Transportation of sublingual antigens across sublingual ductal epithelial cells to the ductal antigen-presenting cells in mice.

BACKGROUND: Sublingual immunotherapy (SLIT) has proven to be safe and efficient for the treatment of type I allergies. However, the mechanisms underlying allergen transportation within the sublingual compartment, the localization of antigens, and the identities of the cells responsible for this immunization remain incompletely understood. OBJECTIVE: In this study, we focused on the sublingual ductal system and analysed the localization and transportation of antigens after their sublingual application. METHODS: In mice given adjuvant-free antigens sublingually, tissues were removed at 0, 0.5, 1, or 2 h after the application and subjected to immunohistochemistry. Cells isolated from the sublingual duct and mucosa were analysed by flow cytometry. RESULTS: Substantial immunoreactivity to ovalbumin (OVA) was evident in sublingual ductal epithelial cells at 30 min and 1 h after sublingual administration of OVA, but it had disappeared at 2 h. The ductal epithelial cells incorporated not only OVA, but also particulate antigens such as latex or silica beads and microbes. MHC class II (MHCII)(+) antigen-presenting cells (APCs) were located around the sublingual ductal system, and MHCII(+) cells were co-localized with, and around, antigen-incorporated sublingual duct cells. CD11b(+) CD11c(-) cells were present among CD45(+) MHCII(+) cells at greater frequency in the sublingual duct than in the sublingual mucosa, and they were the main contributors to the incorporation of OVA in vitro. CONCLUSIONS AND CLINICAL RELEVANCE: This study reveals that sublingual antigens can be transported across sublingual ductal epithelial cells to the ductal APCs. If the system is the same in humans as in mice, the ductal APCs may prove to be important target cells for SLIT.

Significant background rates of HBV and HCV infections in patients and risks of blood transfusion from donors with low anti-HBc titres or high anti-HBc titres with high anti-HBs titres in Japan: a prospective, individual NAT study of transfusion-transmitted HBV, HCV and HIV infections.

BACKGROUND: The Japanese Red Cross (JRC) conducted a prospective study to evaluate the frequency of transfusion-transmitted HBV, HCV and HIV infections to assess the risk of transfusion of blood components routinely supplied to hospitals. STUDY DESIGN AND METHODS: Post-transfusion specimens from patients at eight medical institutes were examined for evidence of infection with HBV (2139 cases), HCV (2091) and HIV (2040) using individual nucleic acid amplification testing (NAT). If these specimens were reactive, pre-transfusion specimens were also examined for the virus concerned by individual NAT. In the event that the pre-transfusion specimen was non-reactive, then all repository specimens from implicated donors were tested for the viruses by individual donation NAT. In addition, a further study was carried out to evaluate the risk of transfusion of components from donors with low anti-HBc titres or high anti-HBc with high anti-HBs titres. RESULTS: Transfusion-transmitted HCV and HIV infections were not observed. One case of post-transfusion HBV infection was identified (rate, 0·0004675; 95% CI for the risk of transmission, 1 in 451-41,841). The background rates of HBV, HCV and HIV infections in patients prior to transfusion were 3·4% (72/2139), 7·2% (150/2091) and 0% (0/2040), respectively. Sixty-four anti-HBc- and/or anti-HBs-reactive blood components were transfused to 52 patients non-reactive for anti-HBc or anti-HBs before and after transfusion (rate, 0; 95% CI for the risk of transmission, <1 in 22). CONCLUSION: This study demonstrated that the current criteria employed by JRC have a low risk, but the background rates of HBV and HCV infections in Japanese patients are significant.

Soymilk-fermented with Lactobacillus delbrueckii subsp. delbrueckii TUA4408L improves immune-health in pigs.

Lactobacillus delbrueckii subsp. delbrueckii TUA4408L has the ability to grow and ferment soymilk and is able to modulate the innate immune response of intestinal epithelial cells in vitro. These two properties prompt us to evaluate whether the soymilk fermented with the TUA4408L strain can induce beneficial immunomodulatory effects in vivo. For this purpose, pigs were selected as a preclinical model. The studies performed here demonstrated that the L. delbrueckii subsp. delbrueckii TUA4408L-fermented soymilk (TUA4408L FSM) reduced blood markers of inflammation and differentially regulated the expression of inflammatory and regulatory cytokines in the intestinal mucosa. These immunological changes induced by the TUA4408L FSM were associated to an enhanced resistance to pathogenic Escherichia coli and an improved grow performance and meat quality of pigs. The experiments and analysis in our study indicate that the immunobiotic TUA4408L FSM could be an interesting non-dairy functional food to beneficially modulate the intestinal immune system, improve protection against pathogens and reduce inflammatory damage. The preclinical study carried out here in pigs could have a better correlation in humans, compared to a rodent model. However, the clinical relevance of these findings still needs to be confirmed by further research, for example, in controlled human challenge studies.

