Comprehensive microRNA expression profiling of the hematopoietic hierarchy.

The hematopoietic system produces a large number of highly specialized cell types that are derived through a hierarchical differentiation process from a common stem cell population. miRNAs are critical players in orchestrating this differentiation. Here, we report the development and application of a high-throughput microfluidic real-time quantitative PCR (RT-qPCR) approach for generating global miRNA profiles for 27 phenotypically distinct cell populations isolated from normal adult mouse hematopoietic tissues. A total of 80,000 RT-qPCR assays were used to map the landscape of miRNA expression across the hematopoietic hierarchy, including rare progenitor and stem cell populations. We show that miRNA profiles allow for the direct inference of cell lineage relations and functional similarity. Our analysis reveals a close relatedness of the miRNA expression patterns in multipotent progenitors and stem cells, followed by a major reprogramming upon restriction of differentiation potential to a single lineage. The analysis of miRNA expression in single hematopoietic cells further demonstrates that miRNA expression is very tightly regulated within highly purified populations, underscoring the potential of single-cell miRNA profiling for assessing compartment heterogeneity.

Prostaglandin E2 down-regulates viable Bacille Calmette-Guérin-induced macrophage cytotoxicity against murine bladder cancer cell MBT-2 in vitro.

The regulatory effect of prostaglandin (PG) E2 and a cyclooxygenase (COX) inhibitor on Bacille Calmette-Guérin (BCG)-induced macrophage cytotoxicity in a bladder cancer cell, MBT-2, was studied in vitro. BCG stimulated thioglycollate-elicited murine peritoneal exudate cells (PEC) to induce cytotoxic activity and to produce cytokines such as interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and PGE2. NS398, a specific COX-2 inhibitor, and indomethacin (IM), a COX-1 and COX-2 inhibitor, enhanced viable BCG-induced cytotoxic activity and IFN-gamma and TNF-alpha production of PEC. However, NS398 and IM did not enhance these activities induced by killed BCG. Enhanced cytotoxicity was mediated by increased amounts of IFN-gamma and TNF-alpha. Exogenous PGE2 reduced cytotoxic activity and IFN-gamma and TNF-alpha production of PEC. These results suggest that PGE2 produced by BCG-activated macrophages has a negative regulatory effect on the cytotoxic activity of macrophages. Accordingly, a PG synthesis inhibitor may be a useful agent to enhance BCG-induced antitumour activity of macrophages.

Unusual development of acute compartment syndrome caused by a suction injury: a case report.

There have been recent reports of acute compartment syndrome secondary to suction injuries of the hands of children. We report the case of a 68-year-old patient who developed an acute compartment syndrome of the forearm after his arm had been sucked into an exhaust port. He was treated by emergency fasciotomies and the wound was closed five days later with a small skin graft. His recovery was uneventful.

Cytotoxic effect of an anti-liver monoclonal autoantibody obtained after neonatal thymectomy in mice.

A monoclonal autoantibody, LSA-1, against murine liver antigen was obtained by fusing spleen cells from a neonatally thymectomized BALB/c mouse with SP2/0 murine myeloma cells. The LSA-1 isotype was IgG2b and kappa. LSA-1 was specific to the liver, especially, to a liver-specific membrane lipoprotein (LSP) fraction. By Western blotting analysis, LSA-1 mainly detected a 100 kDa protein of LSP fraction. LSA-1 stained cytoplasm of the cryostat sections of liver in immunohistochemical analysis. Furthermore, the antigen recognized with LSA-1 was highly expressed on the surface of a murine hepatoma cell line, MH134, slightly on a murine normal liver cell line, C1469, and on freshly prepared hepatocytes, but not on spleen cells. LSA-1 had a cytotoxic activity on liver cell lines in the presence of a complement in vitro. Furthermore, injection of LSA-1 into mice-induced liver injury. These results suggest that anti-liver autoantibody plays an important role in the induction of autoimmune hepatitis. Accordingly, this antibody will be a useful tool for the analysis of the pathogenesis of autoimmune hepatitis.

Phenytoin promotes Th2 type immune response in mice.

