Induction of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic differentiation through combinatorial regulation.

Acute myeloid leukemia (AML) involves a block in terminal differentiation of the myeloid lineage and uncontrolled proliferation of a progenitor state. Using phorbol myristate acetate (PMA), it is possible to overcome this block in THP-1 cells (an M5-AML containing the MLL-MLLT3 fusion), resulting in differentiation to an adherent monocytic phenotype. As part of FANTOM4, we used microarrays to identify 23 microRNAs that are regulated by PMA. We identify four PMA-induced microRNAs (mir-155, mir-222, mir-424 and mir-503) that when overexpressed cause cell-cycle arrest and partial differentiation and when used in combination induce additional changes not seen by any individual microRNA. We further characterize these pro-differentiative microRNAs and show that mir-155 and mir-222 induce G2 arrest and apoptosis, respectively. We find mir-424 and mir-503 are derived from a polycistronic precursor mir-424-503 that is under repression by the MLL-MLLT3 leukemogenic fusion. Both of these microRNAs directly target cell-cycle regulators and induce G1 cell-cycle arrest when overexpressed in THP-1. We also find that the pro-differentiative mir-424 and mir-503 downregulate the anti-differentiative mir-9 by targeting a site in its primary transcript. Our study highlights the combinatorial effects of multiple microRNAs within cellular systems.

Epidemiological and outcome characteristics of major burns in Tokyo.

The Tokyo Burn Unit Association (TBUA) was established in 1983 funded by the Tokyo Metropolitan Government, and is organized by 13 burn units in Tokyo. TBUA covers more than 90% of severe burn patients occurring in Tokyo, and all of the cases are registered according to the burn injury registration format. The purpose of this study is to analyze the registered data and to elucidate epidemiological and outcome characteristics of major burn injuries in Tokyo. The total of 6988 hospitalized patients had data for epidemiological analysis, and 6401 patients had complete data for outcome analysis as well, and were included in this study. The characteristic profiles for the analysis included age, sex, cause of burns, inhalation injury, %BSA, burn index (BI), length of burn unit stay, and outcome, and were analyzed by age groups. The mean age of the patients was 40.4 years, and 63% of them were male. It was noteworthy that 25% of the total patients were elderly patients over 60 years of age. Flame was the most common cause making up 45.6% followed by scalding (32.0%). The overall mortality rate was 15.4%. Inhalation injury was accompanied in 27.3% of burn patients. The mortality rate was 34.6% with inhalation injury, and 8.2% without inhalation injury. Causes of death showed that multiple organ failure made up 36.9% of total mortality, followed by sepsis 25.2 and shock 19.0%. The burn size (%BSA and BI) and inhalation injury were the factors for high mortality rate in all age groups whereas age was a predictor for high mortality in the patients older than 16 years of age. Gender was not a factor for high mortality in any age group. The mortality rate showed mildly decreasing tendency since 1995 for which implementation of skin bank was thought to be responsible.

Aseptic efficacy of prefilled syringes in a polluted environment.

INTRODUCTION: To evaluate the aseptic efficacy of prefilled syringes compared with ampules when used in a polluted environment similar to that at a disaster site. METHODS: The researchers tested epinephrine, 0.1%, atropine sulfate, 0.05%, and lidocaine hydrochloride solutions, 2% (Group A) as well as lidocaine hydrochloride, 10%, sodium bicarbonate, 8.4%, and glucose solutions, 50% (Group B), that frequently are used for intravenous injection and intravenous infusion respectively in Disaster Medicine. Each of these solutions in 10 prefilled syringes (PFSs) and 10 ampules was placed in a box of contaminated soil along with needles and empty syringes for ampules. In the box, each was taken out of its package, all syringes were connected with a needle, and empty syringes were filled with a solution. After this procedure, all syringes were taken out of the box to check their contents for bacterial contamination. RESULTS: No bacterium was observed in any of the 10 PFS samples of Group A and B solutions. In contrast, out of 10 ampule samples, six of the 10 samples containing epinephrine, nine of the 10 containing atropine sulfate, all 10 samples containing lidocaine hydrochloride, 2%, and all of the ampule samples containing Group B solutions tested positive for bacteria. A statistically significant difference was observed between the PFS and ampule samples in all six solutions. CONCLUSION: Results indicate that, in environments with airborne contaminants, the use of prefilled syringes may be useful for preventing bacterial contamination of the medicine inside.

