Safety and immunogenicity of a novel nanoemulsion mucosal adjuvant W805EC combined with approved seasonal influenza antigens.

BACKGROUND: Improving the systemic and mucosal immune response following intranasal vaccination could enhance disease protection against respiratory pathogens. We assessed the safety and immunogenicity of a novel nanoemulsion mucosal adjuvant W(80)5EC combined with approved seasonal influenza antigens. METHODS: This was a first-in-human Phase I study in 199 healthy adult volunteers randomized to receive a single intranasal administration of 5%, 10%, 15% or 20% W(80)5EC, combined with 4 or 10 µg strain-specific Fluzone(®) HA, compared with intranasal PBS, intranasal Fluzone(®), or 15 ug strain-specific intramuscular Fluzone(®). Safety was evaluated by physical examination, laboratory parameters, symptom diaries, and adverse event reports. Serum HAI titers and nasal wash IgA were assessed at baseline as well as 28 and 60 days after vaccination. RESULTS: W(80)5EC adjuvant combined with seasonal influenza antigens was well tolerated without safety concerns or significant adverse events. The highest dose of 20% W(80)5EC combined with 10 µg strain-specific HA elicited clinically meaningful systemic immunity based on increases in serum HAI GMT and ≥ 70% seroprotection for all 3 influenza strains, as well as a rise in antigen-specific IgA in nasal wash specimens. CONCLUSIONS: W(80)5EC adjuvant was safe and well tolerated in healthy adult volunteers and elicited both systemic and mucosal immunity following a single intranasal vaccination.

A novel surfactant nanoemulsion with a unique non-irritant topical antimicrobial activity against bacteria, enveloped viruses and fungi.

A novel non-ionic surfactant nanoemulsion designated 8N8 has been tested for its biocidal activity. One percent 8N8 produced effective bactericidal activity against Bacillus cereus, Bacillus subtilis, Haemophilus influenzae, Neisseria gonorrhoeae, Streptococcus pneumoniae, and Vibrio cholerae in 15 minutes. In contrast, most enteric gram-negative bacteria were resistant to 8N8. One percent 8N8 was also virucidal within 15 minutes for all tested enveloped viruses, including Herpes simplex type 1, influenza A and vaccinia viruses. One percent 8N8 also demonstrated fungistatic activity on Candida albicans. The rapid and non-specific inactivation of vegetative bacteria and enveloped viruses, in addition to its fungistatic activity and low toxicity in experimental animals, makes 8N8 a potential candidate for use as a topical biocidal agent.

2-Methoxyestradiol, an endogenous estrogen metabolite, induces thyroid cell apoptosis.

The etiology of autoimmune thyroid diseases is unclear; however, the extreme female predominance suggests that sex hormones may have a pathogenic role. 2-Methoxyestradiol (2-ME) is present in the serum of women during the ovulatory and luteal phases of the menstrual cycle, and during pregnancy. We investigated the actions of 2-ME and estrogen on thyroid follicular cells. 2-ME induced dramatic changes in cell morphology and decreased the viability of the cells, as well as disrupted the structural integrity of cultured thyroid follicles. Flow cytometric analysis showed that 2-ME halted cell proliferation by arresting the cells in the G2/M cell-cycle compartment. Prolonged exposure to 2-ME led to apoptosis and to increased release of the autoantigen thyroid peroxidase (TPO). 17beta-estradiol failed to produce a similar effect even in 40-fold molar excess to 2-ME. Co-treatment with estrogen receptor antagonists did not alter the 2-ME effect, indicating that 2-ME was not operating through a classic nuclear estrogen receptor. In conclusion, this study indicates that 2-ME induces G2/M cycle arrest, apoptosis and the disruption of thyroid follicles. This process results in the release of thyroid antigens that may play a role in high incidence of thyroid autoantibodies and autoimmune thyroid disease in women.

Prevention of murine influenza A virus pneumonitis by surfactant nano-emulsions.