The effects of chronic administration of phenytoin, a common anticonvulsive drug, on immune responses were studied in mice. Anti-keyhole limpet haemocyanin (KLH) IgE antibody response after KLH-immunization was enhanced in phenytoin-treated mice. Proliferative responses of spleen cells induced with KLH, concanavalin A (ConA), lipopolysaccharide and anti-CD3 antibody were reduced in phenytoin-treated mice. Accessory function of spleen adherent cells on ConA-induced T cell proliferative response was reduced in phenytoin-treated mice. KLH-induced IL-4 production of spleen cells was enhanced, while IFN-gamma production was reduced in phenytoin-treated mice. In addition, production of IL-1 alpha, but not IL-6 and IL-12 by spleen adherent cells from phenytoin-treated mice was reduced. Natural killer cell activity was reduced in phenytoin-treated mice. These results suggest that phenytoin treatment preferentially induces a Th2 type response. We also observed that plasma ACTH and corticosterone levels were increased in phenytoin-treated mice, and speculated that phenytoin might act directly and indirectly, through HPA axis activation, on the immune system to modulate Th1/Th2 balance.

Suppression of macrophage interleukin-12 and tumour necrosis factor-alpha production in mice infected with Toxocara canis.

Toxocara canis induces a predominantly Th2 type response with enhanced amounts of interleukin (IL)-4 and reduced amounts of interferon (IFN)-gamma in infected mice. In this study, we investigated the macrophage function of T. canis-infected mice from the standpoint of cytokine production. C3H/HeN mice were infected with T. canis by oral administration of embryonated eggs. Ten days after infection, macrophages were obtained from spleen and peritoneal cavity, were cultured with lipopolysaccharide, and cytokines in the culture supernatant were evaluated with enzyme-linked immunosorbent assay. Macrophages from T. canis-infected mice produced IL-1 and IL-6 equivalent to macrophages from normal mice. The production of IL-10 and tumour growth factor (TGF)-beta was enhanced, but IL-12 and tumour necrosis factor (TNF)-alpha production was diminished. The addition of IFN-gamma, anti-IL-10 antibody, anti-TGF-beta antibody or indomethacin did not restore the production of IL-12 and TNF-alpha by macrophages from T. canis-infected mice. Furthermore, the stimulation of normal macrophages with T. canis antigen in vitro induced IL-1alpha, IL-6, IL-10 and TGF-beta, but not IL-12 and TNF-alpha. These results indicate that cytokine producing pattern of macrophages is altered by T. canis-infection, and this altered macrophage function may play an important role in the modification of the immune response to T. canis.

Lipomatous change in a brain metastasis from malignant pleural mesothelioma.

We present a case of brain metastasis from malignant mesothelioma with lipomatous change.

Prostaglandin E2 up-regulates macrophage-derived chemokine production but suppresses IFN-inducible protein-10 production by APC.

PGE(2) has been known to suppress Th1 responses. We studied the role of PGE(2) in two representative chemokines, macrophage-derived chemokine (MDC) and IFN-inducible protein-10, production by LPS- or CD40-stimulated spleen cells. The production of MDC, one of the ligands for CCR4 preferentially expressed on Th2, was enhanced in nonstimulated, LPS-, CD40-, or CD3-stimulated spleen cells by the pretreatment with PGE(2), while the production of IFN-inducible protein-10, a representative ligand for CXC chemokine receptor 3 expressed on Th1, was suppressed. MDC production was also enhanced by IL-4, IL-5, and intracellular cAMP-elevating agents such as dibutyryl cAMP and 3-isobutyl-1-methylxanthine, and the effect of IL-4, IL-5, and PGE(2) was additive. However, the pretreatment with IL-6, IL-10, or TGF-beta, or the neutralization of IFN-gamma or IL-12 had no effect on MDC production. B cells, macrophages, and dendritic cells were main producers of MDC, while T cells produced only a small amount of MDC. MDC production by B cells was equally stimulated by LPS and anti-CD40 Ab, while that by macrophages and dendritic cells was more markedly stimulated by anti-CD40 Ab, and PGE(2) further enhanced MDC production by these stimulated cells. These results indicate that PGE(2) regulates Th1/Th2-related chemokine production by B cells, macrophages, and dendritic cells, and that this is a new function of PGE(2) for the regulation of Th2 immune responses at the induction and activation stages.

Role of TGF-beta in the retinoic acid-induced inhibition of proliferation and melanin synthesis in chick retinal pigment epithelial cells in vitro.