Differentially expressed genes during bovine intramuscular adipocyte differentiation profiled by serial analysis of gene expression.

Beef marbling or intramuscular fat deposition is an economically important carcass trait in Japanese Black cattle. To investigate genes involved in intramuscular adipogenesis, differential gene expression during adipogenesis in a clonal bovine intramuscular preadipocyte (BIP) cell line was profiled with serial analysis of gene expression (SAGE). We sequenced 75 283 tags for the proliferation phase (day 0) and 81 878 tags for the differentiation phase (4 days after adipogenic stimulation: day 4). A comparison of the unique SAGE tag frequencies between the day 0- and day 4-libraries revealed that 878 (2.8%) of the 30 989 unique putative transcripts were expressed at significantly different levels (P < 0.05); 401 tags (1.4%) were up-regulated and 477 tags (1.2%) were down-regulated in the day 4-library relative to the day 0-library. We confirmed up-regulation of 10 tags of the genes that were up-regulated in the previous subtraction cloning studies in BIP cells [Animal Science Journal, 76 (2005) 479]. Of the 878 differentially expressed tags, 377 were identified in the bovine RefSeq library and 356 were assigned a bovine draft genomic sequence. Fifteen tags were mapped in previously detected beef marbling quantitative trait loci (QTL) regions [Mammalian Genome, 18 (2007) 125]. These genes may be involved in the adipogenic processes of beef marbling.

Modulation of Toll-like receptor-mediated innate immunity in bovine intestinal epithelial cells by lactic acid bacteria isolated from feedlot cattle.

The ability of lactobacilli isolated from feedlot cattle environment to differentially modulate the innate immune response triggered by Toll-like receptors (TLRs) activation in bovine intestinal epithelial (BIE) cells was evaluated. BIE cells were stimulated with Lactobacillus mucosae CRL2069, Lactobacillus acidophilus CRL2074, Lactobacillus fermentum CRL2085 or Lactobacillus rhamnosus CRL2084 and challenged with heat-stable pathogen associated molecular patterns (PAMPs) from enterotoxigenic Escherichia coli (ETEC) to induce the activation of TLR4 or with polyinosinic:polycytidylic acid (poly(I:C)) to activate TLR3. Type I interferons, cytokines, chemokines and negative regulators of TLR signalling were studied by RT-PCR. L. mucosae CRL2069 significantly reduced the expression of interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 in BIE cells in the context of TLR3 activation. L. mucosae CRL2069 also reduced the expression of tumour necrosis factor-α, IL-ß, MCP-1, and IL-8 in heat-stable ETEC PAMPs-challenged BIE cells. In addition, reduced expressions of IL-6, MCP-1, and IL-8 were found in BIE cells stimulated with L. rhamnosus CRL2084, although its effect was significantly lower than that observed for the CRL2069 strain. The reduced levels of pro-inflammatory factors in BIE cells induced by the CRL2069 and CRL2085 strains was related to their ability of increasing the expression of TLR negative regulators. L. mucosae CRL2069 significantly improved the expression of A20-binding inhibitor of NFκ-B activation 3 (ABIN-3), interleukin-1 receptor-associated kinase M (IRAK-M) and mitogen-activated protein kinase 1 (MKP-1) while L. rhamnosus CRL2084 augmented ABIN-3 expression in BIE cells. The results of this work suggest that among the studied strains, L. mucosae CRL2069 was able to regulate TLR3-mediated innate immune response and showed a remarkable capacity to modulate TLR4-mediated inflammation in BIE cells. The CRL2069 strain induce the up-regulation of three TLR negative regulators that would influence nuclear factor kB and mitogen-activated protein kinases signalling pathways while reducing the expression of pro-inflammatory cytokines and chemokines. Therefore, L. mucosae CRL2069 is an interesting immunobiotic candidate for the protection of the bovine host against TLR-mediated intestinal inflammatory damage.

