Pharmacokinetics, efficacy and safety of daclatasvir plus asunaprevir in dialysis patients with chronic hepatitis C: pilot study.

The aim of this study was to evaluate the pharmacokinetic profile of daclatasvir (DCV) and asunaprevir (ASV) dual therapy in haemodialysis patients infected with hepatitis C virus (HCV). Eighteen haemodialysis patients and 54 patients with normal renal function were treated with DCV and ASV dual therapy for 24 weeks. We evaluated the pharmacokinetic profiles of DCV and ASV and examined the rate of sustained virological response 12 weeks after the end of treatment (SVR12 ) and incidence of adverse events during treatment of haemodialysis patients infected with chronic HCV genotype 1 infection. To adjust for potential differences in baseline characteristics between haemodialysis patients and patients with normal renal function, we used propensity scores case-control matching methods. Area under the plasma concentration time curve from 0 to 6 h (AUC0-6 h ) of DCV was slightly lower in haemodialysis patients than in patients with normal renal function (P > 0.6). AUC0-6 h of ASV was significantly lower in haemodialysis patients (P = 0.012). SVR12 rates were 100% (18/18) for haemodialysis and 96.2% (52/54) for patients with normal renal function. Changes in mean log10 HCV RNA levels and viral response were higher in haemodialysis patients compared to patients with normal renal function. No discontinuations due to adverse events occurred. In conclusion, DCV and ASV dual therapy for HCV infection is effective and safe with similar results in haemodialysis patients compared to patients with normal renal function.

Quantitative assessment of urinary equol levels, equol-producing bacteria, and the faecal microbiota in healthy Japanese individuals.

Equol (4',7-isoflavandiol) has attracted considerable attention for its potential efficacy in treating hormonal diseases. In this study we collected samples from healthy Japanese individuals (n = 91) to observe the relationship between the abundance of equol-producing bacteria in their faeces and the concentration of equol in their urine. Quantitative polymerase chain reaction (qPCR) targeting the dihydrodaidzein reductase gene (dhdr) was used to detect equol-producing bacteria. Equol producers, who were defined as individuals with >1000 nmol/l equol in their urine, exhibited 4-8 log10 copies of dhdr/g faeces of equol-producing bacteria. We assessed the accuracy of these findings by determining the rate of correspondence between possessing equol-producing bacteria and producing urinary equol. Of the 91 participants, 33 were found to be positive for both equol-producing bacteria and urinary equol, 52 were negative for both, one was positive for equol-producing bacteria and negative for urinary equol, and five were negative for equol-producing bacteria and positive for urinary equol. The sensitivity and specificity of the qPCR for detecting equol-producing bacteria were 86.8% and 98.1%, respectively. On the whole, the presence of equol-producing bacteria and urinary equol displayed 93.4% concordance, with a kappa coefficient of 0.862. No apparent correlation was observed between dhdr copy number in the faeces and urinary equol concentrations. Analysis of the faecal microbiota showed that alpha diversity indices (OTU, ACE, Chao1, Shannon) were significantly higher in equol producers. Specifically, the relative abundance of phylum Pseudomonadota was increased in non-equol producers, while abundance of genus Alistipes, Barnesiella, Butyricimonas, Odoribacter, and Ruminococcus, which produce short chain fatty acids and/or hydrogen, were only observed in equol producers. These results suggest that a certain amount of equol-producing bacteria must be present in the intestine to produce detectable levels of equol, and that equol productivity might be affected by other components of the microbiota.

A novel migration pathway for rat dendritic cells from the blood: hepatic sinusoids-lymph translocation.

The migration pathways for dendritic cells (DC) from the blood are not yet completely resolved. In our previous study, a selective recruitment of DC progenitors from the blood to the liver was suggested. To clarify the role of the hepatic sinusoids in the migration of blood DC, relatively immature DC and mature DC were isolated from hepatic and intestinal lymph, and intravenously transferred to allogeneic hosts. It was then possible to detect small numbers of DC within secondary lymphoid tissues either by immunostaining for donor type major histocompatibility complex class I antigen or, at much higher sensitivity, for bromodeoxyuridine incorporated by proliferating cells (mainly T lymphocytes), which responded to the alloantigen presented by the administered DC. The intravenously injected DC accumulated in the paracortex of regional lymph nodes of the liver via a lymph-borne pathway. Intravenously injected fluorochrome-labeled syngeneic DC behaved similarly. In contrast, very few DC were found in spleen sections and were hardly detectable in other lymph nodes or in other tissues. An in situ cell binding assay revealed a significant and selective binding of DC to Kupffer cells in liver cryosections. It is concluded that rat DC can undergo a blood-lymph translocation via the hepatic sinusoids, but not via the high endothelial venules of lymph nodes. Hence the hepatic sinusoids may act as a biological concentrator of blood DC into the regional hepatic nodes. Kupffer cells may play an important role in this mechanism.

