the enhanced expression of Tax-specific CD8 T-cell were observed in the peripheral blood of immunized mice before infection. Furthermore, it was shown that the intranasal inoculation of Tax peptides has comparable effect on the onset of leukemia in the hu-NOG systems. These results suggest that the Tax peptide vaccination can elicit protective immunity against HTLV-1 infection and/or ATL developments.

The Feature of TCR Vβ Subfamily T Cells Expansion in NOD/SCID Mice Transplanted with Human Cord Blood Hematopoietic Stem Cell.

Reconstitute of functional human lymphocytes in some mouse strains has been intensely explored because this approach could provide a valuable means of assessing human immunity or of preclinical testing of vaccines, pathogens, and new therapeuticstrategies. The NOD/SCID mouse strain was found to be an efficient recipient for the reconstitution of human hematopoietic cells. In these xenogenic transplantation models, growth of human lymphocytes has been demonstrated in bone marrow, peripheral blood and spleen of the mice after i.v. inoculation. These mouse models showed the multilineage differentiation of human hematopoietic cells to varying degrees. Moreover, the engraftment of human hematopoietic cells is highly variable. Complete multilineage differentiation, especially T cells, has not been achieved using this strain. Examination of T cell receptor (TCR) gene repertoire is important to analysis the immune status of models, because clonal expansion of T cells permit the identification of specific antigen response of T cells. Little is known about the feature of T-cell immunity in the humanized NOD/SCID mouse model.In order to investigate the distribution and clonal expansion of TCR Vβsubfamily T cells in NOD/SCID mice transplanted with human cord blood hematopoietic stem cell. TCR Vβ repertoire usage and clonality were analyzed. The NOD/SCID mice were sublethally irradiated (60Co, 300cGy) to eliminate residual innate immunity in the host. The experimental mice were transplanted intravenously with cord blood CD34+ cells sorted by MACS. After 6 weeks, RNA was obtained from peripheral blood, bone marrow, thymus of model. The gene expression and clonality of TCR Vβ repertoire were detected by RT-PCR and GeneScan technique. A certain range of TCR Vβ usage was exhibited in the bone marrow of mice, which included TCR Vβ1, 2, 9, 13, 19. Further, oligoclonal expression of some TCR Vβ subfamilies (Vβ9, 13, 19) were identified by GeneScan technique. To investigate the reason involving oligoclonal expansion of TCR Vβ subfamily T cells from cord blood in mouse models, the T-cell culture with tissue-antigen of NOD/SCID mouse was performed in vitro. The cells from peripheral blood mononuclear cells and bone marrow, spleen, thymus in NOD/SCID mice were wiped off by freeze thawing and served as tissue-antigen. Cord blood mononuclear cells were separately cultured with the component from those murine cells for 15 to 20 days. Oligoclonal expression or oligoclonal trend of some TCR Vβ subfamilies (Vβ10, 11 and Vβ2, 15, 16, 19) was detected in T cells after stimulation with tissue-antigen of NOD/SCID mouse. Interesting, the similar clonal expansion of TCR Vβ11 subfamily was found in T cells cultured with peripheral blood, bone marrow and spleen respectively. The results indicate that the TCR Vβ subfamily T cells could be reconstituted in humanized NOD/SCID mouse transplanted with CD34+cells from cord blood. The restricted expression and clonal expansion of some CB T cell clones might be induced by tissue-antigens of NOD/SCID mice.

Glutamate modulation of human lymphocyte growth: in vitro studies

Peripheral blood mononuclear cell (PBMC) proliferation induced by phytohemagglutinin, or by anti-CD3 alone or plus anti-CD28 monoclonal antibodies (mAb) was inhibited by glutamate (Glu) in a concentration-dependent manner. This inhibition was not reproduced by selective ionotropic Glu receptor agonists, whereas it was potentiated by l-buthionine-( S, R)-sulfoximine, which depletes glutathione (GSH) stores, and counteracted by 2-mercaptoethanol, a preserver of cell thiols. The inhibitory effects of Glu were related to depletion of intracellular GSH stores, since it decreased GSH levels in a concentration-dependent manner. Furthermore, Glu modulated cytokine secretion by anti-CD3 mAb activated PBMC: it increased IFN-γ (+44.3 ± 8.2%) and IL-10 (+31.6 ± 9.7%) secretion, whereas that of IL-2, IL-4, IL-5, and TNF-α was not affected. These data suggest that high levels of Glu, which can be reached in damaged tissues, modulate lymphocyte responses to activating stimuli by favouring polarization of the T helper effector response.