The purpose of this study was to investigate the effects of all-trans retinoic acid (RA) on the induction of transforming growth factor-beta (TGF-beta) that is concerned with the proliferation and melanin synthesis of chick retinal pigment epithelial (RPE) cells in vitro. Chick RPE cells were cultured in the presence or absence of RA and anti-TGF-beta antibody for 7 days. The effects of RA and pan-specific TGF-beta antibody on RPE cell proliferation were assessed by counting the number of cells, and their effects on melanin synthesis were evaluated by measuring the melanin content of the cells. TGF-beta activity in the culture supernatant of RPE cells was measured using CCL-64 cells. RA significantly inhibited RPE cell proliferation and increased melanin synthesis. The addition of pan-specific TGF-beta antibody to the culture blocked the inhibition of RPE cell proliferation and the increased melanin synthesis. RA induced TGF-beta production in the culture supernatant of RPE cells. These findings indicate that RA regulates the proliferation and melanin synthesis of RPE cells via induction of TGF-beta.

Effect of mite antigens on antigen presenting cells/macrophages in mice.

The effect of mite antigens on murine lymphocytes and macrophages was studied in vitro. Antigens prepared from Dermatophagoides farinae bodies (Dfb) or recombinant Mag3, glutathione-S transferase (GST)-fused mite antigen, stimulated murine spleen cells to proliferate. The responder cells were B cells, because the response was sensitive to anti-Ig antibody and C treatment, but not to anti-Thy 1 antibody and C treatment. The response was not due to lipopolysaccharide contamination, a representative B-cell mitogen, because polymyxin B column-passed Dfb significantly stimulated B cells, and GST protein alone did not stimulate them. Alloantigen presenting activity was increased in mite antigen-treated B cells and spleen adherent cells. Mite antigens stimulated CD80 and the major histocompatibility complex (MHC) class II molecule expression, but suppressed CD86 expression on B cells and spleen adherent cells that were detected by a flow cytofluorometer. Antibodies to the MHC class II molecules, CD80 and CD86 blocked the alloantigen-presenting activity. Furthermore, mite antigens stimulated B cells and spleen adherent cells to produce cytokines. These results suggest that mite antigens have a stimulating activity on antigen-presenting cells/macrophages and modulate immune responses.

Macrophage-colony stimulating factor inhibits the growth of human ovarian cancer cells in vitro.

The effect of macrophage-colony stimulating factor (M-CSF), which regulates the growth and differentiation of haematopoietic progenitor cells on the growth of ovarian cancer cells was investigated in three ovarian cancer cell lines in vitro. The spontaneous growth of these cells was significantly inhibited by the addition of M-CSF in a concentration-dependent manner over 96 h of culturing. The maximum response was obtained with 10 ng/ml (3857 U/ml) of M-CSF by counting the viable cell number using the trypan blue exclusion assay. [(3)H]-thymidine incorporation by these cells was also suppressed following a 96-h incubation with M-CSF. The inhibitory effect of M-CSF was reversed by the addition of anti-M-CSF monoclonal antibody. Flow cytometric analysis revealed that the treated ovarian cancer cells arrested at the G0/G1 phase of the cell cycle. These cells expressed M-CSF receptors on their surface as detected by Scatchard plot analysis using (125)I-labelled M-CSF. These results indicate that M-CSF has an antitumour activity for ovarian cancer cells and suggest that it can be applied for the treatment of this disease.

Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice.

PGE2 has been shown to play a prominent role in regulating Th1 and Th2 type responses. We studied the role of PGE2 in IFN-gamma production by Staphylococcus aureus Cowan I-stimulated spleen cells from several mouse strains such as BALB/c, C3H/HeN, and C57BL/6. When spleen cells were pretreated with indomethacin (cyclooxygenase (COX)-1 and COX-2 inhibitor) or NS-398 (COX-2-specific inhibitor), S. aureus Cowan I -induced IFN-gamma production was increased more markedly in spleen cells from BALB/c mice than from C3H/HeN and C57BL/6 mouse. However, PGE2 production was not significantly different among spleen cells from three mouse strains. When various concentrations of PGE2 were exogeneously added to spleen cells, PGE2 showed a stronger suppressive effect on IFN-gamma production in spleen cells from BALB/c mice than from other strains of mice. This suppressive effect of PGE2 in BALB/c mice mainly depended on IL-12p70 production by APCs. More PGE2 binding sites were found in BALB/c spleen cells than in C3H/HeN spleen cells, indicating that the sensitivity difference to the suppressive effect of PGE2 was due to the difference of the number of PGE2 receptors. The administration of NS-398 into BALB/c mice enhanced Ag-specific IFN-gamma production, but not IL-4 production. This effect is the same as IL-12 administration in vivo. From these results, we propose that the modulation of PGE2 is important for Th1 activation via IFN-gamma and IL-12p70 production in vitro and in vivo and that PGE2 is one of the pivotal factors in the Th2-dominant immune response in BALB/c mice.

