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.

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.

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.

Orbital varix with a pearly phlebolith. Case report.

A patient is described with an orbital varix arising from the right superior ophthalmic vein, associated with ophthalmoplegia and severe pain, and without proptosis. The varix was detected using computerized tomography and orbital phlebography, and at surgery was verified as a venous aneurysm. During the operation, a pearly phlebolith was found. Histological examination of the varix revealed multiple ectatic venous channels. The etiology of this unusual clinical manifestation and the treatment of the patient are briefly discussed.

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.

Amyloid formation in prolactinoma.

Amyloid deposits within a prolactin-secreting pituitary adenoma were investigated with light and electron microscopy. The appearance of amyloid with Congo red revealed amorphous and round green-yellow birefringence under polarized light. Spheroidal concretions disclosed prolactin-positive areas by immunohistochemistry. The amyloid commonly exhibited a structure of fine fibrils of 10 to 13 nm in width, with a hollow core. The fibrils were usually grouped in compact, stout bundles. Some bundles were present in extracellular space, others were obviously within the adenoma cells. Extracellular amyloid fibrils frequently gathered in a stellate arrangement to make a round body. The presence of intracellular amyloid fibrils in adenoma cells may represent that the source of the amyloid is neoplastic cells rather than mesenchymal cells. In addition, prolactin-positive parts in round bodies presumably exemplify that the amyloid fibrils are formed by hormone-relating proteins.

Symptomatic intrasellar arachnoid cyst: case report.

A case of a large symptomatic intrasellar arachnoid cyst with suprasellar extension is reported. A 53-year-old man was admitted because of decreased visual acuity. Magnetic resonance imaging showed a large intrasellar cyst extending into the suprasellar cistern, with compression of optic nerves. The intensity of the cyst was identical to that of the surrounding subarachnoid space on both T1-, T2-, and proton density-weighted images. Transsphenoidal surgery was performed, but subsequent refilling of the cyst required additional transcranial surgery. Analysis of the cerebrospinal fluid-like cystic fluid revealed high levels of protein and pituitary hormones. Histological study revealed that the cyst wall was composed of connective tissue and arachnoid cells, which were ultrastructurally characterized by a number of desmosomes. Diagnostic, surgical, and pathological features of intrasellar arachnoid cysts are discussed.

Intracranial mycotic aneurysm associated with transsphenoidal surgery to the pituitary adenoma.

A case is reported in which a mycotic aneurysm of the supraclinoidal portion of the internal carotid artery formed as the result of suprasellar infection following transsphenoidal surgery. The source of infection was thought to be a maxillary sinusitis. The aneurysm was treated with systemic antibiotic therapy and disappeared 4 months after the operation. Repeated angiography showed the whole course from the formation to the disappearance of the aneurysm and was valuable in the treatment of intracranial mycotic aneurysms.

Isolation and purification of in vivo-generated 25-hydroxyvitamin D2 by preparative high-performance liquid chromatography.

In order to obtain a standard compound of 25-hydroxyvitamin D2 (25-OH-D2), a method for isolating in vivo-generated 25-OH-D2 from the blood of rats or rabbits was established by using several steps of preparative high-performance liquid chromatography (HPLC). When the unsaponifiable matter of the plasma obtained from rats or rabbits receiving a large dose of vitamin D2 was applied to the preparative HPLC using a Zorbax SIL column, a peak denoted as peak X was observed on the chromatogram. Since the peak X was thought to be due to 25-OH-D2 from the experiments of time course and dose-response, it was purified by subjecting it to successive preparative HPLC using several kinds of columns. From the results of ultraviolet (UV) absorption spectrum, gas chromatography-mass spectrometry (GC-MS) and mass chromatography, the purified peak X compound was confirmed to be 25-OH-D2. The proposed method for isolating in vivo-generated 25-OH-D2 is very convenient, because the time to perform each HPLC is very short though several steps of HPLC are used.

A method for simultaneous determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human plasma by using two steps of high-performance liquid chromatography.