Human lymphocyte growth hormone stimulates interferon gamma production and is inhibited by cortisol and norepinephrine

Lymphocyte growth hormone (L-GH) is distributed throughout the human immune system; however, its biologic role has not been defined. In order to clarify this issue, we determined if Candida, commonly used as a recall antigen, and IL-12, an important monocyte cytokine that stimulates the TH1 cytokine pathway, could stimulate L-GH mRNA synthesis in cultured human peripheral blood mononuclear cells (PBMCs). Both Candida and IL-12 produced significant ( p<0.01) stimulation of L-GH mRNA synthesis and the TH1 cytokine, gamma interferon (IFN-γ). These effects on IFN-γ production were significantly ( p<0.001) inhibited by a GH antagonist, suggesting that L-GH was acting by an autocrine or paracrine mechanism to enhance IFN-γ production in these serum-free cultured cells. Concentrations of norepinephrine and cortisol achievable in humans during stress were able to significantly ( p<0.001) decrease L-GH synthesis in the PBMCs. They also diminished IFN-γ significantly ( p<0.001), but not the TH2 cytokine, IL-10, in the supernatants of the cultured PBMLs. These studies suggest that L-GH plays a role in cellular immune function mediated via the TH-1 pathway.

Glucose and Insulin Responses in Isolated Human Lymphocytes Reflect in Vivo Status: Effects of VLCD Treatment

Human lymphocyte growth response to mitogen was examined in vitro under different conditions to monitor aspects of carbohydrate utilization in three groups: obese nondiabetic, obese/NIDDM, and normal-weight individuals. Although lymphocyte growth capacity in minimal medium for these three groups was found to be similar at a glucose concentration of 72 μg/ml in the absence of insulin or glycerol, differences in mitogen-stimulated growth were observed at lower glucose concentrations or in the presence of insulin or glycerol. Interestingly, these metabolic alterations in lymphocytes can be normalized by treating obese and obese/NIDDM subjects with a very low calorie diet (VLCD) regimen. The results of this study indicate that lymphocyte culture in a defined medium may provide a mechanism to examine different metabolic states and to evaluate treatment regimens (diet, exercise, etc.) for obese and NIDDM subjects.

Inhibitory effect of bestatin on the growth of human lymphocytes

Bestatin at mid to high concentration had inhibitory effect to [3H]thymidine incorporation of human lymphocytes. It also decreased the PHA-P, Con A and PWM-induced mitogenicity of human lymphocytes. On the contrary, bestatin had growth stimulatory effect to murine lymphocytes, and enhanced the Con A, LPS-induced mitogenicity of murine lymphocytes. These growth inhibitory (human) and stimulatory (mouse) effect of bestatin was found to be independent of adherent cells (macrophage and dendritic cell), indicatory directly working to T or B lymphocytes.

Effects of extracellular products from Staphylococcus saprophyticus on human lymphocytes.

Extracellular products from Staphylococcus saprophyticus were isolated and tested for their effect on the lymphoproliferative activity of con A and LPS and on the growth of human lymphocytes. The stimulatory effects of con A or LPS were not affected but a T-cell mitogenic activity was noted after 5 days of incubation. The extracellular material was separated by gel chromatography and the mitogenic activity was shown to be located to the protein fraction.

Nutritional requirements for growth of human lymphocytes.

INTRODUCTION . .... . .. . . . . ....... . . . . . . . . .... . . . . . .. ..... ....... . . . . . . .. ... . ..... . . . . . . .. .. . . . . . ... 8 1 M EDIA FOR GROWTH OF H UMAN LyMPHO CyT ES. . .. . . . . ... . . . . . . .. . ... . . . .. . . . . . . . . 82 Serum-Containing Media . . . ..... ... . . .... . . . . . . ..... . . . . . . ... . . . . . ... . . . . . .... . . . . . ..... . . . .... . 82 Serum-Free Media . . . . . . . . . . . . . .. . ... .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 82 Lymphokines . . . . . . . . . . . . . . . . . . . . . . . . . ....... . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . .... . . . . 84 Chemically Defined Serumand Protein-Free Minimal Media . . . .. . . . . . . . . . . . . . . . . ... . . 85 ACTIVATION AND STIMULATION OF GROWTH . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . 9 1 Lectins .. . . . . . . . .. . . . . . . . . . . . .. . . ..... . . . ..... . . . . . . . . .... . . . . . . . . ..... . . . . . 9 1 Antibodies to SUiface Antigens and Mixed Lymphocyte Culture . . . . . . . . . . . . . . . . . . . . . . . . 9 1 Other Modes o f Activation ... . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1 APPLI CA TIONS FOR M INIMAL M ED IA. . . . . . . . . . . . . . . . . .. ... . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . 92 CON CLUSIONS. . . . ... . .. . . . . . .. . . . . ... . . .... . . . . ...... .. . . . . .... . . . .. .. . ...... . . . . . ..... . ... . ........ 94