Education issues in disaster medicine: summary and action plan.

INTRODUCTION: Change must begin with education. Theme 8 explored issues that need attention in Disaster Medicine education. METHODS: Details of the methods used are provided in the introductory paper. The chairs moderated all presentations and produced a summary that was presented to an assembly of all of the delegates. The chairs then presided over a workshop that resulted in the generation of a set of action plans that then were reported to the collective group of all delegates. RESULTS: Main points developed during the presentations and discussion included: (1) formal education, (2) standardized definitions, (3) integration, (4) evaluation of programs and interventions, (5) international cooperation, (6) identifying the psychosocial consequences of disaster, (7) meaningful research, and (8) hazard, impact, risk and vulnerability analysis. DISCUSSION: Three main components of the action plans were identified as evaluation, research, and education. The action plans recommended that: (1) education on disasters should be formalized, (2) evaluation of education and interventions must be improved, and (3) meaningful research should be promulgated and published for use at multiple levels and that applied research techniques be the subject of future conferences. CONCLUSIONS: The one unanimous conclusion was that we need more and better education on the disaster phenomenon, both in its impacts and in our response to them. Such education must be increasingly evidence-based.

The dimer and trimer of 3-hydroxybutyrate oligomer as a precursor of ketone bodies for nutritional care.

BACKGROUND: Ketone bodies have been considered as a means of providing energy because of their good penetration and rapid diffusion in peripheral tissues. However, because the currently available form of 3-hydroxybu-tyrate is the sodium salt, the sodium load is problematic. To avoid it, a mixture of dimer and trimer has been prepared as a precursor of D-3-hydroxybutyrate. The purpose of this study was to investigate whether and how the solution would be converted to monomers. METHODS: The plasma concentration of 3-hydroxybutyrate monomer was measured in 10 rats during infusion of dimer and trimer. Stepwise dilutions of the solution were incubated with serum and liver homogenates from five rats, serum samples from five volunteers, and a liver sample from one patient with liver injury. The solution also was incubated with carboxylesterase and triacylglycerol lipase. The concentration of monomer in the medium was measured after incubation. RESULTS: The plasma concentration of 3-hydroxybutyrate monomer reached 572 +/- 11 micromol/L 15 minutes after beginning infusion of the mixture at a rate of 25 micromol x kg(-1) x min(-1) and 270 +/- 40 micromol/L at a rate of 12.5 micromol x kg(-1) min(-1). The solution was converted completely to monomers when incubated with rat serum or liver homogenate for 10 minutes. The mixture also was hydrolyzed by human liver homogenate but not by serum. CONCLUSIONS: The dimer and trimer of 3-hydroxybutyrate can be converted rapidly to monomer in rat and human tissues. 3-Hydroxybutyrate oligomers could be an energy substrate for injured patients.

Changes in left ventricular performance in patients with severe head injury during and after mild hypothermia.

OBJECTIVE: To evaluate left ventricular (LV) performance in patients with severe head injury during and after mild hypothermia. PATIENTS AND METHODS: Seven consecutive patients who underwent therapeutic mild hypothermia (age, 15 to 70 years; Glasgow Coma Scale score on admission, 4 to 8). LV performance was assessed by using M-mode, color tissue Doppler imaging tracings and pulsed Doppler echocardiography. LV contraction and relaxation were evaluated by using the peak velocity of LV posterior wall movement during systole (Smax) and diastole (Dmax), respectively, in addition to the conventional echocardiographic indices. RESULTS: Mild hypothermia increased LV ejection time and reciprocally reduced LV filling period as indicated by temperature-dependent shortening of the early diastolic filling and the total diastolic inflow time. The indices depending on temporal factors such as ejection time, Smax, or Dmax were significantly affected by mild hypothermia, whereas those depending on spatial factors such as fractional shortening or stroke volume index were not. The attenuated Smax was compensated for the prolonged ejection time resulting in the relatively consistent fractional shortening regardless of body temperature. There was no compensatory mechanism for the decreased Dmax during diastole. CONCLUSION: The effect of mild hypothermia seemed to be predominantly negatively chronotropic. LV diastolic function was more vulnerable to mild hypothermia than LV systolic function was.

Involvement of phosphatidylinositol 3-kinase in Fc gamma receptor signaling.

