Deficiency in WT1-targeting microRNA-125a leads to myeloid malignancies and urogenital abnormalities.

The Wilms' tumor gene WT1 is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3'UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (-/-) mice than in miR-125a (+/-) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.

Detection of Corynebacterium kutscheri in the faeces of subclinically infected mice.

Mice were infected experimentally and subclinically with Corynebacterium kutscheri to recover the organism from mice faeces. The faeces were then cultured using selective furazolidone-nalidixic acid-colimycin agar. The number of C. kutscheri per gram of fresh faeces varied from mouse to mouse, but once established in the intestine, the organism was excreted in the faeces for at least five months. Viable bacteria were detected in most of the faecal samples, including those stored in the animal room for five days. The number of organisms in the stored faeces decreased gradually but did not differ significantly from those in the fresh faeces until they had been stored for more than three days. Many infected mice excreted between 10(4.77) and 10(5.37) colony forming units (CFU) of C. kutscheri per day in their faeces, and one mouse even excreted 10(3.74) CFU at eight weeks postinfection. These values showed little daily variation. Our present study showed that subclinically infected mice discharged the organism continuously and persistently in their faeces. Therefore, faecal samples would be useful for monitoring infection with C. kutscheri in living mice in a manner that is not stressful for the animals.

Cytoprotection by pyruvate through an anti-oxidative mechanism in cultured rat calvarial osteoblasts.

Although we have previously shown drastic cell death by pyruvate deficiency in osteoblasts at the proliferative stage, the exact mechanism remains unclear so far. Cell survivability was significantly decreased in rat calvarial osteoblasts cultured for 0 to 3 days in vitro (DIV) following replacement of the eutrophic alpha-modified minimum essential medium (alpha-MEM) with Dulbecco's modified eagle medium (DMEM) for cultivation. The addition of pyruvate enriched in alpha-MEM, but not in MEM, entirely prevented cell death induced by the medium replacement throughout a culture period from 0 to 3 DIV. Both cysteine and reduced glutathione protected cell death in cells cultured for 3 DIV without significantly affecting that in cells cultured for 1 DIV, however, while none of lactate, acetate and insulin significantly prevented the cell death irrespective of the culture period up to 3 DIV. A marked increase was detected in intracellular reactive oxygen species (ROS) levels 4 h after the medium replacement. In osteoblasts cultured in alpha-MEM for 3 DIV, but not in those for 7 DIV, hydrogen peroxide (H2O2) markedly decreased cell survivability when exposed for 2 to 24 h. Furthermore, H2O2 was effective in significantly decreasing cell survivability in osteoblasts cultured in DMEM for 7 DIV. Pyruvate at 1 mM not only prevented cell death by H2O2, but also suppressed the generation of intracellular ROS in osteoblasts exposed to H2O2. These results suggest that pyruvate could be cytoprotective through a mechanism associated with the anti-oxidative property rather than an energy fuel in cultured rat calvarial osteoblasts.

Overexpression of bcl-2 in transient abnormal myelopoiesis associated with Down syndrome.

Transient abnormal myelopoiesis (TAM) is a haematological complication found in Down syndrome. To determine the mechanisms of sustained proliferation of TAM cells, we studied the expression of apoptosis-related proteins, such as bcl-2, Fas (APO-1/CD95) and p-53, in peripheral blood cells from a new-born infant with Down syndrome and TAM. Using flow cytometry, peripheral blood mononuclear cells (PBMCs), consisting mostly of blast cells, showed marked expression of bcl-2 protein but not of Fas or p-53 products. DNA gel electrophoresis of PBMCs, cultured in the absence of serum factors, revealed no marked fragmentation. Our findings suggest that bcl-2 overexpression may be associated with prolonged cell survival of TAM cells.

Utility of liposomes coated with polysaccharide bearing 1-amino-lactose as targeting chemotherapy for AH66 hepatoma cells.

The cell recognition element is very important for drug delivery systems. We synthesized cholesteryl pullulan (CHP) bearing 1-aminolactose (1-AL) and introduced a saccharide, cholesteryl pullulan bearing 1-aminolactose (1-AL/CHP), to an outer layer of the conventional liposome as a cell recognition element. Lectin recognized the beta-galactose by aggregation of 1-AL/CHP coated liposome (1-AL/CHP liposome). The uptake of this liposome to AH66 rat hepatoma cells was greater than in liposomes without 1-aminolactose in vitro. Furthermore, 1-AL/CHP liposomal adriamycin showed a stronger antitumor effect in comparison with other types of liposomal adriamycin in vitro. When in vivo tumor-targeting efficacy was investigated in AH66 tumor transplanted mice using 3H-liposome, the tumor/serum radioactivity ratio in mice injected with 1-AL/CHP liposome was higher than that of mice injected with other liposomes. These observations suggest that 1-AL is effective as a cell recognition element. As a result, 1-AL/CHP liposome is considered to be a good carrier of anticancer drugs for the active targeting of tumor cells.

Clinical features and treatment of children with hemolytic uremic syndrome caused by enterohemorrhagic Escherichia coli O157:H7 infection: experience of an outbreak in Sakai City, 1996.