Non-ionic surfactant nano-emulsions have extensive anti-microbial activity and are biocompatible with skin and mucous membranes at effective concentrations. Two nano-emulsion formulations (8N8 and 20N10) made from soybean oil, tributyl phosphate and Triton X-100, were tested for their ability to prevent murine influenza virus pneumonia in vivo. In the initial study, CD-1 mice were administered various dilutions of the nano-emulsions intranasally, and safe dosages and concentrations were determined. Non-toxic concentrations of the nano-emulsions were then mixed with influenza virus and applied to the nares of mice. Animals receiving mixtures of two different emulsions (8N8 or 20N10) and a LD50 of virus survived the challenge without evidence of viral infection. To determine if the nano-emulsions could prevent influenza virus infection in vivo when used as a prophylactic treatment, the nano-emulsions (8N8 at 1.0% and 20N10 at 1.0% or 0.2%) were applied to mouse nares 90 min before exposure to 5x10(5) p.f.u./ml virus by nebulized aerosol. Animals pretreated with the nano-emulsions had significantly decreased clinical signs of infection. Only 26.0% (8N8 at 1.0%), 31.25% (20N10 at 1.0%) and 37.0% (20N10 at 0.2%) of animals pretreated with nano-emulsion died from pneumonitis, whereas >80.0% of mock pretreated animals succumbed to infection (P<0.005). These findings suggest that non-ionic surfactant nano-emulsions have therapeutic potential for the prevention of influenza virus infection in vivo.

Fas (CD95) expression is up-regulated on papillary thyroid carcinoma.

Thyrocyte apoptosis signaled through the Fas receptor has been proposed as a mechanism for the cytotoxicity observed in thyroiditis, but the role the Fas pathway plays in thyroid cancer is not known. We examined Fas expression in thyroid tissue derived from patients with papillary carcinoma and follicular cancer. More intense immunohistological staining for the Fas protein was observed on papillary cancer cells as compared with adjacent normal follicles. To further characterize the expression of Fas in papillary cancer, paired normal and cancerous thyroid tissues were obtained at thyroidectomy from several donors, digested, and placed into cell culture. Messenger RNA was analyzed by ribonuclease protection assays, and protein was identified by flow cytometry. Fas expression was detected at levels up to 3-fold higher in cancerous thyrocytes compared with paired normal cells. To determine whether the expressed Fas antigen was functional, thyrocytes were treated with a monoclonal IgM anti-Fas antibody (clone CH11; Upstate Biotechnology, Inc., Lake Placid, NY) in the presence of interferon-gamma and cycloheximide. Whereas both normal and cancerous thyrocytes were induced to die after this treatment, the cancerous thyrocytes were more sensitive to anti-Fas antibody. This work demonstrates that the Fas antigen is expressed and functional on papillary thyroid cancer cells and this may have potential therapeutic significance.

Characterization of FAP-1 expression and function in thyroid follicular cells.

Human thyrocytes are resistant to Fas-mediated programmed cell death (PCD). It has been reported that a labile protein inhibitor is involved in the protection of thyrocytes from PCD, and its action can be reversed by incubation of thyrocytes with cycloheximide (CHX) during treatment with agonist anti-Fas Ab. Fas-associated phosphatase-1 (FAP-1) is a protein that has been shown to interact with the negative regulatory domain of Fas and block Fas-mediated apoptosis in FAP-1 transfected Jurkat cells. We investigated the possibility that FAP-1 might be involved in protection against Fas-mediated PCD in human thyrocytes. FAP-1 mRNA was detected in primary thyrocytes using a ribonuclease protection assay. The presence of FAP-1 protein was confirmed by immunohistochemical staining and flow cytometry using a polyclonal anti-FAP-1 Ab. FAP-1 protein also disappeared from thyroid cells in response to CHX. To determine whether FAP-1 is a functional inhibitor of PCD in thyrocytes, we incubated thyrocytes with synthetic SLV (Ac-SLV) tripeptide to compete with Fas for interaction with FAP-1. Thyrocytes treated with Ac-SLV tripeptide showed significantly increased cell death as compared to cells treated with control tripeptide. In addition, in the presence of a suboptimal concentration of CHX, the Ac-SLV tripeptide yielded a strong, synergistic increase in Fas-mediated PCD as compared to thyrocytes treated with control tripeptide. These results implicate FAP-1 as a regulator of Fas-induced PCD in thyrocytes.

TRAIL death pathway expression and induction in thyroid follicular cells.