A life stage of particle-laden rat dendritic cells in vivo: their terminal division, active phagocytosis, and translocation from the liver to the draining lymph.

Initiation of an adoptive immune response against pathogenic organisms, such as bacteria and fungi, may involve phagocytic activity of dendritic cells (DC) or their immature precursors as a prelude to antigen processing and presentation. After intravenous injection of rats with particulate matter, particle-laden cells were detected in the peripheral hepatic lymph. Since it has been known there is a constant efflux of DC from nonlymphoid organs into the draining peripheral lymph, we examined whether these particle-laden cells belonged to the DC or macrophage lineage. The majority of particle-laden cells in lymph showed immature monocyte-like cytology, and the amount of ingested particles was small relative to typical macrophages. We identified these particle-laden cells as DC based on a number of established criteria: (a) they had a phenotype characteristic of rat DC, that is, major histocompatibility complex class Ihigh+ and IIhigh+, intercellular adhesion molecule 1+ and 80% positive with the rat DC-specific mAb OX62; (b) they showed strong stimulating capacity in primary allogeneic mixed leukocyte reaction; (c) in vitro, they had little phagocytic activity; and (d) the kinetics of translocation was similar to that of lymph DC in that they migrated to the thymus-dependent area of the regional nodes. Furthermore, bromodeoxyuridine feeding studies revealed that most of the particle-laden DC were recently produced by the terminal division of precursor cells, at least 45% of them being <5.5 d old. The particle-laden DC, defined as OX62+ latex-laden cells, were first found in the sinusoidal area of the liver, in the liver perfusate, and in spleen cell suspensions, suggesting that the site of particle capture was mainly in the blood marginating pool. It is concluded that the particle-laden cells in the hepatic lymph are recently produced immature DC that manifest a temporary phagocytic activity for intravascular particles during or after the terminal division and that the phagocytic activity is downregulated at a migratory stage when they translocate from the sinusoidal area to the hepatic lymph.

The mutations of Th1 cell-specific T-box transcription factor may be associated with a predominant Th2 phenotype in gastric cancers.

Gastric cancer is a serious public health cancer and causes nearly 1 million deaths a year worldwide. Th1 cells play critical roles in orchestrating the adaptive immune responses against gastric cancer. T-bet, a member of the T-box family of transcription factors, is the Th1 master regulator and up-regulated during Th1 differentiation. Polymorphisms have also been shown to exist in T-bet. Some reports indicated that some tumours were associated with the drift of Th1 and Th2. In the present work, we investigated the drift of Th1/Th2 by detecting the expression levels of T-bet, IFN-gamma, IL-4, and GATA-3 in peripheral blood mononuclear cell of gastric cancer patients by real-time PCR, explored the relationship between the polymorphism of T-bet gene and drift of Th1/Th2 by gene sequence, western blot, and gene transfection. Our results indicated that a predominant Th2 phenotype was existence. T-bet gene mutations may be associated with Th2-dominated condition in gastric cancers.

Four novel resistance integron gene-cassette occurrences in bacterial isolates from zhenjiang, china.

Integrons, which are widely distributed among bacteria and are strongly associated with resistance, are specialized genetic elements that are capable of capturing, integrating, and mobilizing gene cassette. In this work, we investigated classes 1, 2, and 3 integrons associated integrases genes in 365 bacteria isolates, amplified and analyzed the structure of class 1 integron, detected 8 resistant gene cassettes [dfr17, aadA5, aadA1, aadA2, dhfrI, aadB, aac(6')-II, and pse-I], and found four novel gene-cassette arrays. We also found that commensal bacteria in the common microenvironment had the same integron gene cassette, which provided direct evidence that integron was an important horizontal transmission element.

Rapid detection of Brucella spp. by the loop-mediated isothermal amplification method.

