Lymphocyte isolation

In vivo studies showed that in vitro proliferation of lymphocytes isolated from marijuana smokers is suppressed, especially with heavy marijuana smoking, and that the relative proportion of lymphocyte subpopulations was also altered. Concentrations of serum IgG are decreased and IgE concentrations are increased in marijuana smokers. Furthermore, phagocytic and bactericidal activity of alveolar macrophages from heavy marijuana smokers are decreased. These effects translate into reduced host resistance following administration of cannabinoids, including THC, in both humans and animals (Table 30.2). Increased susceptibility has been demonstrated to opportunistic microbes including HIV, Herpes simplex virus, Friend leukemia virus Listeria, Treponema pallidum, and Legionella. 

To obtain MABs, lymphocytes isolated from the spleen of immunized mice (1) are fused with mouse tumor cells (myeloma cells, 2). This is necessary because antibody-secreting lymphocytes in culture have a lifespan of only a few weeks. Fusion of lymphocytes with tumor cells gives rise to cell hybrids, known as hybridomas, which are potentially immortal. 

We developed a PEEK-WC hollow-fiber (HF) membrane bioreactor for the maintenance of human peripheral lymphocytes as a model system for the in-vitro investigation of disease pathogenesis, chemical effects and individual drug sensitivity. Peripheral lymphocytes isolated from the donor s human buffy coat were cultured in the shell compartment of the PEEK-WC-H F bioreactor. Lymphocytes in the PEEK-WC-HF membrane bioreactor produced IL-2 and IL-10 throughout the culture period of 14 days (Figure 19.5). 

The obtained results demonstrated that a PEEK-WC-H F membrane bioreactor is able to support the proliferation and functions of human peripheral lymphocytes isolated from the buffy coat of healthy individuals. Therefore, the lymphocyte HF membrane bioreactor can be used as a valuable tool to maintain viable and functional lymphocytes and as an in-vitro model for pharmacological and adoptive immunotherapy. 

Vacutainer CPT cell preparation tubes (Becton Dickinson) can be used as an alternative to the standard Ficoll-Paque protocol for lymphocyte isolation. This allows for sterile blood collection and cell separation using a single centrifugation step. 

Hybridomas (or fused cell culture lines) are produced by fusing lymphocytes isolated from the spleen of an immunized animal with the HPRT deficient malignant plasma cells. The lymphocytes. The unfused cells die, since spleen cells survive only a few days, while the malignant plasma cells lack HPRT. Fused cells, however, are HPRT positive as well as immortal these hybridomas, or hybrid cells, retain the Ab-producing characteristics of the spleen cells. Hybrid cell lines that produce a specific antibody are then cloned from the single cells and cultured. Each clone created in this manner produces antibodies of a single epitope specificity ... 

Murthy SK, Sin A, Tompkins RG, Toner M (2004) Effect of flow and surface conditions on human lymphocyte isolation using microfluidic chambers. Langmuir 20(26) 11649-11655... 

Cush, J. J. and Lipsky, P. E. (1988) Phenotypic analysis of synovial tissue and peripheral blood lymphocytes isolated from patients with rheumatoid arthritis. Arthritis Rheum. 31,1230-1238. 

The effect of stavudine-based HAART on mitochondrial respiratory transport chain function in CD4-I- peripheral blood lymphocytes has been studied in HTV-positive patients in Australia [73. CD4-I-lymphocytes isolated from asymptomatic stavudine-treated patients had reduced basal mitochondrial oxygen consumption compared with untreated HIV-infected patients and uninfected controls. Cells from the stavudine-treated patients also had reduced maximal oxygen consumption. There were no significant differences in citrate synthase activities, mitochondrial volume, or mitochondrial DNA copy numbers per cell between the groups. These findings support an association between stavudine and mitochondrial dysfunction and suggest that the alteration in function does not solely result from the... 

Mouri, T., T. Murahara, H. Kayama, S. Tsutsui, T. Kurokawa, Y. Shibata, N. IsHiDA, S. Kakimoto, F. Asakura, H. Shirahama, and T. Matsumoto New inhibitors for the blastgenation of human lymphocytes. Isolation from edible mushrooms. Agric. Biol. Chem. 42, 2179 (1978). 

Surfactant protein D" " mice demonstrated increased numbers of airway- and vessel-associated lymphocytes without increases in interstitial lymphocytes (Fisher et al. 2002). There was increased proliferative activity of lymphocytes isolated by enzymatic dissociation of minced lung. There was marked T-cell activation in the lungs of surfactant protein D" mice, as reflected by an increased percentage of both CD4 and CD8 T cells expressing CD69 and CD25. 

In order to systematically investigate the mechanism(s) of fragile chromosome expression in folate deficient media we developed a basic culture medium. The medium is deficient in folic acid and a number of metabolites produced by folate mediated one-carbon transfer reactions (Fig. 1). These include the amino acids, serine, glycine and methionine the purine source hypoxanthine and the thymidylate source thymidine. Previous studies of fragile site expression have used leucocytes isolated in autologous plasma and cultured in medium supplemented with undialyzed serum. In order to eliminate serum sources of folate, amino acids and nucleotide precursors, we have used washed lymphocytes isolated from ficoll-hypaque banding and medium containing dialyzed human AB serum. 

In most cases, the spleen is the best choice for antigen-specific lymphocyte isolation. The immunization regime used to immunize mice is identical in every respect to that used for conventional hybridoma production. In particular about ten days after each injection, test bleeds should be carried out to test for the presence of specific antibodies and only animals giving the best antiserum should be selected. Do not expect to recover high affinity antibodies from phage antibody libraries that are not foimd in the test serum. Three to five days before harvesting the spleen for RNA extraction, the immunized mouse is given a final boost. This is best achieved by an intravenous injection done concurrently with an intraperitoneal injectiou... 

In contrast to the above studies, no increase in proliferation response to mitogen was seen in peripheral blood lymphocytes isolated from healthy elderly persons taking large doses of vitamin C along with other vitamins (Goodwin and Garry, 1983). The same persons, however, did show improved reactivity in a skin test. 

At least three lectin species have been detected in lymphocytes isolated from the thymus and spleen of BALB/c mice. Lectins specific towards jS-o-galactosides and 2-acetamido-2-deoxy-D-galactosides are found mainly in thymus extracts, and those specific towards a-D-mannosides in spleen extracts. 

This test can be performed with the total peripheral blood lymphocyte population or with individual lymphocyte subpopulations (B lymphocytes, T4 and Tg lymphocytes) isolated from the total human lymphocyte fraction by monoclonal antibody separation. The rate of lymphocyte proliferation under influence of mitogens is measured through determination of the rate of incorporation of H-thymidine into the DNA of the lymphocytes. 

Employing the conventional proliferation assay using lymphocytes isolated from spleen (mouse, rat), lymph nodes (mouse, rat), or peripheral human blood and... 

JARRY A, CERF-BENSUSSAN N, BROUSSE N,SELZ F, GUY-GRAND D (1990), Subsets of CD3+ (T cell receptor alpha/beta or gamma/delta) and CD3-lymphocytes isolated from normal human gut epithelium display phenotypical features different from their counterparts in peripheral blood , Eur J Immunol, 20,1097-103. 

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