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Primary central nervous system lymphoma (PCNSL) is a distinct subtype of extranodal lymphoma with aggressive clinical course and poor outcome. As increased IL‐10/IL‐6 ratio is recognized in the cerebrospinal fluid (CSF) of PCNSL patients, we hypothesized that PCNSL might originate from a population of B cells with high IL‐10‐producing capacity, an equivalent of “regulatory B cells” in mice. We intended in this study to clarify whether Tim‐1, a molecule known as a marker for regulatory B cells in mice, is expressed in PCNSL. By immunohistochemical analysis, Tim‐1 was shown to be positive in as high as 54.2% of PCNSL (26 of 58 samples), while it was positive in 19.1% of systemic diffuse large B‐cell lymphoma (DLBCL) samples (17 of 89 samples; P

Infections caused by Mycobacterium wolinskyi have rarely been reported, and essentially all were cellulitis and/or osteomyelitis related with traumatic event or surgical wound. Here, we present the 1st case of septic complication due to this organism in a patient with chronic myelogenous leukemia of the 1st but late chronic phase.

mRNA of cytochrome P450 21-hydroxylase (P450c21) is expressed in the brain, but little is known about the enzymatic properties of P450c21 in the brain. In the present study, we showed, by using various recombinant cytochrome P450 (CYP)2D enzymes and anti-CYP2D4- or P450c21-specific antibodies, that rat brain microsomal steroid 21-hydroxylation is catalyzed not by P450c21, but by CYP2D isoforms. Rat CYP2D4 and human CYP2D6, which are the predominant CYP2D isoforms in the brain, possess 21-hydroxylation activity for both progesterone and 17α-hydroxyprogesterone. In rat brain microsomes, these activities were not inhibited by anti-P450c21 antibodies, but they were effectively inhibited by the CYP2D-specific chemical inhibitor quinidine and by anti-CYP2D4 antibodies. mRNA and protein of CYP2D4 were expressed throughout the brain, especially in cerebellum, striatum, pons, and medulla oblongata, whereas the mRNA and protein levels of P450c21 were extremely low or undetectable. These results support the idea that CYP2D4, not P450c21, works as steroid 21-hydroxylase in the brain. Allopregnanolone, a representative γ-aminobutyric acid receptor modulator, was also hydroxylated at the C-21 position by recombinant CYP2D4 and CYP2D6. Rat brain microsomal allopregnanolone 21-hydroxylation was inhibited by fluoxetine with an IC50 value of 2 μm, suggesting the possibility that the brain CYP2D isoforms regulate levels of neurosteroids such as allopregnanolone, and that this regulation is modified by central nervous system-active drugs such as fluoxetine.

Following oral administration of empagliflozin (1000 mg/kg/day) to male and female CD-1 mice for 2 years, renal tubular injury was identified in male mice. Renal injury was not detected in male mice (≤300 mg/kg/day), in female mice (1000 mg/kg/day), or in male or female Han Wistar rats (700 mg/kg/day). Using transfected HEK293 cells and Xenopus oocytes, empagliflozin was found to be a substrate of various mouse and rat organic anion transporters (oat/Oat) and organic anion transporting polypeptide (oatp/Oatp) transporters: mouse oat3, rat Oat3, mouse oatp1a1, and rat Oatp1a1. However, using isolated kidney slices from male and female mice and rats, no sex-based difference in the extent of uptake of empagliflozin occurred. Metabolism studies using hepatic and renal microsomes from male and female mice, rats, and humans revealed a hemiacetal metabolite of empagliflozin (M466/2), predominantly formed in male mouse kidney microsomes. Formation of M466/2 in male mouse kidney microsomes was 31-fold higher compared to that in female mouse kidney microsomes and was ∼29- and ∼20-fold higher compared to that in male and female mouse liver microsomes, respectively. M466/2 is unstable and degrades to form a phenol metabolite (M380/1) and 4-hydroxycrotonaldehyde (4-OH CTA). Formed 4-OH CTA was trapped by reduced GSH, and the structure of the GSH adduct was confirmed by mass spectrometry. Stoichiometric formation of M380/1 from M466/2 was observed (93-96% at 24 h); however, formation of 4-OH CTA was considerably lower (∼17.5% at 40 h), which is consistent with 4-OH CTA being a highly reactive species. These data represent a highly selective tissue-, species-, and sex-specific lesion in male CD-1 mice associated with a cytotoxic metabolite product, 4-OH CTA. In humans, glucuronidation of empagliflozin is the most prevalent metabolic pathway, and oxidation is a minor pathway. Thus, renal toxicity due to the formation of 4-OH CTA from empagliflozin is not expected in humans.