Deciphering the influence of paraimmunobiotic bifidobacteria on the innate antiviral immune response of bovine intestinal epitheliocytes by transcriptomic analysis.

Previously, we reported that the non-viable immunomodulatory Bifidobacterium infantis MCC12 and Bifidobacterium breve MCC1274 strains (paraimmunobiotic bifidobacteria) were able to increase the protection against rotavirus infection in bovine intestinal epithelial (BIE) cells. In order to gain insight into the influence of paraimmunobiotic bifidobacteria on the innate antiviral immune response of BIE cells, their effect on the transcriptomic response triggered by Toll-like receptor 3 (TLR3) activation was investigated. By using microarray technology and qPCR analysis, we obtained a global overview of the immune genes involved in the innate antiviral immune response in BIE cells. Activation of TLR3 by poly(I:C) in BIE cells significantly increased the expression of interferon (IFN)-α and IFN-ß, several interferon-stimulated genes, cytokines, and chemokines. It was also observed that both paraimmunobiotic bifidobacteria differently modulated immune genes expression in poly(I:C)-challenged BIE cells. Most notable changes were found in genes involved in antiviral defence (IFN-ß, MX1, OAS1X, MDA5, TLR3, STAT2, STAT3), cytokines (interleukin (IL)-6), and chemokines (CCL2, CXCL2, CXCL6) that were significantly increased in bifidobacteria-treated BIE cells. B. infantis MCC12 and B. breve MCC1274 showed quantitative and qualitative differences in their capacities to modulate the innate antiviral immune response in BIE cells. B. breve MCC1274 was more efficient than the MCC12 strain to improve the production of type I IFNs and antiviral factors, an effect that could be related to its higher ability to protect against rotavirus replication in BIE cells. Interestingly, B. infantis MCC12 showed a remarkable anti-inflammatory effect. The MCC12 strain was more efficient to reduce the expression of inflammatory cytokines and chemokines (IL-16, IL-20, CX3CL1) when compared with B. breve MCC1274. These results provided valuable information for the deeper understanding of the antiviral immune response of intestinal epithelial cells as well as the host-paraimmunobiotic interaction in the bovine host.

Development of immune and microbial environments is independently regulated in the mammary gland.

Breastfeeding is important for mammals, providing immunological and microbiological advantages to neonates, together with the nutritional supply from the mother. However, the mechanisms of this functional diversity in the mammary gland remain poorly characterized. Here, we show that, similar to the gastrointestinal tract, the mammary gland develops immune and microbial environments consisting of immunoglobulin A (IgA) and the microflora, respectively, both of which are important for protecting neonates and the mother from infectious diseases. The IgA production and microflora development are coordinated in the gastrointestinal tract but seem to be independently regulated in the mammary gland. In particular, the chemokine (C-C motif) ligand 28 and poly-Ig receptor, crucial molecules for the IgA production in milk, were expressed normally in germ-free lactating mice but were almost undetectable in postweaning mothers, regardless of the microflora presence. Our findings offer insights into potentially improving the quality of breastfeeding, using both immunological and microbiological approaches.

Development of an in vitro immunobiotic evaluation system against rotavirus infection in bovine intestinal epitheliocytes.

The bovine intestinal epithelial cell line (BIE cells) expresses the Toll-like receptor (TLR)3 and is able to mount an antiviral immune response after the stimulation with poly(I:C). In the present study, we aimed to further characterise the antiviral defence mechanisms in BIE cells by evaluating the innate immune response triggered by rotavirus (RV) infection. In addition, we attempted to determine whether immunobiotic bifidobacteria are able to confer protection of BIE cells against RV infection by beneficially modulating the antiviral immune response. RV OSU (porcine) and UK (bovine) effectively infected BIE cells, while a significant lower capacity to infect BIE cells was observed for human (Wa) and murine (EW) RV. We observed that viral infection in BIE cells triggered TLR3/RIG-I-mediated immune responses with activation of IRF3 and TRAF3, induction of interferon beta (IFN-ß) and up-regulation of inflammatory cytokines. Our results also demonstrated that preventive treatments with Bifidobacterium infantis MCC12 or Bifidobacterium breve MCC1274 significantly reduced RV titres in infected BIE cells and differentially modulated the innate immune response. Of note, both strains significantly improved the production of the antiviral factor IFN-ß in RV-infected BIE cells. In conclusion, this work provides comprehensive information on the antiviral immune response of BIE cells against RV, that can be further studied for the development of strategies aimed to improve antiviral defences in bovine intestinal epithelial cells. Our results also demonstrate that BIE cells could be used as a newly immunobiotic evaluation system against RV infection for application in the bovine host.