AIMS: To develop a rapid and sensitive method for detecting Brucella spp. METHODS AND RESULTS: Two sets of six Brucella-specific primers for loop-mediated isothermal amplification (LAMP) were designed from the sequence of the Brucella abortus BCSP31 gene. The specificity and sensitivity were examined for six Brucella species (22 strains) and 18 non-Brucella species (28 strains). The LAMP assay was specific to Brucella spp. in 35 min at 63 degrees C and sensitive (detected 10 fg of genomic DNA). The assay was also applied for the detection of Brucella DNA in contaminated milk and infected mouse organs. CONCLUSIONS: We developed a sensitive and specific LAMP assay for Brucella spp., with the test appearing to be useful for the detection of the pathogen from clinical and food samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the development of LAMP for the detection of Brucella spp. As the LAMP assay can be performed at a constant temperature and its reactivity is directly observed with the naked eye without electrophoresis, our assay should be useful for the diagnosis of brucellosis as well as the detection of the bacteria in environmental or food samples.

Levels of calcium, magnesium and zinc in urine among adult women in relation to age with special reference to menopause.

OBJECTIVES: This study was initiated to examine, on a basis of large-scale epidemiology, if urinary calcium (Ca), magnesium (Mg) and zinc (Zn) levels change as a function of age and menopause. METHODS: Spot urine samples were collected from adult women, and analyzed for the minerals. Additional information e.g. on smoking habits was obtained by questionnaires, so that cases were classified into 10,464 never-smokers, 1,351 current smokers and 343 past smokers. The mineral concentrations were evaluated as observed (e.g. Ca-U(ob)), and after correction for creatinine (CR) concentration (e.g. Ca-U(cr)) or specific gravity (SG) (e.g. Ca-U(sg)). RESULTS: Analyses with never-smokers showed that age-dependent changes in Ca-U(ob), Mg-U(ob) and Zn-U(ob) were minute. Menopause induced a small increase in Ca-U(ob) and a small decrease in Zn-U(ob). Values after CR or SG correction were increased in accordance with both age and menopause, possibly due to age- and menopause-associated decreases in urine density. CONCLUSIONS: Ca-U(ob), Mg-U(ob) and Zn-U(ob) did not vary substantially throughout life. Ca-U(ob) and Zn-U(ob) were slightly higher and lower, respectively, in post-menopausal women than in pre-menopausal women, but such changes were too small to affect life-long stabilities. Thus, the urinalyses did not suggest need of additional supply of Ca, Mg or Zn at advanced ages. Correction for CR or SG may induce a bias in evaluation of age-dependent changes in mineral concentrations, because CR and SG decrease in accordance with age.

Low-level cadmium exposure in Toyama City and its surroundings in Toyama prefecture, Japan, with references to possible contribution of shellfish intake to increase urinary cadmium levels.

OBJECTIVES: This study was initiated to examine if exposure to cadmium (Cd) was high also outside of the previously identified Itai-itai disease endemic region in the Jinzu River basin in Toyama prefecture in Japan. METHODS: Morning spot urine samples were collected in June-August 2004 from 651 adult women (including 535 never-smokers) in various regions in Toyama prefecture, and subjected to urinalyses for cadmium (Cd), alpha1-microglobulin (alpha1-MG), beta2-microglobulin (beta2-MG), N-acetyl-beta-D-glucosaminidase (NAG), specific gravity (SG or sg) and creatinine (CR or cr). Three months later, the second urine samples were collected from those with elevated Cd in urine (e.g., > or =4 microg/g cr), together with answers to questionnaires on shellfish consumption. RESULTS: The geometric mean (GM) Cd, alpha1-MG, beta2-MG and NAG (after correction for CR) for the total participants were 2.0 microg/g cr, 2.4 mg/g cr, 104 microg/g cr and 2.8 units/g cr, respectively; further analysis with never-smoking cases only did not induce significant changes in these parameters. Analyses of the second urine samples from the high Cd subjects showed that there was substantial decrease (to about a half) in Cd in the 3-month period, and that the decrease was accompanied by reduction in alpha1-MG and NAG (beta2-MG did not show elevation even in the first samples). The urinalysis results in combination with the results of the questionnaire survey suggest that the high urinary Cd was temporary and might be induced by intake of shellfish that is edible whole. CONCLUSIONS: The overall findings appear to suggest that Cd exposure in Toyama populations (outside of the Itai-itai disease endemic region) was at the levels commonly observed on the coast of the Sea of Japan, and that the Cd level in urine might be modified by the intake of some types of seafood. Further studies are necessary to elucidate the relation of urinary Cd with seafood intake.