The existence of cytochrome P450 2D isoforms in the brain has been demonstrated, although their physiological functions remain to be elucidated. In this study we demonstrated that recombinant rat cytochrome P450 2D1 and 2D4 and human cytochrome P450 2D6 possess progesterone 6 beta- and 16 alpha- hydroxylation activities; 2 beta- and 21-hydroxylation activities; and 2 beta-, 6 beta-, 16 alpha- and 21-hydroxylation activities, respectively. Cytochrome P450 2D4 had the lowest K(m) value and the highest maximum velocity value toward these activities. Progesterone 2 beta- and 21-hydroxylation activities were also detected in rat brain microsomes, and these activities were completely inhibited by anticytochrome P450 2D antibodies. The presence of endogenous 2 beta- and 21-hydroxyprogesterones in rat brain tissues was also demonstrated. The mRNAs of cytochrome P450 2D4, CYP11A, and 3 beta-hydroxysteroid dehydrogenase were detected in the rat brain, suggesting that progesterone was generated from cholesterol by CYP11A and 3 beta-hydroxysteroid dehydrogenase and then underwent hydroxylation to hydroxyprogesterones by cytochrome P450 2D4 in rat brain. Collectively, our findings support the idea that cytochrome P450 2D may be involved in the regulation (metabolism and/or synthesis) of endogenous neuroactive steroids, such as progesterone and its derivatives, in brain tissues.

The absorption, distribution, and excretion of radioactivity were investigated following a single oral administration of 14C-pramipexole to rats at a dose of 0.5 mg/kg. The protein binding of the drug was also investigated in vitro and in vivo. 1. The total radioactivity in plasma reached the maximum at about 1.5 hr after oral administration of 14C-pramipexole to male and female rats. The half-lives of radioactivity in plasma were about 48 and 41 hr in male and female rats, respectively, as determined from the data at 24 to 72 hr after administration. The Cmax was decreased in the presence of food, but total absorption of radioactivity remained unaffected. 14CPramipexole was well absorbed throughout the duodenum, jejunum and ileum, but not in the stomach. 2. The radioactivity in most tissues after oral administration of 14C-pramipexole to male rats reached Cmax at 2 hr, being higher in liver, kidney, hypophysis and salivary gland. The radioactivity in the whole brain was higher than that in plasma at 2 and 6 hr after administration. At 48 hr after oral administration, the radioactivity in most tissues except blood had decreased to below 1/10 of the respective maximum concentration. The ratio of the concentration in blood cells to that in plasma was in the range of 1.63-7.15 at 0.5 to 48 hr after oral administration. 3. The binding of 14C-pramipexole at concentrations of 10-1000 ng/ml to rat plasma protein in vitro was about 15-18% and that at concentrations of 0.5-50 ng/ml to human serum protein in vitro was about 17-26%. The percentage of radioactivity bound to rat plasma protein within 2 hr after oral administration of 14Cpramipexole to male rats was below 20%. After 2 hr, the ratio of plasma protein binding increased with time, and then the percentage of radioactivity bound to rat plasma protein was about 67% at 48 hr. 4. The amount of radioactivity excreted in urine and feces within 168 hr after oral administration of 14C-pramipexole to male rats were 59.0% and 44.0% of the dose, respectively. Within 48 hr after intraduodenal injection of the bile obtained from other rats which had been administered 14C-pramipexole (0.1 mg/kg) intravenously, about 4% of the injected radioactivity was excreted in the bile. 5. The radioactivity in milk after oral administration to lactating rats was 2-5 times higher than that in plasma at all times from 0.5 to 48 hr, but the half-life of radioactivity in milk was shorter than that in plasma.

The absorption and distribution of radioactivity were investigated following a 14-day period of daily oral administration of 14C-pramipexole (0.5 mg/kg/day) to male rats. 1. When measured 24 hr after each of 14 repeated daily administrations of 14C-pramipexole to male rats, the level of radioactivity in plasma rose as the number of doses increased, and reached a steady state after 12 or 13 doses. The elimination of radioactivity from the plasma after the last dose was similar to that after a single administration. 2. At 1 hr after the 14th administration, the level of radioactivity reached the maximum in most tissues. High levels were observed in the liver and kidney, being about 22 and 14 times higher than that in the plasma, respectively. At 168 hr after the last dose, the levels of radioactivity in the liver and kidney were very much higher than that in plasma. The elimination of radioactivity from most tissues after the last dose was parallel to that from plasma, except the spleen and kidney, where an accumulation was observed. 3. After the last administration, the ratio of the concentration in blood cells to that in plasma gradually increased with the lapse of time. The elimination of radioactivity from blood cells was very much slower than that from plasma. The relative increase of radioactivity in blood cells at the late stage after drug administration may be attributed to metabolites of pramipexole.