To determine whether programmed cell death in thyroid follicular cells can be related to activation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway, we examined the expression and function of this pathway in primary thyroid follicular cells and a papillary thyroid carcinoma cell line in vitro. Despite the expression of TRAIL receptors death receptor 4 and death receptor 5, purified TRAIL could not induce programmed cell death (PCD) in any of the thyroid follicular cells examined. However, pre-incubation with cycloheximide before TRAIL facilitated the induction of rapid and massive PCD. This suggested that despite the presence of a labile inhibitor of the TRAIL pathway, TRAIL could mediate PCD under appropriate conditions. To determine whether there were sources of TRAIL in the thyroid that could interact with thyroid follicular cell TRAIL receptors, RNase protection assays were used to determine TRAIL mRNA expression. TRAIL message was expressed in intrathyroidal lymphocytes isolated from a patient with thyroiditis, and unexpectedly, thyroid follicular cells themselves could be induced to express abundant TRAIL message in the presence of the inflammatory cytokines interferon gamma, tumor necrosis factor alpha, and interleukin 1beta. Furthermore, the papillary thyroid carcinoma cell line could be induced to kill the TRAIL-sensitive lymphoma cell line BJAB through a TRAIL-dependent mechanism.

Inflammatory cytokine regulation of Fas-mediated apoptosis in thyroid follicular cells.

The occurrence of apoptosis in thyroid follicular cells induced by Fas activation has been a subject of much debate. This is due, in part, to the fact that no physiologically relevant treatment conditions have been reported to cause rapid and extensive Fas-mediated apoptosis in thyroid cells, whereas treatment with the protein synthesis inhibitor cycloheximide prior to Fas activation allows for massive cell death. This indicates that the Fas signaling pathway is present but that its function is blocked in the overwhelming majority of cultured thyroid cells. To reconcile the conflicting reports, we set out to identify physiologically relevant conditions in which rapid, massive thyroid cell apoptosis in response to Fas activation could be demonstrated. We determined that susceptibility to Fas-activated apoptosis could be influenced by certain combinations of inflammatory cytokines. Although no single cytokine was effective, pretreatment of thyroid cells with the combination of gamma-interferon and either tumor necrosis factor-alpha or interleukin 1beta allowed for massive Fas-mediated apoptosis. Susceptibility to Fas-induced death correlated with an increase in expression of a tunicamycin-inhibitable high molecular weight form of Fas but not with aggregate expression of Fas.

Optimization of in situ cellular ELISA performed on influenza A virus-infected monolayers for screening of antiviral agents.

Viral susceptibility testing has been traditionally performed by the plaque reduction assay (PRA) which is laborious, time consuming, relatively expensive, and requires subjective input by the reader. An in situ cellular enzyme-linked immunosorbent assay (ELISA) has been developed with the potential to overcome many of the limitations of PRA and has been applied to a variety of viruses. This study establishes the specific conditions necessary for susceptibility testing of influenza A virus to antiviral agents such as amount of inoculum size, duration of incubation, fixative type, and cell number; factors which are critical to the performance of the in situ cellular ELISA. In situ cellular ELISA was found to correlate strongly with the plaque assay (PA) (R2 = 0.997, P < 0.002). Both assays were applied to test the susceptibility of influenza A virus to a new antiviral emulsion agent and yielded comparable data. The optimized in situ cellular ELISA can serve as a reliable assay for the rapid screening of large numbers of antiviral agents.

Inhibition of viral adhesion and infection by sialic-acid-conjugated dendritic polymers.

Multiple sialic acid (SA) residues conjugated to a linear polyacrylamide backbone are more effective than monomeric SA at inhibiting influenza-induced agglutination of red blood cells. However, "polymeric inhibitors" based on polyacrylamide backbones are cytotoxic. Dendritic polymers offer a nontoxic alternative to polyacrylamide and may provide a variety of potential synthetic inhibitors of influenza virus adhesion due to the wide range of available polymer structures. We evaluated several dendritic polymeric inhibitors, including spheroidal, linear, linear-dendron copolymers, comb-branched, and dendrigraft polymers, for the ability to inhibit virus hemagglutination (HA) and to block infection of mammalian cells in vitro. Four viruses were tested: influenza A H2N2 (selectively propagated two ways), X-31 influenza A H3N2, and sendai. The most potent of the linear and spheroidal inhibitors were 32-256-fold more effective than monomeric SA at inhibiting HA by the H2N2 influenza virus. Linear-dendron copolymers were 1025-8200-fold more effective against H2N2 influenza, X-31 influenza, and sendai viruses. The most effective were the comb-branched and dendrigraft inhibitors, which showed up to 50000-fold increased activity against these viruses. We were able to demonstrate significant (p < 0.001) dose-dependent reduction of influenza infection in mammalian cells by polymeric inhibitors, the first such demonstration for multivalent SA inhibitors. Effective dendrimer polymers were not cytotoxic to mammalian cells at therapeutic levels. Of additional interest, variation in the inhibitory effect was observed with different viruses, suggesting possible differences due to specific growth conditions of virus. SA-conjugated dendritic polymers may provide a new therapeutic modality for viruses that employ SA as their target receptor.

