Phenotypic and functional alterations of Vγ2Vδ2 T cell subsets in patients with active nasopharyngeal carcinoma

Introduction  Human Vγ2Vδ2 T cells play important role in immunity to infection and cancer by monitoring self and foreign isoprenoid metabolites with their γδ T cell antigen receptors. Like CD4 and CD8 αβ T cells, adult peripheral Vγ2Vδ2 T cells represent a pool of heterogeneous cells with distinct functional capabilities. Purpose  The aim of this study was to characterize the phenotypes and functions of various Vγ2Vδ2 T cell subsets in patients with nasopharyngeal carcinoma (NPC). We sought to develop a better understanding of the role of these cells during the course of disease and to facilitate the development of immunotherapeutic strategies against NPC. Results  Although similar total percentages of peripheral blood Vγ2Vδ2 T cells were found in both NPC patients and normal donors, Vγ2Vδ2 T cells from NPC patients showed decreased cytotoxicity against tumor cells whereas Vγ2Vδ2 T cells from normal donors showed potent cytotoxicity. To investigate further, we compared the phenotypic characteristics of Vγ2Vδ2 T cells from 96 patients with NPC and 54 healthy controls. The fraction of late effector memory Vγ2Vδ2 T cells (T_{EM RA}) was significantly increased in NPC patients with corresponding decreases in the fraction of early memory Vγ2Vδ2 T cells (T_CM) compared with those in healthy controls. Moreover, T_{EM RA} and T_CM Vγ2Vδ2 cells from NPC patients produced significantly less IFN-γ and TNF-α, potentially contributing to their impaired cytotoxicity. Radiotherapy or concurrent chemo-radiotherapy further increased the T_{EM RA} Vγ2Vδ2 T cell population but did not correct the impaired production of IFN-γ and TNF-α observed for T_{EM RA} Vγ2Vδ2 T cells. Conclusion  We have identified distinct alterations in the Vγ2Vδ2 T cell subsets of patients with NPC. Moreover, the overall cellular effector function of γδ T cells is compromised in these patients. Our data suggest that the contribution of Vγ2Vδ2 T cells to control NPC may depend on the activation state and differentiation of these cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

PCR-mediated screening and cloning of a malate synthase genefrom Streptomyces griseus NCIMB 9001

Malate synthase is an essential metabolic enzyme of the glyoxylate bypass that makes possible the replenishment of carbon intermediates to cells grown on acetate. A polymerase chain reaction (PCR)-based molecular screening investigation of full-length malate synthase genes from Streptomyces spp. was initiated by our group. To this end, consensus primers were designed based on known streptomycete malate synthase sequences and successful amplification was obtained for Streptomyces griseus, S. fimbriatus and S. lipmanii. The putative full-length malate synthase gene from S. griseus was subsequently cloned, sequenced and expressed. Sequence analysis of this gene showed very high identity with other streptomycete malate synthase genes. Furthermore, high malate synthase activity was detected after heterologous expression in Escherichia coli, thus demonstrating successfully the rapid cloning and functional verification of a streptomycete malate synthase gene. Growth studies of S. griseus revealed that malate synthase activity was induced by the presence of acetate, which is a two-carbon source. Interestingly, the activity peaked during late growth phase when the biomass was declining, suggesting that the enzyme may have a late role in metabolism.     

Establishment of <Emphasis Type="Italic">Cyperus aromaticus</Emphasis> cell suspension cultures for the production of Juvenile hormone III

Cell suspension cultures of Cyperus aromaticus were established from the yellow friable callus derived from the root explants of in vitro plantlets. Four callus cell lines were selected based on their growth index from two populations of callus cultures originated from the mother plants grown in two different locations. The selected four cell lines (Z1, Z6, P4, P9) showed uniform cell growth but produced different amounts of juvenile hormone III (JHIII). The Z1 cell line possessed fast-growing characteristics, produced a high JHIII content, and was chosen as the elite cell line for an optimization study of C. aromaticus cell suspension cultures. An inoculum cell mass of 0.3 g from 12-d cultures in 30 ml culture medium was found to be the optimum inoculum size and culture age for establishing the cell suspension culture of C. aromaticus. MS basal medium supplemented with 4.5 mg/l 2,4-D and 5.5 mg/l NAA was found to be the best medium for production of maximum cell biomass and JHIII. These results indicated that JHIII can be produced from suspension culture of C. aromaticus using a single-stage cell-culture system.     

Auxin, Cytokinin and Ethylene Differentially Regulate Specific Developmental States Associated with Shoot Bud Morphogenesis in Leaf Tissues of Mangosteen (Garcinia mangostana L.) Cultured in Vitro

Hormonal regulation of de novo shoot bud formation in leaf explantsof mangosteen has been studied from a developmental perspective.This analysis indicates that at least three discrete, experimentallydistinguishable developmental states, namely, morphogenic competence,caulogenic determination and organ differentiation, were expressedduring shoot bud morphogenesis. The state of morphogenic competencein leaf tissues was expressed maximally between days 10 and12 of leaf development. Competent cells in explants requireda minimum of 6 days of BA treatment (20 µM) to becomecaulogenically determined. Such determined cells would continueshoot organogenesis on medium devoid of growth regulators. Delayingof BA exposure for as short as 2 days caused a dramatic declinein tissue competence. The state of competence and the processof caulogenic determination were adversely affected by IAA,but were insensitive to ethylene or its precursor, ACC. Shootbud differentiation was greatly enhanced by BA, but selectivelydelayed by ethylene. IAA also showed an inhibitory effect onshoot bud differentiation, but not mediated through ethylene.The distinct roles of auxin, cytokinin and ethylene on the regulationof shoot bud development in mangosteen leaf explants have beendiscussed on the basis of the current understanding of the conceptof tissue competence, determination and differentiation. (Received August 12, 1996; Accepted October 31, 1996)     

Passive mechanical behavior of human neutrophils: effects of colchicine and paclitaxel.

The role of microtubules in determining the mechanical rigidity of neutrophils was assessed. Neutrophils were treated with colchicine to disrupt microtubules, or with paclitaxel to promote formation of microtubules. Paclitaxel caused an increase in the number of microtubules in the cells as assessed by immunofluorescence, but it had no effect on the presence or organization of actin filaments or on cellular mechanical properties. Colchicine at concentrations <1.0 microM caused disruption of microtubular structures, but had little effect on either F-actin or on cellular mechanical properties. Higher concentrations of colchicine disrupted microtubular structure, but also caused increased actin polymerization and increases in cell rigidity. Treatment with 10 microM colchicine increased F-actin content by 17%, the characteristic cellular viscosity by 30%, the dependence of viscosity on shear rate by 10%, and the cortical tension by 18%. At 100 microM colchicine the corresponding increases were F-actin, 25%; characteristic viscosity, 50%; dependence of viscosity on shear rate, 20%; and cortical tension, 21%. These results indicate that microtubules have little influence on the mechanical properties of neutrophils, and that increases in cellular rigidity caused by high concentrations of colchicine are due to a secondary effect that triggers actin polymerization. This study supports the conclusion that actin filaments are the primary structural determinants of neutrophil mechanical properties.