Establishment of a high throughput EST sequencing system using poly(A) tail-removed cDNA libraries and determination of 36,000 bovine ESTs.

We determined 36,310 bovine expressed sequence tag (EST) sequences using 10 different cDNA libraries. For massive EST sequencing, we devised a new system with two major features. First, we constructed cDNA libraries in which the poly(A) tails were removed using nested deletion at the 3'-ends. This permitted high quality reading of sequences from the 3'-end of the cDNA, which is otherwise difficult to do. Second, we increased throughput by sequencing directly on templates generated by colony PCR. Using this system, we determined 600 cDNA sequences per day. The read-out length was >450 bases in >90% of the sequences. Furthermore, we established a data management system for analyses, storage and manipulation of the sequence data. Finally, 16,358 non-redundant ESTs were derived from approximately 6900 independent genes. These data will facilitate construction of a precise comparative map across mammalian species and isolate the functional genes that govern economic traits. This system is applicable to other organisms, including livestock, for which EST data are limited.

Variants of a human monocytic leukemia cell line (THP-1): induction of differentiation by retinoic acid, interferon-gamma, and T-lymphocyte-derived differentiation-inducing activity.

Induction of terminal differentiation of two variant sublines derived from the human monocytic leukemia cell line THP-1 by all-trans-beta-retinoic acid (RA) was studied. One of the variants was approximately 10 times more resistant to RA and the other, approximately 10 times more sensitive than the parent THP-1. Differentiation of the RA-resistant variant could be induced by immune interferon (IFN-gamma) and a T-lymphocyte-derived lymphokine of a differentiation-inducing activity (DIA), alone or in combination with 10 nM RA. These induced cells showed many of functional monocyte and/or macrophage characteristics, such as superoxide anion production, phagocytosis, and antibody-dependent cellular cytotoxicity. RA suppressed the expression of Fc receptors, phagocytosis, and 5'-nucleotidase activity of the resistant cells, although not the expression of the sensitive variant. These results indicate that RA-resistant cells can be fully induced to differentiate by the combination of RA and IFN-gamma or DIA.

Modulation of porcine intestinal epitheliocytes immunetranscriptome response by Lactobacillus jensenii TL2937.

In order to evaluate probiotic strains applicable for the beneficial immunomodulation of the porcine gut (immunobiotics), we previously developed a porcine intestinal epitheliocyte cell line (PIE cells). Here, transcriptomic studies using PIE cells were performed considering that this information would be valuable for understanding the mechanisms involved in the protective activity of the immunobiotic strain Lactobacillus jensenii TL2937 against intestinal inflammatory damage in pigs. In addition, those studies would provide criteria for selecting biomarkers for the screening of new immunobiotic strains. We performed microarray analysis to investigate the transcriptomic response of PIE cells to the challenge with heat-stable enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs) and, the changes induced by L. jensenii TL2937 in that response. The approach allowed us to obtain a global overview of the immune genes involved in the response of PIE cells to heat-stable ETEC PAMPs. We observed that L. jensenii TL2937 differently modulated gene expression in ETEC PAMPs-challenged PIE cells. Microarray and RT-PCR analysis indicated that the most remarkable changes in PIE cells transcriptomic profile after heat-stable ETEC PAMPs challenge were observed in chemokines, adhesion molecules, complement and coagulation cascades factors. In addition, an anti-inflammatory effect triggered by TL2937 strain in PIE cells was clearly demonstrated. The decrease in the expression of chemokines (CCL8, CXCL5, CXCL9, CXCL10, and CXCL11), complement (C1R, C1S, C3, and CFB), and coagulation factors (F3) by L. jensenii TL2937 supports our previous reports on the immunoregulatory effect of this strain. These results provided clues for the better understanding of the mechanism underlying host-immunobiotic interaction in the porcine host. The comprehensive transcriptomic profiles of PIE cells provided by our analyses successfully identified a group of genes, which could be used as prospective biomarkers for the screening and evaluation of new anti-inflammatory immunobiotics for the prevention of inflammatory intestinal disorders in pigs.

The regulation of oxytocin receptor gene expression during adipogenesis.