A method for simultaneous determination of 25-hydroxyvitamin D2 (25-OH-D2) and 25-hydroxyvitamin D3 (25-OH-D3) in human plasma has been developed by using two steps of high-performance liquid chromatography (HPLC). Lipids extracted from 0.51 ml of human plasma were first subjected to the preparative HPLC using a Nucleosil 5C18 column (reversed-phase type) and a 25-OH-D fraction containing 25-OH-D2 and 25-OH-D3 was separated. The separated fraction was subsequently subjected to the analytical HPLC using a Zorbax SIL column (straight-phase type). Since the peaks corresponding to 25-OH-D2 and 25-OH-D3 were clearly separated from one another on the chromatogram of the analytical HPLC, the metabolites could be simultaneously determined by estimating the respective peak heights. When the fractions corresponding to the respective peaks were separately collected by repeatedly applying rather large quantities of human plasma and were subjected to gas chromatography-mass spectrometry (GC-MS), they were identified as containing 25-OH-D2 and 25-OH-D3, respectively. The proposed method was applied to plasma samples of human adults taking 400 I.U./day of vitamin D2 for 8 weeks and the values were 22.5 +/- 8.1 ng/ml for 25-OH-D3 and 11.5 +/- 1.8 ng/ml for 25-OH-D2 (mean +/- S.D.), respectively.

Plasma levels of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in maternal, cord and neonatal blood.

A high-performance liquid chromatographic method for simultaneous assay of 25-hydroxyvitamin D2 (25-OH-D2) and 25-hydroxyvitamin D3 (25-OH-D3) proposed in a previous paper (1) was applied to determine the plasma levels of the metabolites in perinatal and postnatal periods. The plasma samples of maternal, cord and neonatal blood were collected in summer and winter seasons. 25-Hydroxyvitamin D3 was detected in all the samples. The plasma levels of the metabolite in mothers, cords and newborn infants (at life within 24 hr) in summer were 33.9 +/- 12.5, 18.9 +/- 8.4 and 16.6 +/- 6.4 (mean +/- S.D.) ng/ml, respectively, while those in winter were 15.8 +/- 6.6, 8.8 +/- 3.4 and 7.7 +/- 3.2 ng/ml, respectively. The data in summer were significantly higher than the respective data in winter and there were high significant correlations between the mothers and cords and between mothers and newborns. In both seasons, the plasma levels of mothers were about two times higher than the respective data of cords and newborns which were nearly identical with one another. The similar tendency was always observed in the individual data of mothers-cords-newborns pair samples. In this study, many plasma samples from mothers, cords and newborns were examined, but 25-OH-D2 was detected in only few samples (6/41 for mothers, 3/36 for cords and 2/34 for newborns). However, the metabolite began to appear in all the samples during nursing with vitamin D2-fortified dry milk to show 4.6 +/- 1.3 and 4.8 +/- 1.2 ng/ml in the summer and winter samples of neonates at life of 5-6 days, respectively. When the variation of plasma 25-OH-D2 and 25-OH-D3 levels was examined in postnatal periods until one month, the levels of exogenous 25-OH-D2 was increased while those of endogenous 25-OH-D3 was decreased. The sum of vitamin D2 intake from fortified dry milk was highly significantly correlated with the plasma levels of 25-OH-D2, which indicates that daily uptake of exogenous vitamin D2 is very important in nutrition during postnatal periods of bottle-fed infants.

Lack of evidence for existence of vitamin D and 25-hydroxyvitamin D sulfates in human breast and cow's milk.

The presence of water-soluble vitamin D and 25-OH-D sulfates in human breast and cow's milk was studied. We first confirmed that synthetic vitamin D2 and D3 sulfates could not be hydrolyzed by alkali but by acid. Breast or cow's milk was separated into milk whey containing water-soluble components and milk curd containing crude proteins and lipophilic components. The separated milk whey and curd were hydrolyzed by acid or alkali and each lipid extract was subjected to HPLC analysis. Neither peak due to vitamin D and 25-OH-D was observed in the chromatograms of acid- and alkali-hydrolyzed milk whey, whereas the peaks due to vitamin D3 and 25-OH-D3 were found in the chromatograms of both acid- and alkali-hydrolyzed milk curd and there was no significant difference between the respective peak heights. The eluates corresponding to the respective peaks observed on the latter's chromatograms were collected and subjected to UV, HPLC, GC-MS and GLC to identify the existence of vitamin D3 and 25-OH-D3, respectively. We concluded from these results that neither breast nor cow's milk contained water-soluble vitamin D and 25-OH-D sulfates, whereas they contained fat-soluble vitamin D3 and 25-OH-D3. The concentrations of vitamin D3 and 25-OH-D3 in breast milk were about 125 and 350 ng/liter, while those in cow's milk were about 420 and 270 ng/liter, respectively. The experiments on the transfer of 3H-D3 and 3H-25-OH-D3 perorally dosed to lactating rats into suckling pups through their milk also supported the above conclusion.

Variation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 levels in human plasma obtained from 758 Japanese healthy subjects.