Nutritional Requirements For Growth Of Human Lymphocytes

Nutritional Requirements For Growth Of Human Lymphocytes

Suppression of growth of PHA-stimulated human lymphocytes by a novel steroid (RU 28 362) lacking the 21-OH group

RU 28 362 is a novel synthetic steroid (lacking the 21-OH group) which reacts exclusively with the glucocorticoid receptor. It is therefore chemically different from the other natural and synthetic glucocorticoids in common use which also bind to a lesser extent to receptors for other classes of steroids. Aspects of the immunosuppressive effect of RU 28 362 were studied by comparing its suppressive effects on PHA-stimulated growth of human lymphocytes with those of hydrocortisone, betamethasone and the inactive analogue, 21-deoxyhydrocortisone. RU 28 362 was more potent than either hydrocortisone or betamethasone, but all three gluococorticoids had a parallel dose — response curve in suppression of the increase in cell volume that occurs with activation during the first day in culture. In studies on the time of appearance and density of IL-2 receptors during the first 3 days in culture, RU 28 362 was also somewhat more inhibitory and had a steeper dose — response curve than hydrocortisone or betamethasone. RU 28 362 was much more inhibitory and had a steeper dose — response curve than either hydrocortisone or betamethasone in inhibiting [ 3H]-TdR incorporation by 3-day PHA stimulated cultures. 21-deoxyhydrocortisone did not inhibit any aspect of PHA-stimulated lymphocyte growth.

Chemically defined medium for the growth of lymphocytes

This chapter describes the composition and preparation of a chemically defined medium, CFBI 1000, that promotes growth of human lymphocytes in the absence of serum or added proteins. It also discusses other serum-free but not protein-free media used for serum-free experimentation. The CFBI 1000 medium is a buffered solution containing amino acids, vitamins, adenine, choline, glucose, inositol, pyruvate, EDTA, and the ions sodium, potassium, phosphate, chloride, sulfate, calcium, iron, and magnesium. This defined medium can be utilized to test effects of any metabolite not present in the medium for a deficit in biosynthesis or utilization. In addition, a defined medium also has broad applications in the study of the immune response mediated by the lymphocyte population (T and B cells). For media preparation, the composition of medium CFBI 1000 is presented in tabular form in the chapter. Medium CFBI 1000 is prepared from solid materials and a series of sterile stock solutions. The stock solutions are sterilized by filtration and stored in aliquots at -20° (except phenol red is stored at 5°, and MgSO4 and CaCl2 stock solutions are stored at 25°).

BrdU-Hoechst flow cytometry reveals regulation of human lymphocyte growth by donor-age-related growth fraction and transition rate.

An improved BrdU-Hoechst flow assay was applied to cell kinetic studies of human lymphocyte cultures during a 24-96 hr interval after PHA stimulation. The assay shows that the duration of the initial lag phase and the proportions of noncycling cells increase as a function of donor age, whereas the rates of transition from each cell cycle compartment to the next decrease. Cell cycle arrest occurs in the first S and G2 phase after stimulation of lymphocytes from a 75-year-old donor but not from younger donors. The data are consistent with several models of cell cycle kinetics, so long as these models are modified to include a fraction of noncycling cells in each cell cycle compartment.

Idoxuridine induction of micronuclei containing the long or short arms of human chromosome 9.

PHA-stimulated growth of human lymphocytes in the presence of idoxuridine (IUdR) results in chromosomal decondensation and fragility of large heterochromatic regions. This instability is especially evident in the heterochromatic region of chromosome 9 (9h). A high frequency of micronuclei is seen in all IUdR-treated cultures. By a combination of chromosomal localization of induced aberrations, analysis of metaphases with prematurely condensed micronuclear chromatin, and specific staining of 9h in interphase micronuclei, it can be shown that 80-90% of all micronuclei contains 9h material. This pattern is found whether the heterochromatic region is situated on the long arm or the short arm of chromosome 9. These observations suggest that IUdR-induced micronucleation may be a valuable method for separation of the long and short arms of human chromosome 9.