Although it has been reported that oxytocin stimulates lipolysis in adipocytes, changes in the expression of oxytocin receptor (OTR) mRNA in adipogenesis are still unknown. The present study aimed to investigate the expression of OTR mRNA during adipocyte differentiation and fat accumulation in adipocytes. OTR mRNA was highly expressed in adipocytes prepared from mouse adipose tissues compared to stromal-vascular cells. OTR mRNA expression was increased during the adipocyte differentiation of 3T3-L1 cells. OTR expression levels were higher in subcutaneous and epididymal adipose tissues of 14-week-old male mice compared to 7-week-old male mice. Levels of OTR mRNA expression were higher in adipose tissues at four different sites of mice fed a high-fat diet than in those of mice fed a normal diet. The OTR expression level was also increased by refeeding for 4 h after fasting for 16 h. Oxytocin significantly induced lipolysis in 3T3-L1 adipocytes. In conclusion, a new regulatory mechanism is demonstrated for oxytocin to control the differentiation and fat accumulation in adipocytes via activation of OTR as a part of the hypothalamic-pituitary-adipose axis.

Effects of cold exposure on insulin and glucagon secretion in sheep.

The effectiveness of cold exposure on the secretion of insulin and glucagon were examined using five adult sheep. Endocrine responses were studied in a warm environment and after cold exposure (0 C) from 4-19 days. Compared to levels at room temperature, basal plasma glucose levels were elevated during cold exposure, but basal levels of plasma insulin and glucagon were unchanged. Cold exposure significantly decreased the early insulin response to a primed iv infusion of glucose. Plasma glucose and glucagon levels during glucose infusion were unaffected by cold exposure. The decrease in plasma glucose after iv insulin injection (0.2 U/kg BW) was greater during cold exposure than at room temperature. Butyrate injection (0.625 mmol/kg, iv) resulted in a significantly lower secretion of both insulin and glucagon in the cold than in the warm environment. The glucagon response to arginine infusion (0.5 g/kg over 30 min, iv) was elevated by cold exposure, whereas the insulin response to arginine tended to be reduced. Propranolol infusion (20 micrograms/kg . min, iv) caused a slight inhibition of insulin secretion in the cold environment, but did not affect glucagon levels in either the cold or warm environment. Phentolamine infusion (20 micrograms/kg . min, iv) inhibited glucagon secretion, particularly in the cold environment, and caused a markedly greater stimulation of insulin secretion in the cold. It is concluded that cold exposure insufficient to cause hypothermia decreases insulin secretion in response to a variety of stimuli. Effects of cold on glucagon secretion depend upon the stimulating agent used.

Role of natural killer cells in intraocular melanoma metastasis.

The authors studied the role of natural killer (NK) cells in spontaneous metastasis of murine intraocular melanoma by transplanting murine B16 melanoma cells into the anterior chamber of the eye in syngeneic C57BL/6 mice and determining the number of metastatic lung tumor colonies after 40 days. Depletion of NK activity by anti-asialo GM1 serum dramatically enhanced metastasis and augmentation of NK activity by interferon inhibited it. The strong correlation between host NK activity and intraocular melanoma metastasis indicated that NK cells have an important role in spontaneous metastasis of intraocular melanoma.

Assessment of myocardial fibrosis in cardiomyopathic hamsters with gadolinium-DTPA enhanced magnetic resonance imaging.

RATIONALE AND OBJECTIVES: The authors investigated whether magnetic resonance (MR) imaging enhanced with gadolinium (Gd)-DTPA would be useful for assessment of myocardial fibrosis in cardiomyopathy. METHODS: The authors compared MR images of the excised heart after Gd-DTPA injection with histopathologic findings in 33 hamsters with cardiomyopathy of the Bio 14.6 strain and 26 healthy hamsters of various age groups and assessed localization of Gd-14C-DTPA by autoradiography in the myocardium of three hamsters with cardiomyopathy. RESULTS: The mean signal intensity ratios for the entire myocardium in hamsters with cardiomyopathy relative to that in healthy hamsters was significantly higher in a younger age group than in an older age group (1.30+/-0.09 versus 1.03+/-0.08, P < 0.001, respectively). This myocardial enhancement was more obvious in areas containing massive fibrosis in the early and mid stages than in the late stage. Autoradiograms of hamsters with cardiomyopathy showed patchy or linear increases in uptake, corresponding to the areas of myocardial fibrosis. Gadolinium-14C-DTPA radioactivity ratios of myocardial fibrosis to healthy myocardium were significantly higher in the early and mid stages than in the late stage (P < 0.01). CONCLUSIONS: Myocardial fibrosis with high cellularity and proliferation of vessels was delineated as an area enhanced with Gd-DTPA on MR images, and its signal intensity decreased with the late stage of myocardial fibrosis.