No meaningful increase in urinary tubular dysfunction markers in a population with 3 microg cadmium/g creatinine in urine.

The critical Cd exposure level to induce tubular dysfunctions is a focus of public concern among general populations in Japan. To answer this question, one group each (about 1000 adult women/area) in nonpolluted areas with high (Area H) and low Cd exposure (Area L) was obtained, and 742 strictly age-matched pairs of never-smoking adult women were selected for comparison. Cd, alpha1-MG (microglobulin) and beta2- MG in urine were taken as markers of exposure and tubular dysfunction, respectively. Geometric mean Cd levels as corrected for creatinine (Cdcr) was greater than three times higher in Area H (2.8 microg/g cr) than in Area L (0.8 microg/g cr). Nevertheless, beta2-MGcr did not differ between the two areas (125 microg/g cr for Area H vs 118 microg/g cr for Area L). alpha1-MGcr was only marginally higher in Area H (2.8 mg/g cr) than in Area L (2.1 mg/g cr), with no biomedical significance. Results were essentially the same when analyses were conducted with noncorrected observed values or values corrected for a specific gravity. Thus, the effects of Cd exposure in Area H on renal tubular function should be essentially nil.

Presentation of tumor antigens by phagocytic dendritic cell clusters generated from human CD34+ hematopoietic progenitor cells: induction of autologous cytotoxic T lymphocytes against leukemic cells in acute myelogenous leukemia patients.

The use of antigen-presenting dendritic cells (DCs) is currently proposed for tumor immunotherapy through generation of CTLs to tumor antigens in cancer patients. In this study, DCs were differentiated using granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha from CD34+ hematopoietic progenitor cells that had been mobilized into the peripheral blood. To use the phagocytic activity of DCs for processing and presentation of tumor antigens, we established DC clusters containing immature DCs by preserving proliferating cell clusters without mechanical disruption. After an 11-day culture, the developed clusters contained not only typical mature DCs but also immature DCs that showed active phagocytosis of latex particles, suggesting that the clusters consisted of DCs of different maturational stages. These heterogeneous clusters could present an exogenous protein antigen, keyhold limpet hemocyanin, to both CD4+ and CD8+ T lymphocytes. Furthermore, in three acute myelogeneous leukemia patients, clusters pulsed with autologous irradiated leukemic cells could also induce antileukemic CTLs. The mechanical disruption of clusters abrogated the induction of CTLs to leukemic cells as well as to hemocyanin. This observation gives an important information for the use of heterogeneous DC clusters derived from autologous peripheral blood CD34+ cells in the case of immunotherapy for leukemia.

Interferon-inducible gene family 1-8U expression in colitis-associated colon cancer and severely inflamed mucosa in ulcerative colitis.

Persistent severe inflammation in colonic mucosa is thought to cause the development of colon cancer in patients with ulcerative colitis (UC). However, predisposing genetic abnormalities have not been identified in this sequence. Using differential display PCR, we isolated cDNA fragments corresponding to mRNAs that were differentially expressed in colitis-associated cancer tissues and mucosa with mild inflammation in the colon of five UC patients. This molecular screening approach identified 60 cDNA fragments, and we sequenced 34 fragments. One cDNA fragment, which is identical to IFN-inducible gene family 1-8U, was strongly expressed in all five UC-associated cancers. 1-8U was also expressed in sporadic colon cancer tissues and colon cancer cell lines, but not in normal mucosa. This gene was strongly expressed in severely inflamed colonic mucosa of UC without colitis-associated colon cancer, although 1-8U expression was not related to the extent and duration of the disease. However, 1-8U was expressed in the colonic mucosa of all patients with chronic, continuously severe inflammation. These results indicated that IFN-inducible gene family 1-8U expression in inflamed colonic mucosa might be used as a preferential marker of colitis-associated colon cancer in UC.

Dendritic cell dynamics in the liver and hepatic lymph.