1Alpha,25-dihydroxyvitamin D3 up-regulates Bcl-2 expression and protects normal human thyrocytes from programmed cell death.

Apoptosis is thought to play an important role in the pathogenesis of autoimmune thyroid disease. 1alpha,25-dihydroxyvitamin D3 (VD3) has been shown to suppress several autoimmune diseases. However, the mechanism by which VD3 has these effects is not known. We evaluated the alterations in apoptosis, induced by VD3. Thyrocytes were treated with VD3, and the expression of the Bcl-2 family molecules was studied at both the messenger RNA and protein levels. It was found that VD3 significantly induced the expression of Bcl-2 messenger RNA and protein in thyrocytes but had no effect on the expression of Bcl-xl and Bax. The increase in Bcl-2 expression, mediated by VD3, correlated with protection of thyrocytes against the induction of apoptosis by either staurosporine or UV irradiation. VD3-induced increases in the expression of Bcl-2 could be mimicked by VD3 analogs with high nuclear receptor affinity, but not by analogs only with nongenomic actions. These data indicate a role for Bcl-2 in the regulation of apoptosis in thyrocytes and raise the possibility that VD3 or its agonists may have therapeutic benefit in thyroid disorders.

Anaphylaxis after ingestion of carmine colored foods: two case reports and a review of the literature.

Two patients with adverse food reactions to foods colored with carmine dye are presented, along with a review of the medical literature addressing adverse reactions to carmine colorant. This review summarizes the mounting evidence suggesting that adverse reactions to carmine colorant are the result of an IgE mediated mechanism.

Studies on binding of HIV-1 p24gag peptide to HLA-Cw3+ cells.

Human major histocompatibility complex class I antigens, HLA-C, are expressed on the cell surface at approximately a tenfold lower level than HLA-A and -B. We hypothesized that the expression of HLA-C is limited by the quantity of high affinity peptides which bind to these molecules, thus allowing only a small fraction of HLA-C molecules to be transported and/or to remain stable on the cell surface. If this assumption is correct, then the addition of exogenous peptide should increase cell surface HLA-C expression. To verify the hypothesis, we pulsed lymphoblastoid cell line PAJ (HLA-Cw3+) with synthetic HIV-1 p24gag 145-152 peptide, known to be presented to T-lymphocytes by HLA-Cw3 molecule. PAJ (HLA-Cw3+) cells bound approximately two times more of the peptide than HAJ (HLA-Cw3-), and four times more than 500/C9 (HLA-Cw3-) cells. Accordingly, overnight pulsing of PAJ cells with the p24gag 145-152 peptide caused an increase in class I HLA expression detected on the cell surface by flow cytofluorimetric analysis with anti-HLA-B,C monoclonal antibodies but not by anti-HLA-A antibody. In contrast, HLA-Cw3- cells treated in the same manner did not show any increase of HLA class I expression. Our data suggest that low concentration of high affinity peptides within the cell may be one of the factors limiting cell surface expression of HLA-C molecules.

The level of lipopolysaccharide-binding protein is significantly increased in plasma in patients with the systemic inflammatory response syndrome.

Currently, there is no way to predict with a high degree of sensitivity and specificity which patients are likely to develop systemic inflammatory response syndrome (SIRS) following systemic infection, trauma, organ rejection, or blood loss. The level of human lipopolysaccharide-binding protein (LBP) was determined in the plasma of 22 patients with a clinical diagnosis of early SIRS. Twenty-nine plasma samples from healthy volunteers were used as controls. The mean level of LBP in the plasma of healthy volunteers was 7.7 micrograms/ml (standard deviation, 6.2 micrograms/ml). Twenty-one of 22 patients (95%) with SIRS had an LBP level on admission at least 2 standard deviations above the mean LBP level for a healthy volunteer control group (range, 4.9 to 114.2 micrograms/ml; mean, 36.6 micrograms/ml; standard deviation, 22.2 micrograms/ml; P < 0.0001). The level of LBP in the plasma of the majority of patients with early SIRS is significantly increased compared to that in healthy controls. The sensitivity, specificity, and predictive value of elevated plasma LBP levels in patients with SIRS remain to be determined.