Autoradiographic distribution of radioactivity from (14)C-GABA in the mouse.

We investigated the distribution of radioactivity from (14)C-labeled gamma-aminobutyric acid (GABA) in the mouse by in vivo autoradiography to clarify the tissues that show GABA uptake and/or GABA binding. Male mice were injected intravenously with (14)C-GABA in both the absence and presence of an excess of unlabeled GABA, baclofen and isoguvacine. Whole-body autoradiography of (3)H-baclofen, a GABA(B) receptor agonist was also performed. At short intervals after (14)C-GABA injection ( 3 and 6 minutes), very high radioactivity was detected in the kidney cortex, liver, pineal gland, hypophysis, median eminence of the hypothalamus, and cervical ganglion. The hyaline cartilage and glandular part of the stomach showed moderate radioactivity. In the presence of an excess amount of unlabeled GABA, radioactivity in most of tissues decreased significantly, but no significant difference in radioactivity was observed in the presence of baclofen and isoguvacine, agonists of GABA(A) and GABA(B) receptors, respectively. Autoradiography of (3)H-baclofen showed that the kidney had high level of radioactivity, whereas the activity in other tissues and organs was similar or lower than in the blood except for the content of the urinary bladder and the pancreas at 15 minutes after injection. These data indicate that radioactivity from incorporated (14)C-GABA into a variety of cells is much higher than that from bound (14)C-GABA to the receptor sites. Our results suggest that GABA can be quickly localized in many organs of the mouse body after 3 minutes following injection, and GABA may serve multiple functions in those organs.

Immunomodulating activity of seaweed extract on human lymphocytes in vitro.

Effect of eight kinds of seaweed extract (SWE) on human lymphocytes was studied in vitro. The extracts of Hizikiafusiformis and Meristotheca papulosa (green) markedly stimulated human lymphocytes to proliferate, whereas Eucheuma muricatum and Meristotheca papulosa (red) weakly stimulated proliferation. The responder cells are T cells, because T cells purified by sheep red blood cell (SRBC) rosette-formation were significantly stimulated with SWE, but B cells were not. These extracts enhanced the induction of cytotoxic T lymphocyte (CTL) activity, but failed to enhance natural killer (NK) cell activity. These extracts had a stimulatory effect on immunoglobulin (Ig) production by B cells and tumor necrosis factor (TNF) production by monocytes. The activity of Hizikia fusiformis associated with polysaccharides which were extracted with ethanol and purified by ion-exchange and gel filtration chromatography, whose molecular weight was about 100 kDa. These results suggest that SWE has an immunomodulating activity on human lymphocytes and this ability might be useful for clinical application to treat several diseases such as tumors.

Expression and localization of cysteine dioxygenase mRNA in the liver, lung, and kidney of the rat.

The expressions of cysteine dioxygenase (CDO) gene in the liver, lung, skeletal muscle, and kidney were studied by in situ hybridization with a cDNA probe from rat liver CDO under normal conditions. Significant expression of the CDO gene was detected in the liver, lung, and kidney, but not skeletal muscle. In the liver, the signal was confined to the cytoplasm of the hepatocytes. Furthermore, the signal was stronger in the periportal than that in the perivenous areas. In the lung, an intensive signal was found in the bronchiolar epithelium. As to the kidney, an intensive signal was observed in the distal convoluted tubules, while no signal was found in the proximal convultions.

Effect of maternal lifestyle on cord blood IgE factor.

During recent decades much interest has been focused on the possibility of predicting and preventing atopic diseases during pregnancy. The idea of being able to detect a predisposition early and take suitable environmental measures in order to avoid overt allergy is an attractive position. Elevated cord IgE of around 1.0 IU/ml has been proposed as a predictor in western children. However, there remains no information about the effect of maternal lifestyle during pregnancy on these levels. Total IgE levels were therefore determined using Pharmacia CAP system and PRIST, with sensitivities of 0.01 kU/l and 0.25 kU/l, respectively, from serum samples taken from 1138 Japanese pairs of cord blood and pregnant women responding to a questionnaire regarding 17 health practices, intake of 32 food allergens and 5 environmental factors. Of these, 28 (2.5%) pairs of samples were excluded from further analysis because of high contamination of IgA (> 15.4 mg/ml) in cord blood. Median cord blood IgE was 0.286 kU/l and geometric mean IgE was 66.25 kU/l in maternal sera using CAP system; there was no significant correlation between maternal log (IgE) and cord blood IgE. Similar results were obtained from PRIST, whose correlation with CAP system was significant (r = 0.884, p < 0.001 for maternal and r = 0.765, p < 0.001 for cord blood). Multiple logistic analysis demonstrated that avoidance of simultaneous exposure to hens' eggs and cow's milk (relative risk = 1.3, p < 0.05) as well as soy beans (relative risk = 2.8, p < 0.01) should be advised to mothers with positive allergic histories and/or high total IgE (> 400 IU/ml), especially in women aged more than 35 years who are pregnant with a male child. However, maintenance of healthy lifestyles, especially taking proper exercise and sleeping, and avoidance of inhalant allergens during late pregnancy may be a more important strategy for the reduction of cord blood IgE levels.