Dendritic cells (DC) are bone-marrow-derived cells that function as professional antigen-presenting cells (APC). Liver is an essential organ for a host defense. It not only is armed with a powerful macrophage system but also is constantly surveyed by a heavy traffic of DC and lymphocytes. In case of emergency, such as infection and inflammation, DC traffic in the liver is accelerated. DC in the liver (interstitial DC) capture and process antigens, enter the draining lymph (DC in hepatic lymph) and accumulate in the T-cell area of hepatic lymph nodes (LN). DC in the LN present antigens to T and B cells to initiate immune responses. In accelerated states, DC precursors are recruited to the liver and soon translocate to hepatic lymph. Even mature lymph DC can undergo a blood-lymph translocation from the liver to hepatic LN after i.v. injection into normal rats. Rat Kupffer cells in the hepatic sinusoids are capable of selectively trapping DC from the blood in vivo and in vitro, suggesting involvement of certain adhesion molecules. Kupffer cells presumably elaborate chemokines to attract and trap the recruited DC via selective adhesion, leading to DC extravasation. The accelerated traffic and the presence of blood-lymph translocation would induce rapid and efficient immune responses and thus contribute to the local defense to antigens within liver tissues as well as systemic defense to blood-borne antigens. DC progenitors are also present in the liver, and these may play an important role in tolerance induction in liver transplantation.

In vivo roles of donor and host dendritic cells in allogeneic immune response: cluster formation with host proliferating T cells.

Possible roles of dendritic cells (DCs) in allogeneic immune responses in host lymphoid tissues were characterized in situ by using rat DC transfer and cardiac transplantation models. When allogeneic DCs were intravenously injected, these cells selectively migrated to the T-cell area of hepatic lymph nodes, with peak accumulation at 18 h after injection. Donor DCs and proliferating host T cells formed clusters (rosettes) in which the T-cell proliferative response started. The donor DCs were CD80(+) CD86(+) and, ultrastructurally, were in intimate contact with lymphoblasts within the rosettes. As a novel finding, some of the migrated donor DCs were quickly phagocytosed by putative host interdigitating DCS: By 48 h, the remaining donor DCs had disintegrated within the rosettes. Host interdigitating DCs also formed rosettes throughout the T-cell area, and their kinetics correlated well with that of the T-cell proliferation. In the cardiac allograft model, a few donor DCs selectively migrated to the host spleen and hepatic nodes. Rosette formation by donor and host DCs, phagocytosis of donor DCs, and the T-cell proliferative response occurred in much the same fashion as they did in the first experiment. We conclude that the donor rosettes at the early stage represent the sites of direct allosensitization and those at the late stage represent donor-DC killing. Host rosettes are the sites of T-cell proliferation. In this structure, phagocytosed donor-DC-derived antigens are presumably indirectly presented.

Effects of estrogen on hepatic hemopoiesis in the adult mouse.

After a single pharmacologic dose of estriol (E3) treatment, an increase in number of focal areas of hepatic hemopoiesis was observed in adult mice. When syngeneic bone marrow cells were transfused into E3-treated mice, focal hepatic hemopoiesis was increased further. In a single experiment, using a single cell suspension, the concentration of CFUs in the liver was increased 5 days after E3-treatment, while that in the blood decreased. In addition, 51Cr-labeled bone marrow cells selectively accumulated in the E3-treated mouse liver. These results suggest, but in not sense prove, that circulating hemopoietic stem cells are trapped in the E3-treated mouse liver, and that E3-activated Kupffer cells may play a central role in focal hemopoiesis in the liver.

5 alpha-reductase type 2 is constitutively expressed in the dermal papilla and connective tissue sheath of the hair follicle in vivo but not during culture in vitro.

Recent studies suggest that 5 alpha-reductase type 2 (5 alpha R2) rather than 5 alpha R1 plays a key role in the pathogenesis of male-pattern baldness. To clarify the localization of the androgen receptor (AR), 5 alpha R1, and 5 alpha R2 in the hair follicle, we investigated the expression of the corresponding genes by RT-PCR using microdissected hair follicles. AR and 5 alpha R1 mRNAs were expressed in all portions of the hair follicle. By contrast, 5 alpha R2 mRNA was expressed only in mesenchymal portions that included the dermal papilla and connective tissue sheath, and hardly any was expressed in epithelial portions. The intensity of expression of these genes in each portion of the hair follicles did not differ between follicles from balding and nonbalding scalp. We also examined the expression of these genes in cultured fibroblasts derived from the dermal papilla and connective tissue sheath. Although expression of AR and 5 alpha R1 mRNAs was easily detected, there was no obvious expression of 5 alpha R2 mRNA in either type of cell. Type-specific inhibition of 5 alpha R activity by MK386 and MK906 confirmed these patterns of expression of 5 alpha R mRNA. Thus, the expression of 5 alpha R2 mRNA seems to be characteristic of freshly microdissected mesenchymal portions of the hair follicle, but such expression might not be maintained in culture.