Wortmannin, a potent and selective inhibitor of phosphatidylinositol (PI) 3-kinase (Okada, T., Sakuma, L., Fukui, Y., Hazeki, O., and Ui, M. (1994) J. Biol. Chem. 269, 3563-3567), prevented Fc receptor for IgG (Fc gamma R)-dependent phagocytosis of the human monocytic cell line U937 or guinea pig neutrophils. Cross-linking of Fc gamma R on the surface of U937 cells increased PI 3-kinase activity that was immunoprecipitated with antibody against phosphotyrosine or antibody against the 85-kDa regulatory subunit of PI 3-kinase. Specific cross-linking of Fc gamma R subclass Fc gamma RI or Fc gamma RII, using monoclonal antibodies against each receptor subclass and the F(ab')2 fragment of goat antibody against mouse IgG, increased anti-phosphotyrosine-precipitable PI 3-kinase activity. Treatment of cells with anti-Fc gamma RIII antibody plus the same F(ab')2 did not affect the activity, reflecting the lack of Fc gamma RIII in U937 cells. Fcy gamma R stimulation triggered prominent tyrosine phosphorylation of several proteins, among which the 115-kDa peptide showed strong association with PI 3-kinase. Thus, Fc gamma R appears to be coupled functionally, via a tyrosine kinase, to PI 3-kinase, which may regulate the phagocytotic activity of the cells.

Small renal cell carcinoma: pathologic and radiologic correlation.

The authors correlated the radiologic and pathologic findings of 36 patients with small renal cell carcinoma (less than or equal to 3 cm in diameter). Tumors were discovered incidentally or by means of mass surveys with ultrasound (US). Computed tomography (CT) and angiography were performed before surgery. Of the 36 tumors, 24 were of solid (alveolar) architecture, five were of papillary architecture, three were of tubular architecture, and four were of multilocular cystic architecture. Cell arrangement was closely correlated with radiologic appearance, especially in regard to tumor vascularity and echogenicity. Histologically homogeneous tumors of solid architecture were hypoechoic at US and hypervascular at angiography and contrast medium-enhanced CT. Tumors of papillary, tubular, and multilocular cystic architecture were hyperechoic at US and hypovascular at angiography. There was no correlation of cell differentiation and cell architecture with echogenicity or tumor vascularity. Tumors with hemorrhage showed marked hyperattenuation at CT. A tumor capsule was observed in 25 patients (69%); this was demonstrated as a rim at US or angiography.

Large-scale, high-density freezing of hybridomas and its application to high-density culture.

Large-scale, high-density freezing of hybridomas was studied to apply frozen cells to start high-density culture. We showed here that hybridomas can be frozen at 1.5 x 10(8) cells/mL, without decrement in viability and proliferating activity. Blood transporting bags were used for large-scale freezing to store 25 mL of cell suspension with a cell density, 1.5 x 10(8)/mL. The number of cells stored in a bag (3.0 x 10(9) cells) was enough to start a high-density culture at a 10 times higher cell density (6.0 x 10(6) cells/mL) than normal inoculation, and the cells proliferated to 10(7) cells/mL within 2 days. These results indicate that the large-scale freezing method is useful for large-scale culture of mammalian cells.

[Salvage chemotherapy with a combination of VP-16, ifosfamide, procarbazine, prednisolone, bleomycin and methotrexate (VIPP-BM) for refractory malignant lymphoma].

Twenty patients with refractory malignant lymphoma were treated with a combination of VP-16, ifosfamide, procarbazine, prednisolone, bleomycin and methotrexate (VIPP-BM) as salvage chemotherapy. These patients were either resistant to front-line therapy or refractory in their relapses. Two patients (10%) achieved a complete remission and eleven patients (55%) attained a partial remission. An overall response rate was 65%. Major toxicities were myelosuppression, nausea and vomiting, and mucositis. However they were well tolerated. This regimen has been effective in the treatment for the patients with refractory lymphoma.

Renal handling of carnitine in children with carnitine deficiency and hyperammonemia associated with valproate therapy.

Free and acylcarnitine in serum and urine samples were measured in five patients with hyperammonemia associated with anticonvulsant therapy including sodium valproate, of whom three had a Reye-like syndrome. All had considerable reduction in serum free carnitine and slight increase of acylcarnitine concentrations, suggesting increased conversion of free to acylcarnitine by valproate administration. Urinary excretion of both free and acylcarnitine was increased, accompanied by depressed reabsorption of free carnitine and decreased acylcarnitine/free carnitine clearance ratio. These results indicate a decreased threshold for free carnitine. The combination of these several factors may be responsible for carnitine deficiency in patients with hyperammonemia taking valproate.