In vitro bromodeoxyuridine labeling of malignant neoplasms. A comparative study with flow cytometry cell-cycle analysis.

Proliferative fraction has been defined as an independent prognostic marker for some malignant neoplasms. An estimate of the proliferative fraction can be obtained by cell-cycle analysis of flow cytometric DNA measurements. However, overlapping DNA distributions of aneuploid neoplasms are difficult to analyze, even with sophisticated computer programs, and results are not always reproducible. Bromodeoxyuridine (BrdUrd) labeling followed by immunohistochemical detection has been proposed as an alternative, simple, and accurate method for identifying and counting DNA synthesizing cells. In vitro BrdUrd labeling was performed on 87 tumors, including 35 lung cancers, 25 breast carcinomas, and 27 tumors of other origin. Results were compared with flow cytometric S-phase estimates in 46 cases. Mean BrdUrd labeling of lung tumors was 8.5% +/- 5.2%, compared with a mean flow cytometric S-phase fraction of 20% +/- 18.4%. Mean BrdUrd labeling of breast carcinomas was 6.8% +/- 3.8%, compared with a mean flow cytometric S-phase fraction of 12.5% +/- 9.9%. In both tumor types, BrdUrd labeling correlated well with histologic grade. Correlation between BrdUrd labeling and flow cytometric S-phase estimates were generally poor, particularly with aneuploid tumors.

DNA stainability in aneuploid breast tumors: comparison of four DNA fluorochromes differing in binding properties.

The aim of this study was to evaluate whether or not the differences in chromatin structure between diploid stromal cells or lymphocytes, which are often used as DNA ploidy standard, and aneuploid breast tumor cells can significantly affect the estimates of the DNA index of these tumors. To this end, the DNA content estimates of 34 aneuploid breast tumors, differing in size, degree of differentiation, and presence or absence of estrogen and progesterone receptors and metastases, were compared using four common DNA fluorochromes: DAPI, Hoechst 33342, propidium iodide, and acridine orange. These dyes differ in their mode of interaction with DNA (binding to minor groove or intercalation) and for each of them binding to DNA is restricted to a different degree by nuclear proteins. It was expected, therefore, that if differences in chromatin structure play a role in DNA content estimates, the DNA index of the measured tumors may vary depending on the dye. The cell nuclei were isolated from the tumors using a detergent-based procedure and stained with each of the dyes and the DNA index was estimated using peripheral blood lymphocytes as a DNA content standard. For each of the tumors, the DNA index estimates with all four dyes correlated very well. When the results obtained with individual dyes were compared in pairs, the correlation coefficients (r) of DNA indices were all above 0.96 (correlation at p less than 0.001). The best concordance was seen between specimens stained with Hoechst 33342 and DAPI (r = 0.99), and the least between those stained with Hoechst 33342 and propidium iodide (r = 0.96). The data indicate that DNA content analysis of unfixed nuclei, utilizing the above fluorochromes, is not significantly biased by differences in chromatin structure of the measured cells.

Increase in acridine orange (AO) fluorescence intensity of monocytes cultured in plastic tissue culture plates as measured by flow cytometry.

Although the green-red fluorescence of AO is an accepted measure of DNA-RNA content, respectively, it is actually a measure of the fluorescence of dye bound to nucleic acids, and may vary with changes in accessibility to the dye. It has been shown for example that extraction of nuclear proteins results in a marked increase in DNA stainability. Moreover, in certain cell systems the binding of fluorochromes correlates with structural modifications in chromatin that accompany cell differentiation. We report here that changes in green & red fluorescence intensity also occur in long-term monocyte cultures. The increased red fluorescence intensity observed in cultured monocytes may reflect ribosomal RNA synthesis and the increased green fluorescence enhanced AO accessibility to DNA due to changes in chromatin organization. We compared cultured monocytes from bladder cancer patients and healthy donors. The results indicate a small but statistically significantly greater increase in mean green & red fluorescence of cultured monocytes from the cancer patients. These fluorescence variations may indicate differences in the immunologic status of cancer patients and/or be related to disease state.