Progressive multifocal leukoencephalopathy (PML) is a rare and fatal demyelinating disease of the central nervous system caused by JC polyomavirus (JCV) reactivation in an immunocompromized host. We describe a case of PML in a 76-year-old woman with myelodysplastic syndrome, who had been treated with azathioprine for a pure red cell aplasia-like condition. PML was diagnosed based on the neurologic symptoms, the magnetic resonance imaging patterns and the detection of JCV DNA in the cerebrospinal fluid. She died ten months after the diagnosis. An autopsy confirmed the diagnosis, and JCV DNA was detected in the cerebrum. Azathioprine might have triggered PML.

The development of autoimmune disease after autologous stem cell transplantation (ASCT) is very rare in multiple myeloma (MM). We describe the first case of Evans syndrome after ASCT for MM. A 60-year-old man with MM received ASCT and subsequently developed Evans syndrome following two febrile episodes. The syndrome was refractory to conventional therapies but it was managed with a second ASCT. This unique complication was thought to have been triggered by an infection during the recovery of the immune system. We assumed that reconstructing the immune system via ASCT might eliminate infection-induced autoantibodies to platelets and erythrocytes.

Telmisartan, a nonpeptide angiotensin II receptor antagonist, is selectively distributed to liver. In the present study, we have characterized the contribution of organic anion transporting polypeptide (OATP) isoforms to the hepatic uptake of telmisartan by isolated rat hepatocytes, human cryopreserved hepatocytes, and human transporter-expressing cells. Because it is difficult to evaluate the transport activity of telmisartan because of its extensive adsorption to cells and culture materials, we performed the uptake study in the presence of human serum albumin. The saturable uptake of telmisartan into isolated rat hepatocytes took place in a Na(+)-independent manner and was inhibited by pravastatin, taurocholate, and digoxin, which are Oatp substrates and inhibitors, but not by organic cation, tetraethylammonium, indicating the involvement of Oatp isoforms in its uptake into rat hepatocytes. To identify which human OATP transporters are important for the hepatic uptake of telmisartan, the uptake assay was carried out using OATP1B1- and OATP1B3-expressing human embryonic kidney 293 cells and cryopreserved human hepatocytes. The uptake of telmisartan by OATP1B3-expressing cells was saturable (K(m) = 0.81 microM) and significantly higher than that by vector-transfected cells. In contrast, no significant uptake was observed in OATP1B1-expressing cells. We also observed the saturable uptake of telmisartan by human hepatocytes. Thirty micromolar estrone-3-sulfate, which can selectively inhibit OATP1B1-mediated uptake compared with OATP1B3, did not inhibit the uptake of telmisartan in human hepatocytes, whereas it could inhibit the uptake of estradiol 17beta-d-glucuronide mediated by OATP1B1. These results suggest that OATP1B3 is predominantly involved in the hepatic uptake of telmisartan in humans.

CYP2D1, 2D2, 2D3, and 2D4 are major CYP2D isoforms expressed in the rat. In humans, only CYP2D6 is expressed. In rat brain, the mRNA for CYP2D4 is most abundant in cerebellum, striatum, pons and medulla oblongata. In human brain, CYP2D6 mRNA expression was detected in all regions with highest levels observed in cerebellum. CYP2D isoforms are involved in the metabolism of not only xenobiotics such as antidepressants, beta-adrenergic blockers, antiarrhysthmics, and antihypertensives, but also endogenous compounds such as trace amine and neurosteroids. Among 11 isoforms of human recombinant P450s, only CYP2D6 exhibited an ability to efficiently convert tyramine which exists in the brain, to dopamine. CYP2D4 and CYP2D6 which are the predominant CYP2D isoforms in the rat and human brain, respectively, possess 21-hydroxylation activity for both progesterone and allopregnanolone. CYP2D4, not P450c21, works as a steroid 21-hydroxylase in the brain. These results suggested that CYP2D in the brain may be involved in the metabolism of neuronal amines and steroids and in the regulation of the central nervous system.

