Selective Lympholysis: A Unique Method Using the Double-Dye Technique.

Many methods are utilized to destroy mononuclear cells (primarily lymphocytes) either for neoplasm or immunosuppression. Neither radiation nor chemotherapy are truly selective or completely successful. The concept of “Double-Dye” treatment of blood for transfusion has been developed in order to inactivate parasites, bacteria and viruses (J Thromb Haemost2003; 1 Supplement 1 July: P1114). In recent studies, it has been observed that this same “Double-Dye” concept presents the possibility of very selectively eliminating lymphocytes (mononuclear cells) without affecting neutrophils in whole blood. To demonstrate the selectivity of dyes for lymphocytic/mononuclear cell types, two sets of experiments were performed. In the first, 0.3% (w/v) of the “Double Dye’ solution was added to several normal citrated whole blood samples to assess the effect on normal cells, compared to an untreated control. At 24hours post treatment, the lymphocyte count in the treated sample had dropped more than 80%, while little effect on neutrophils was noted. The control counts showed little change for either lymphocytes or neutrophils. Table 1. Lymphocyte reduction by 0.3%(w/v) Double-dye solution (units: cells/mm3). 0hr Control 0.3% Dye WBC 6850 ± 560 6920 ± 630 Neut 5030 ± 375 5100 ± 420 Lymph 1485 ± 220 1490 ± 265 24hr Control 0.3% Dye WBC 6700 ± 480 5300 ± 505 Neut 5025 ± 360 5025 ± 420 Lymph 1474 ± 240 275 ± 75 In the second series of experiments, 0.3%(w/v) “Double-Dye” solution or 0.15%(w/v) Crystal Violet or 0.15%(w/v) Methylene Blue were added to two T-cell leukemia lines (Jurkat, L1210), with a non-malignant, non-lymphocytic cell line (WISH) for the control. The combination of dyes showed the most potent activity against the lymphocytic lines, while the control was virtually unaffected. Table 2: Viability of cell lines after 24 hour exposure to dye solutions. Jurkat L1210 WISH Control 100% ± 2% 100% ± 4% 99% ± 2% 0.3% Double Dye 37% ± 5% 12% ± 4% 88% ± 9% 0.15% Meth. Blue 58% ± 12% 52% ± 9% 100% ± 3% 0.15% Cr. Violet 90% ± 12% 92% ± 10% 99% ± 4% The novel use of these dyes reported here coincided with the recent interest in utilizing methylene blue to increase transfusion safety, but recognizing that the concurrent need to photoactivate was too toxic to certain proteins and didn’t inactivate all pathogens (Transfusion2003; 43(9): 1238–47). Studies investigating the in-vivo efficacy of these novel immunosuppressive and chemotherapeutic methods are currently underway.

Activation of the NF-κB transcription factor in a T-lymphocytic cell line by hypochlorous acid

Reactive oxygen species (ROS) such as hydrogen peroxide serve as second messengers in the induction of the transcription factor NF-κB, and hence in the activation and replication of human immunodeficiency virus type 1 (HIV-1) in human cells. During inflammatory reactions, many oxidative species are produced, one of which is hypochlorous acid (HOCl), which is responsible for the microbicidal effects of activated human polymorphonuclear leukocytes. Treatment of a T-lymphocytic cell line with micromolar concentrations of HOCl promoted the appearance of transcription factor NF-κB (the heterodimer p50/p65) in the nucleus of the cells, even in the absence of de novo protein synthesis. Western blot analysis of the NF-κB inhibitory subunits (IκB) demonstrated that both IκB-α proteolysis and p105 processing were induced by the treatment. NF-κB activation was very effective when cells were subjected to hyperthermia before being treated with HOCl. Various antioxidants, such as pyrrolidine dithiocarbamate, p-bromophenacyl-bromide and nordihydroguaiaretic acid could strongly reduce NF-κB translocation, demonstrating the importance of oxidative species in the transduction mechanism. Moreover, ACH-2 cells treated with HOCl or H2O2 released tumour necrosis factor-α (TNF-α) in the supernatants. The importance of TNF-α release in NF-κB induction by HOCl or H2O2 was demonstrated by the fact that: (1) the nuclear appearance of NF-κB was promoted in untreated cells; and (2) synergism between TNF-α and HOCl was detected. Collectively, these results suggest that HOCl should be considered as an oxidative species capable of inducing NF-κB in a T-lymphocytic cell line through a transduction mechanism involving ROS, and having a long-distance effect through subsequent TNF-α release.