Differences in the action and metabolism between retinol and retinoic acid in B lymphocytes.

We have previously reported on the dependency of activated B lymphocytes for retinol. Here we confirm and extend these findings that cells deprived of retinol perish in cell culture within days, displaying neither signs of apoptosis nor of cell cycle arrest. Cell death can be prevented by physiological concentrations of retinol and retinal, but not by retinoic acid or three synthetic retinoic acid analogues. To exclude the possibility that retinoic acid is so rapidly degraded as to escape detection, we have tested its stability in intra- and extracellular compartments. Contrary to expectation, we find that retinoic acid persists for longer (t 1/2 3 d) in cultures than retinol (t 1/2 1 d). Furthermore, despite the use of sensitive trace-labeling techniques, we cannot detect retinoic acid or 3,4-didehydroretinoic acid among retinol metabolites. However, retinol is converted into several new retinoids, one of which has the ability to sustain B cell growth in the absence of an external source of retinol, supporting the notion of a second retinol pathway. We have also determined which of the known retinoid-binding proteins are expressed in B lymphoblastoid cells. According to results obtained with polymerase chain reaction-assisted mRNA detection, they transcribe the genes for cellular retinol- and cellular retinoic acid-binding proteins, for the nuclear retinoic acid receptors, RAR-alpha, -gamma, and RXR-alpha, but not RAR-beta. Our findings that B cells do not synthesize retinoic acid or respond to exogenous retinoic acid on the one hand, but on the other hand convert retinol to a novel bioactive form of retinol, suggest the existence of a second retinoid pathway, distinct from that of retinoic acids.

Effect of swainsonine on interleukin-2 alpha chain receptor expression and proliferation of human lymphocytes.

Swainsonine, an inhibitor of mannosidase II, involved in N-linked glycoprotein processing, modifies expression of cell surface receptors. This alkaloid has strong anti-metastatic and immunomodulatory activity; it enhances stimulation of lymphocytes triggered by concanavalin A (ConA) but suppresses stimulatory effects of phytohaemagglutinin (PHA). We presently observe that swainsonine decreases expression of the interleukin-2 (IL-2) receptor (IL-2R) on PHA-stimulated human peripheral blood lymphocytes, measured by binding of a monoclonal antibody that recognizes the 55-kD glycoprotein subunit (alpha) of this receptor. Proliferation of the PHA-stimulated lymphocytes is suppressed by swainsonine, which manifests in the decreased proportion of both cells entering G1 (from G0) and those progressing through S. G2 and M. This suppression can be overcome by addition of IL-2 into cultures. In contrast, swainsonine has no effect on IL-2R expression and stimulation (cell cycle progression) of lymphocytes triggered by the monoclonal antibody OKT3. The data suggest a possibility that the observed swainsonine effects on lymphocytes stimulated by PHA are mediated via surface receptors other than IL-2R. These receptors may appear prior to IL-2R and be also involved in cell stimulation by PHA but not by other mitogens.

Retention of the mitochondrial probe rhodamine 123 in normal lymphocytes and leukemic cells in relation to the cell cycle.

The cationic fluorochrome rhodamine 123 (R123) is specifically taken up by mitochondria of live cells where it is retained due to the mitochondrial transmembrane potential. After pulse exposure of human normal quiescent or proliferating lymphocytes, human lymphocytic leukemic MOLT cells, and mice leukemic L1210 cells to 10 micrograms/ml of R123, the dye release was studied using flow cytometry. Two distinct phases of R123 release, each following first-order kinetics, were apparent; the half-time of retention for the rapidly and slowly released fractions of R123 was 0.8-1.1 and 2.8-4.2 h, respectively. Simultaneous supravital cell staining with R123 and Hoechst 33342 made it possible to correlate retention of R123 with cell position in the cell cycle. No significant differences were observed in the rate of R123 release from cells in G1 vs S or vs G2 + M phases of the cycle. The data rule out a possibility that the release of R123 is due to periodic depolarization of the mitochondria in the cell as may be postulated by cell cycle models that assume a transient passage of cells through resting phase following division. The observed similar rates of R123 release regardless of cell type or cell cycle phase suggest that the factors affecting the exchange are similar in normal lymphocytes vs leukemic cells and unrelated to cell proliferation rate or phase of the cell cycle. Two distinct rates of R123 release indicate the presence of two kinds of binding sites differing in affinity to the dye.