The haemolytic plaque reduction technique to measure cytotoxicity with hybridoma cells as a target.

The use of hybridoma cells as targets to measure cell-mediated cytotoxicity has been established. The ability of target hybridoma cells to form haemolytic plaques has been used as an indicator of target viability and therefore, cytotoxicity has been detected by plaque reduction (PR). Allogeneic responses, spontaneous cytotoxic responses and anti-hapten responses have been measured by the PR assay. Compared with the standard [51Cr]chromate release assay, small numbers of target cells (400 or less) can be used in the PR assay and this results in a greatly enhanced sensitivity in detecting small amounts of cytotoxicity. The ability to construct a range of hybridoma targets with the appropriate cell surface determinants presents a new approach, of general applicability, to the sensitive detection of cytotoxic lymphocytes.

The adaptation of the plaque reduction assay for measuring specific cytotoxic cells in limiting dilution cultures.

The use of the original haemolytic plaque reduction technique to measure cytotoxic T lymphocytes (CTL) has been developed further as a rapid screening assay, particularly suitable for limiting dilution analyses. Using hybridoma cells as targets, the cytotoxicity has been measured by the loss of haemolytic plaque formation and by the reduction of the amount of haemolytic monoclonal antibody secreted from viable target cells into the assay supernatants. The assessment of large numbers of cytotoxic samples has been greatly facilitated by quantitating the amount of haemoglobin released in the assay with an automated microELISA multiscanner and by scoring visually using a modification of the spot test. Using these new techniques, relatively high frequency estimates of cytotoxic cell precursors in an allogeneic response (1 in 462 spleen cells) and an anti-fluorescein response (1 in 3970 spleen cells) were obtained.

Trafficking of host- and donor-derived dendritic cells in rat cardiac transplantation: allosensitization in the spleen and hepatic nodes.

BACKGROUND: Kinetics and role of host and donor dendritic cells (DCs) in transplantation immunity are still ill-defined. Using a rat cardiac transplantation model, we studied DC trafficking and sites for allosensitization. METHODS: Host and donor DCs were defined as host- or donor-type class II major histocompatibility complex antigen single-positive cells by double-immunostaining. Proliferative response of both donor and host cells were also analyzed. RESULTS: Host DCs were recruited to the graft soon after transplantation. These cells represented definitive precursors because of high labeling index by a continuous bromodeoxyuridine infusion, their small round shape, and their putative bone marrow origin. Donor interstitial DCs showed a significant self-replicating capability. Both recruited host DCs in a regraft experiment and donor DCs preferentially performed blood-borne migration to the T-cell area of host spleen. Furthermore, they also migrated to the T-cell area of hepatic lymph nodes after executing the sinusoids-lymph translocation as a novel pathway for these DCs. Selectively at their migration sites, a strong T-cell proliferative response occurred, which preceded that in the graft tissues. Removal of spleen and hepatic lymph nodes significantly prolonged the mean graft survival time. CONCLUSION: We conclude that allogeneic heart transplantation induces the recruitment of host DC precursors to the graft tissues and the blood-borne migration of both recruited host and donor DCs to the host spleen and hepatic nodes where effector cells are predominantly sensitized.

Isolation of dendritic cells in the rat liver lymph.

Lymphadenectomy of the regional lymph nodes of the rat liver resulted in the direct influx of peripheral hepatic lymph into the thoracic duct after regeneration of lymphatic vessels. Thus, we could obtain the dendritic cells in the hepatic lymph by cannulating the thoracic duct. By the double immunostaining, dendritic cells in cytosmears could be easily determined as non-B, non-T, and MHC class II-positive cells. The yield of dendritic cells after enrichment by the metrizamide density gradient was about 5 x 10(5)/first 16 hr collection/rat, with viability of more than 95% and purity of more than 70%. About 80% of dendritic cells were positive for OX62, which recognized the rat dendritic cell subpopulation. They showed strong stimulating activity in the primary allogeneic mixed leukocyte reaction. The method presented here should be applicable to studies of the roles of liver dendritic cells, especially in transplantation immunity.