Primary central nervous system lymphoma (PCNSL) is a subtype of non-Hodgkin’s lymphoma that arises within the central nervous system (CNS) as a primary lesion, most of which demonstrate diffuse large B-cell lymphoma (DLBCL) histology. However, CNS is recognized as an “immune sanctuary”, and it is not clear in what mechanism B cells develop tumor at this immunoprivileged site. In the past mouse models of multiple sclerosis and cerebral infarction, regulatory B cells, a population of B cells with high IL-10 producing capacity, were reported to have a function to migrate to CNS and suppress inflammation. As the IL-10 level is typically increased in the cerebrospinal fluid (CSF) of PCNSL patients, we hypothesized that PCNSL might originate from B cells that have a physiological role to produce IL-10 for suppressing unfavorable inflammation in CNS, such as regulatory B cells in mice. Recently, a cell surface molecule T cell immunoglobulin domain and mucin domain protein 1 (Tim-1) has been reported to be specifically expressed in the majority of regulatory B cells in mice. Tim-1 was originally identified as a costimulatory molecule on T cells that negatively regulates cellular immune response. Regulatory B cells in mice with defective Tim-1 mutation were reported to demonstrate a profound defect in IL-10 production, suggesting that Tim-1 plays an essential role in their IL-10 production. However, there has been no previous report on Tim-1 expression on human B-cells or B-cell lymphomas, or what function they may serve if it is present. We performed immunohistochemical staining of Tim-1 in various formalin-fixed paraffin embedded lymphoma samples. We observed strong expression of Tim-1 in PCNSL samples, in contrast to its lower expression in other DLBCL and follicular lymphoma samples. Expression of Tim-1 is also detected in a cell line derived from PCNSL (TK), as well as in several other B-cell lymphoma cell lines, by RT-PCR and western blot. As we detected spontaneous shedding of the ectodomain of Tim-1 in the culture media of Tim-1-expressing B-cell lymphoma cell lines, we examined whether Tim-1 can also be detected in the CSF of PCNSL patients. By ELISA, we detected soluble Tim-1 in the CSF of PCNSL patients with active disease, and found it undetectable after the successful treatment with chemo/radiotherapy. The level of soluble Tim-1 in the CSF was positively correlated with IL-10, and it is suggested that these two molecules are functionally related also in humans. In a patient with continuous Tim-1 detection in the CSF after chemotherapy while radiological examination could no longer detected any abnormality, subsequently manifested relapse in the brain. According to these findings, soluble Tim-1 in CSF may be expected to serve as a sensitive biomarker for PCNSL. To clarify the biological role of soluble Tim-1 in tumor microenvironment, we examined its effect on T cell function. We stimulated CD4+ and CD8+ T cells with or without soluble Tim-1 in vitro and compared their cytokine production. The result showed that, in the presence of soluble Tim-1, both IL-2 and IFN-g production was suppressed in CD8+ T cells. In conclusion, Tim-1 is expressed in PCNSL and shedding of extracellular domain of Tim-1, in addition to IL-10, may contribute to the immunosuppressive microenvironment of PCNSL. Disclosures No relevant conflicts of interest to declare.

Abstract 416 In B-cell lymphomas, several chromosomal translocations are associated with specific histological subtypes. CCND1/IGH is detected in more than 90 % of the cases with mantle cell lymphoma (MCL), and BCL2/IGH is characteristically observed in follicular lymphoma and diffuse large B-cell lymphoma (DLBCL) of germinal center (GC) B-cell origin. Although these strong correlations are clinically recognized, their biological mechanism is not clearly explained so far. According to the results of translocation breakpoint mapping, both CCND1 and BCL2 translocations are considered to be generated by an error during physiological VDJ rearrangement of the IGH gene in the precursor-B cell stage. We hypothesized that the occurrence of these translocations in the immature B-cell stage, probably as an initial genetic event, should have a special impact on the determination of resulting lymphoma histology. We generated a mouse model mimicking human lymphoma with CCND1 or BCL2 translocation by lentivirally introducing these genes into Tp53+/− B6 mouse bone marrow cells and transplanting them to lethally irradiated wild-type B6 mice. In this model, CCND1 or BCL2 is expressed from immature to mature B cell stage under the control of the CD19 promoter, and subsequently mutations accumulate in the background of Tp53 haploinsufficiency. Both mice developed B-cell lymphomas several months after the transplantation, but their tumors showed some different features. CCND1-Tp53+/− mice developed B220lowCD5+CD23− tumors that expand into the B-cell area with sparing the GC, whereas BCL2-Tp53+/− mice preferentially developed B220highCD5−CD23+ tumors that tend to localize in the GC. Additionally, somatic hypermutation (SHM) analysis of the IGH gene of the tumor cells revealed obviously higher mutation frequency in the BCL2-Tp53+/− mice than in the CCND1-Tp53+/- mice (p There has been a debate whether B-1 cell population exists in humans, but it is recently proposed that the phenotype of human B-1 cells is CD20+CD27+CD43+CD70− by testing sort-purified B cell fractions for fundamental B-1 cell functions based on mouse studies (J Exp Med 2011;208:67–80). Interestingly, flow cytometric analysis of the human MCL tumor cells has shown that they mostly express this provisional B-1 cell phenotype, supporting the idea that human MCL is also derived from B-1 cells. Our mouse model precisely reproduces the link between CCND1 and BCL2 translocations and the resulting lymphoma subtypes in humans. It is assumed that these translocations trigger the cell expansion of different B-cell subgroups, which consequently leads to the development of lymphoma of distinct histology. Our findings provide new insights into the mechanism of lymphoma subtype determination. Disclosures: No relevant conflicts of interest to declare.