Expression of mRNA encoding IFN alpha in macrophages stimulated with Lactobacillus gasseri.

The ability of Lactobacillus gasseri, a dairy lactic acid bacterium, to induce interferon (IFN) was investigated in murine macrophage cultures. IFN alpha was substantially induced by some strains of L. gasseri and the titers were the highest at a concentration of 100 micrograms ml-1 of L. gasseri DSM20243T. The expression of mRNA encoding IFN alpha was detected in spleen-macrophages (SP-M phi) and Peyer's patch-adherent cells stimulated with L. gasseri DSM20243T. Actinomycin D and cycloheximide added to SP-M phi cultures showed that the mRNA was synthesized by 0.5 h, and that IFN alpha was produced within 3 to 6 h after the stimulation with L. gasseri DSM20243T. The results support the notion that dairy products containing L. gasseri can be 'physiologically functional foods'.

Intrathecal synthesis of immunosuppressive acidic protein (IAP) in patients with multiple sclerosis and other inflammatory neurological diseases.

Immunosuppressive acidic protein (IAP), an immunosuppressive substance produced mainly by macrophages, has previously been shown to increase in the serum of patients with inflammatory neurological diseases. In this study we assayed the IAP levels in cerebrospinal fluid (CSF) by a passive hemagglutination-inhibition test. CSF-IAP levels increased significantly in patients with multiple sclerosis (MS) during both the active and inactive stages and in patients with Guillain-Barré syndrome (GBS) or Miller Fisher syndrome (MFS) as compared with those of control patients, but not in patients with amyotrophic lateral sclerosis or spinocerebellar degeneration. During the active stage of MS, increased CSF-IAP levels together with the elevated IAP% (CSF-IAP/total CSF protein) and IAP index [(CSF-IAP/serum IAP)/(CSF albumin/serum albumin)] suggested the intrathecal synthesis of IAP. In contrast, in patients with GBS or MFS, increased CSF-IAP levels without elevation of IAP% could be attributed largely to increased total CSF protein levels. In patients with neuro-Behçet's disease, CSF-IAP levels and IAP% were elevated during the active stage, but remained normal levels during the inactive stage. Assaying CSF-IAP could provide useful information about inflammatory or immunopathological events within the central nervous system.

Distribution of carbon-11 labeled methamphetamine and the effect of its chronic administration in mice.

[11C]Methamphetamine, a psychotropic agent, was synthesized by N-methylation of amphetamine with [11C]CH3 I in hopes that it could be applied in the near future to assist positron emission tomography (PET) in the imaging of its distribution in the human brain. The regional distribution of [11C]methamphetamine was investigated in the mice brain at various intervals after an intravenous (i.v.) injection. Radioactivity was higher in the hypothalamus, cortex, striatum and hippocampus. Furthermore, in chronically administered mice, the uptake of [11C]methamphetamine was higher in the striatum than those in other regions. The regional differences in the distribution of methamphetamine in the mice brain may enable the imaging of its distribution by PET using [11C]methamphetamine.

The distribution of [11C]cocaine in normal and cocaine-sensitization mice.

[N-11C-methyl]-cocaine ([11C]cocaine), synthesized by N-methylation of norcocaine with [11C]CH3I, was used to assist in imaging the variety of local distribution by positron emission tomography (PET). The radiochemical yield and the radiochemical purity after purification of [11C]cocaine by high performance liquid chromatography (HPLC) at a sp. act. of 814 GBq/mmol were 47-58% and > 99%, respectively. The time required for synthesis including the purification was 25-30 min from the end of [11C]CH3I trapping. The physical distribution of [11C]cocaine in organ was also investigated in mice at various time after i.v. injection. The main accumulation of radioactivity occurred in the lung, kidney and brain within 1 min after the injection. In the brain, no differences in the organ were observed except the radioactivity level in each section increased for the first 5 min, since then radioactivity decreased dramatically. Furthermore, in the behavioral sensitization model of cocaine, the peak of [11C]cocaine uptake in each brain area was shown to be 5-15 min.