Dysfunction of immune system and induction of autoantibodies to liver antigens by neonatal thymectomy in mice.

We examined the development of autoantibodies to liver proteins and hepatitis in BALB/c mice thymectomized 2 days after birth and attempted to characterized the immune function of these mice. Autoantibodies to crude liver proteins detected by ELISA were found in 21 (84%) of 25 mice thymectomized 2 days after birth. In these mice, sera of 11 animals showed reactivity with both liver specific proteins (LSP) and the second fraction of crude liver proteins; sera of 3 mice showed reactivity with only the second fraction but sera showed reactivity with only LSP. By Western-immunoblotting, sera of BALB/c mice which showed high autoantibody level to liver proteins detected a strong band around 150kD in the second fraction of crude liver proteins. Still more, hepatic inflammation; mononuclear cell infiltration in the portal area, was induced in mice with apparently high autoantibody level to crude liver proteins. These results in BALB/c mice corresponded with our previous reports which employed C3H/HeN mice. Next, we examined immune functions of mice thymectomized 2 days after birth. In thymectomized mice, the proportion of Thy-1, L3T4 and Lyt-2 positive cells (T cells) decreased and the proportion of B220 positive cells (B cells) increased. The proliferative response of lymph nodes lymphocytes cultured with mitomycin C-treated syngeneic spleen cells was lower, and the total IgG level in the sera was higher when compared with control normal mice. Anti-nuclear antibody (ANA) also appeared in the sera of thymectomized mice 2 days after birth. All these results suggest that the dysfunction of T cell and polyclonal activation of B cell were induced in neonatally thymectomized mice and resulted in the production of ANA and autoantibodies to liver proteins.

[Studies on cerebrospinal fluid penetration of cefpirome in adult with meningitis].

A patient with intracerebral hematoma suffered from postoperative bacterial meningitis. Staphylococcus aureus was found from CSF. The organism was multiple drug resistant and refractory to antibiotics including piperacillin (PIPC), cephalexin (CEX), cefotaxime (CTX), ceftazidime (CAZ) and latamoxef (LMOX). It was susceptible to cefpirome (CPR). Treatment with CPR resulted in clinical improvement associated with clearing of the organism from CSF. Serum level of CPR was high enough and CPR penetration into the CSF was satisfactory. The results suggest that CPR is an extremely effective antibiotic for meningitis caused by CPR-susceptible bacteria. Evaluation of the CPR penetration into the CSF of adult meningitis was rarely reported. The result we obtained was important in the treatment for the adult meningitis.

[Parasagittal meningioma growing in the superior sagittal sinus presenting intracranial hypertension: a case report].

Parasagittal meningiomas often invade the superior sagittal sinus (SSS), but rarely grow inside the SSS as a primary tumor. The authors report a case of parasagittal meningioma growing mainly inside the SSS and presenting papilledema. The SSS is invisible behind the tumor end on the right carotid angiogram but still patent on the left carotid angiogram. The superficial cortical vein on the opposite side works as a collateral pathway. The tumor may have originated from the right wall of the SSS, grew inside the sinus and covered the entrances of the right ascending cerebral veins. V-P shunt was performed after removal of the mass outside the sinus for resolving headache and visual symptoms.

Ultrastructural relation between nerve terminals and dentine bridge formation after pulpotomy in human teeth.

Close association between nerve terminals and preodontoblasts, odontoblasts and predentine was observed during healing after pulpotomy. The nerve terminals frequently contained large numbers of synaptic vesicles. Terminals with many vesicles tended to be fewer in the predentine than in the odontoblastic layer. The distribution of terminals was more dense at the stage before the regenerated odontoblasts became arranged regularly beneath the predentine. It is suggested that these terminals have some efferent role(s), especially during collagen synthesis at the early stage of dentinogenesis. The nerves may release their abundant synaptic vesicles, in addition to serving a sensory role for monitoring the increased sensitivity in the injured areas.