Plasma levels of 25-hydroxyvitamin D3 (25-OH-D3) and 25-hydroxyvitamin D2 (25-OH-D2) in 758 Japanese healthy subjects (most of them adults) were determined by a high-performance liquid chromatographic method previously reported (6) and the following results were obtained: The mean and standard deviation (M +/- SD) of the assayed values of 25-OH-D (sum of 25-OH-D3 and 25-OH-D2) was 23.8 +/- 10.1 ng/ml. 25-OH-D3 was detected in all the samples and the M +/- SD was 23.0 +/- 10.1 ng/ml. The plasma levels clearly showed the seasonal variation that the levels in summer were significantly higher than those in winter. Moreover, the plasma levels were significantly correlated with the amounts of UV light in solar radiation. These results strongly suggested that 25-OH-D3 in plasma mainly originated from endogenous vitamin D3 formed by photo-conversion of 7-dehydrocholesterol in skin. 25-OH-D2 was detected only in 18.3% of the plasma samples and the M +/- SD in the detected samples was 4.4 +/- 2.9 ng/ml which was much lower than those of 25-OH-D3. The results suggested that few healthy Japanese are taking daily exogenous vitamin D2 from multivitamin preparations or others. The M +/- SD values of 25-OH-D3 plasma levels in men and women were 26.2 +/- 10.4 and 19.3 +/- 8.0 ng/ml, respectively. The formers were significantly higher than the latters. The results were thought to be due to the reason that men might be outdoors for longer periods than women. When age variation of plasma 25-OH-D3 levels was examined, the levels in the twenties were significantly lower than the other generations. This was confirmed to be due to the low values observed in the female twenties group, but the detailed reason is unclear at the present time. When 4 healthy volunteers were orally administered 400 I.U./day of vitamin D2 every day for 8 weeks, maximum levels (average: 11.5 ng/ml) were observed at the 8 weeks and the levels gradually decreased after stopping the administration. The results suggested that the half life of 25-OH-D2 in plasma might be 4-5 weeks.

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.

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.

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.

[Ceftazidime in the treatment of meningitis caused by multiresistant Serratia marcescens].

A patient of subarachnoid hemorrhage was treated with spinal CSF drainage. Serratia marcescens meningitis occurred because of the spinal CSF drainage. The organism was multiresistant and refractory to antibiotics including piperacillin, imipenem, gentamicin and cephaloridine. It was sensitive to ceftazidime (CAZ). Treatment with CAZ resulted in clinical improvement associated with rapid clearing of the organism from CSF. CAZ serum level was high enough and CAZ penetration into the CSF was satisfactory. According to the evaluation of CAZ concentrations in serum and CSF, two regimens of treatment were recommended. One is an administration of CAZ 1 g x 4 times/day. Another is a combination with CAZ administration 2 g x 2 times/day and followed by 1 g x 4 times/day. The results suggest that CAZ is an extremely effective antibiotic for meningitis caused by CAZ-susceptible bacteria.

[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.

[A case report of a 6-year-old boy with intracranial yolk sac tumor treated by VAB-6 regimen].

Several clinical trials have demonstrated that cisplatin-based chemotherapy for primary intracranial germ-cell tumors is effective as a neoadjuvant chemotherapy. In this report, we describe a 6-year-old boy, Down syndrome and Hirschsprung's disease with intracranial pure yolk sac tumor treated by combined chemotherapy with cisplatin, vinblastine, bleomycin and cyclophosphamide (modified VAB-6 regimen). He had been admitted to our hospital because of intractable vomiting, and left facial nerve palsy since 1 month before. An MRI revealed an enlarged mass, 4cm in diameter, in the left cerebello-pontine angle with uniformal enhancement by Gd-DTPA, and bilateral ventricular dilatation. He was found to have increased serum alpha-fetoprotein level (AFP 11, 786ng/ml), but not human chorionic gonadotropin beta-subunit. After a partial resection of the tumor, diagnosed as pure yolk sac tumor, and ventriculo-peritoneal shunt, three courses of combined chemotherapy with cisplatin, bleomycin, vinblastine and cyclophosphamide (modified VAB-6 therapy) were carried out. The serum AFP level returned to normal, and the tumor mass entirely disappeared (a complete response) on MRI after the second course of chemotherapy. However, cisplatin-induced vomiting and mild neutropenia and renal tubular injury developed after the third course of chemotherapy. Irrespective of administration of recombinant human G-CSF and broad spectrum antibiotics, he suffered from pneumonia and died of septic shock and multiple organ failure. Autopsy showed microscopic residual tumors. The combination chemotherapy with cisplatin, bleomycin, vinblastine and cyclophosphamide is effective for initial treatment of childhood intracranial yolk sac tumor. It is necessary, however, to reevaluate the cisplatin dosage and treatment schedule in order to reduce such side effects as bone marrow suppression and renal damage.