Demonstration of acid alpha-glucosidase in different types of Pompe disease by use of an immunochemical method.

The nature of mutant acid alpha-glucosidase (AAG) in muscle was studied in 6 patients with Pompe disease, consisting of 2 each of the infantile, childhood and adult types. Anti-human liver AAG rabbit antibody prepared in the present study was confirmed to be monospecific by immunodiffusion, immunotitration and immunohistochemical methods. It was found by the immunodiffusion and enzyme immunoassay methods using this antibody that the mutation produced a normal amount of enzyme protein but the latter was an inactive form, suggesting structural gene mutation in 5 of the 6 cases. In the remaining childhood type case there was no detectable amount of enzyme protein, suggesting that the mutation causes a reduction in the amount of the enzyme protein or synthesis of unstable enzyme protein. Similarly, the enzyme activity of AAG was markedly reduced in all patients, but that of neutral alpha-glucosidase was the least reduced in the adult type, medium in the childhood type, and the most reduced in the infantile type.

[Laboratory and clinical studies of cefotiam in digestive diseases].

Experimental and clinical studies on cefotiam (CTM), a new synthetic cephalosporin, were performed in digestive diseases. Among the 7 cases of 'cholelithiasis and cholangitis', 6 cases (86%) showed better response than "effective". Among the 9 cases of infections except 'cholelithiasis and cholangitis' which had no connection with cancers, 6 cases (67%) showed better response than "effective". Among the 15 cases of 'infections following advanced cancers', 7 cases (47%) showed better response than "effective". This inferior result was due to the relation to factors coming from cancers. CTM was effective in not only infections of the aged but that which resisted antibiotics of penicillins and the other cephems. Sensitivity of E. coli, Klebsiella and S. aureus was made a comparative study of by MIC and disc test. It was found as a result that CTM had the equal or superior antimicrobial activity to other antibiotics of cephems, penicillins and aminoglycosides.

[Treatment of multiple myeloma. High-dose intermittent administration of cyclophosphamide and prednisolone--follow up study of 10 years period].

Twenty patients with plasma cell myeloma were treated with high-dose (10 mg/kg) intermittent administration of cyclophosphamide for remission induction and maintenance therapy. High-dose intermittent administration of glucocorticosteroid hormone was also performed usually in parallel with large-dose of cyclophosphamide. A significant objective response was obtained in 12 of 20 patients (60.0%). The median survival was 32 months in all cases; 47 months in the responding cases. The response rate was not affected by the class of M-protein, the type of light-chain and/or the presence of bone lesions. All responders were on the 3rd stage. High-dose intermittent administration of cyclophosphamide was evaluated as very useful since it showed equivalent effects obtained by combination chemotherapy regimens, hardly producing bone marrow suppression.

Myopathy due to glycogen storage disease: pathological and biochemical studies in relation to glycogenosome formation.

Ten cases of myopathy caused by glycogen storage diseases of type II, III, and V, and phosphorylase b kinase deficiency are reported. So-called "abnormal lysosomes" or glycogenosomes which contain abundant glycogen were found in cases of type II, and in some numbers, in cases of type III, and in one case of phosphorylase b kinase deficiency which revealed a moderate decrease in debranching enzyme (amylo-1,6-glucosidase) activity. In these cases of type III and phosphorylase b kinase deficiency, the glycogenosomes are formed through deposition of abnormal glycogen (limit dextrin structure glycogen).

Glycogen storage disease. Studies related to the mechanism of glycogenosome formation.

Glycogen storage diseases of type I, II, III, IV, V and the other muscle types, were examined electron microscopically, biochemically and physicochemically. Glycogenosomes (glycogen containing vacuoles) were found in the affected tissues of type II, type III variant of muscle glycogen storage disease, type IV and muscle type phosphorylase b kinase deficiency (disorder of the phosphorylase b kinase activation mechanism). The acid alpha-glucosidase activity was decreased only in the case of type II glycogen storage disease (Pompe's disease). The other types of glycogen storage disease showed no decrease in acid alpha-glucosidase activity. Moreover, one patient with type II disease also revealed a decrease in neutral alpha-glucosidase activity. In all cases where glycogenosomes were found, the extracted glycogen macromolecules showed some molecular abnormality or deviation when compared with normal native glycogen macromolecules.