Close correlation between initiator methionyl-tRNA level and rate of protein synthesis during human lymphocyte growth cycle.

Close correlation between initiator methionyl-tRNA level and rate of protein synthesis during human lymphocyte growth cycle.

Human beta-globulin factors affecting growth of human lymphocytes and mouse fibroblasts.

Evidence is presented to show the existence in human beta-globulin (Cohn fraction III) of 2 growth-promoting factors that stimulate DNA synthesis and cell division in human lymphocytes and 3T3 B mouse fibroblasts. After extraction of human beta-globulins at pH 3.0 in 0.1 M NaCl followed by sieve chromatography on Sephadex, 2 distinct fractions were obtained containing the biological activity; one of polypeptide nature and molecular weight of approx. 10 000 Daltons and the other consisting of a homogeneous ribonucleic acid, as revealed by polyacrylamide gel electrophoresis. The above-mentioned factors appear to lack both cell and species specificity, since they are mitogenic agents for cells as diverse as mouse fibroblasts and human lymphocytes.

Selective toxicity of purine deoxynucleosides for human lymphocyte growth and function

A role for the enzymes adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyl-transferase, EC 2.4.2.1) in the functional maturation of lymphoid cells has been revealed by the association of inherited deficiencies of these enzymes and profound immune deficiency. Previous studies have suggested that the selective toxicity for lymphocytes may be mediated by the accumulation of toxic deoxynucleoside metabolites, likely through the action of specific kinases enriched in lymphoid cells. In order to study possible mechanisms whereby lymphocyte function may be impaired in these disorders, we have studied the effect of nucleosides and their deoxy analogues on both T and B lymphocyte growth and function. In the presence of deoxyguanosine, there was marked inhibition of T lymphoblast growth, phytohem-agglutinin-induced cell proliferation, and T suppressor cell activity. T helper cell activity and the differentiation of B cells to an antibody-secreting stage were unaffected. Deoxyadenosine was much less inhibitory, but in the presence of an inhibitor of adenosine deaminase, its effects on lymphocyte growth and function were markedly potentiated. The addition of deoxycytidine prevented deoxyadenosine toxicity in all assays, whereas it only interfered with deoxyguanosine effects on T lymphoblast growth. These studies provide some initial understanding for the selective loss of specific lymphocyte functions in individuals with inborn errors of purine metabolism.

Abortive growth of human lymphocytes carrying a dormant Epstein-Barr viral genome.

When P3HR-1 lymphoblastoid cells expressing Epstein-Barr virus (EBV)-related antigens at very low frequency were cocultivated with human umbilical cord blood lymphocytes and the cell-free mixed culture fluid was applied to fresh cord lymphocytes, cells morphologically distinct from normal lymphocytes became evident after one to two weeks' exposure. The abnormal cells became abundant after one month and were easily identified by B-cell markers and a variety of morphologic abnormalities. Such abnormal B-lymphocytes appeared to be negative for EBV-determined nuclear antigen (EBNA), but when the immunofluorescence-negative cells were exposed to pokeweed mitogen and 5-iododeoxyuridine, a striking EBNA induction occurred. The growth of these abnormal cells was limited and they could be maintained for no more than three months. The implications of these findings are discussed in relation to the biological activity of EBV.

Plasma membrane glycosphingolipids (GSLs) of the human lymphoblastoid cell-line BRI 8 and differences between the GSLs of BRI 8 cells and those of peripheral lymphocytes

Plasma membrane of the human lymphoblastoid cell-line BRI 8 contained about tenfold (expressed relative to per mg of protein) more hematoside and about three fold more GL 2, GL 3 and GL 4, but less GL 1 than the whole cell homogenate. Human peripheral blood lymphocytes contained small quantities of hematoside but were two to three times more rich in neutral GSLs than BRI 8 cells. GL 3 was the major GSL of BRI 8 cells cultured in media supplemented with piglet or human serum. This GLS was not detected in human peripheral lymphocytes which contained mostly GL 2. The results suggest that growth of human lymphocytes causes activation of enzymes which mediate the elongation of the GL 2 oligosaccharide chain.