Presented is a study of 15 patients (seven males and eight females ranging between 5 and 10 years of age) with hemolytic uremic syndrome (HUS) associated with hemorrhagic colitis that was caused by enterohemorrhagic Escherichia coli (EHEC) O157:H7, encountered during the outbreak in Sakai City in July, 1996. The complete form of HUS, which includes the three characteristics hemolytic anemia, thrombocytopenia and acute renal dysfunction, was noted in eight patients, while an incomplete form of HUS, which did not include all three characteristics, was noted in seven patients. Regarding treatment, intravenous gamma-globulin was administered in nine patients and dialysis was performed in five patients (two males and three females) with the complete form of HUS. In three of these five patients, plasma exchange was also performed. Weaning from dialysis was accomplished by the 15th day of disease in all patients. Some patients developed pancreatitis, central nervous system symptoms, fundal hemorrhage and elevation of transaminase, although these abnormalities subsided uneventfully. Renal biopsy, which was performed in two patients who recovered from acute renal failure but still had mild proteinuria and a decrease in creatinine clearance, showed moderate changes in the glomeruli and tubulointerstitium. One year after onset of disease, hematological and urological findings were within normal limits in all patients except one with the complete form of HUS, who still had slightly decreased creatinine clearance.

[Influence for cultured renal cell growth ability by toxins of Escherichia coli].

There have been many reports indicating that Escherichia coli from pyelonephritis may exhibit several specific phenotypes. The purpose of the present study was to examine the effects of various toxins from Escherichia coli on cultured renal cell growth. alpha-hemolysin suppressed growth of Mardin Darby canine kidney (MDCK) cells in a dose-dependent manner. A low dose of O-antigen suppressed MDCK cell growth, while a high dose of the antigen increased the cell growth slightly. K:1-antigen had no effect on MDCK cell growth. Mesangial cell growth was not affected by alpha-hemolysin. A significant increase in mesangial cell growth was recognized by the O- or K:1-antigen. From these results it can be speculated that alpha-hemolysin from Escherichia coli may play a leading role, and O- or K-antigen may play a supporting role in the pathogenesis of pyelonephritis.

[The drug sensitivity of enterohemorragic Escherichia coli and antibiotics treatment for hemorrhagic enterocolitis--from an outbreak of enterocolitis in Sakai city].

In July, 1996, a massive outbreak of hemorrhagic enterocolitis involving more than 5,000 people was caused by enterohemorragic Escherichia coli (EHEC) O157:H7 occurred mainly among elementary school children of Sakai City, Japan. The antibacterial activities in vitro against EHEC from stool specimens were determined. Norfloxacin showed the highest antibacterial activity, and fosfomycin, kanamycin, ampicillin, cefaclor were considered as effective drugs. But doxycycline showed lower antibacterial activities compared to other examined drugs, and it appears necessary to take antibiotic resistance of Escherichia coli into consideration when a treatment regimen is determined. As a result of the oral administration of fosfomycin to 95 patients of hemorrhagic enterocolitis and carrier, no patients developed complications with hemolytic uremic syndrome (HUS). However, a study of 17 patients with HUS demonstrated the fact that most of them were subjected to intravenous administration of fosfomycin. It may be needed to consider oral administration route of effective antibiotics in the treatment of enterocolitis in order to maintain high concentrations of a drug in the intestine.

Antigenic phenotypes of Escherichia coli in urine from patients with urinary tract infections.

Various antigenic phenotypes of Escherichia coli in urine were analysed using monoclonal antibody against pyelonephritis-associated P-pili (PAP-pili), and polyvalent O- and K1-antisera, and the results were compared with the clinical diagnosis. PAP-pili, O1- and K1-positive E. coli were isolated more frequently in urine from patients with acute pyelonephritis. E. coli found in urine from patients with recurrent pyelonephritis were frequently PAP positive. Based on the antigenic phenotypes of strains in urine, it is suggested that pyelonephritopathogenic strains may originate from a small number of clones.

Production and characterisation of monoclonal antibodies against pyelonephritis-associated P-pili of Escherichia coli.

Pyelonephritis-associated P-pili (PAP) of Escherichia coli O6,H(-),K1(-),F12,haemolysin(-) were purified by salt precipitation and affinity chromatography using Synsorb P1. Purified PAP showed a single band with a molecular weight of 18 kDa by electrophoretic analysis. A monoclonal antibody (mAb) was produced by fusion of the PAI myeloma cell line with splenic lymphocytes from BALB/c mice immunised with the purified PAP. The mAb was of IgM class with kappa light chains and reacted with a 18-kDa moeity of the salt precipitate; the epitope was present near the apical part of the pilus filaments. The mAb reacted with PAP in both immunofluorescence and haemagglutination tests when 108 strains isolated from urine samples were tested; the two tests were in agreement for 202 of 204 strains isolated from faecal samples.

Nickel sulfate-specific suppressor T cells induced by nickel sulfate in drinking water.

Suppressor T cell function was studied in nickel sulfate (NiSO4) delayed type hypersensitivity (DTH). NiSO4 in drinking water administered orally to normal mice for 10 weeks elicited no significant footpad swelling. However, after drinking water for 7 weeks, suppression of footpad swelling response was not detected. Suppression of footpad swelling response was mediated by CD4-8+ T cells. However, these suppressor T cells did not overcome CD4+8- helper T cells by co-transfer to recipient mice. Unresponsiveness to NiSO4 by oral administration of antigen was due to suppressor T cells.

Analysis of responsive cells in tolerance by the oral administration of ovalbumin.

The induction of immune tolerance is the most common consequence of protein feeding, i.e., "oral tolerance". In this study we investigated the genetic basis of oral tolerance using various kinds of recombinant and congenic mice, and the cells involved in the development of this phenomenon in mice. The footpad swelling response to ovalbumin (OVA) was inhibited in mice that were orally fed OVA 7 days before sensitization. No effect of strain of mouse was seen in this inhibition. This inhibition could be transferred by Peyer's patch cells. The CD4-8+ T cells were responsible for the inhibition of footpad swelling. The number of CD4+ cells from OVA-fed tolerant mice decreased significantly, but CD8+ cells did not. The number of CD4-8+ T cells was increased in Peyer's patches of OVA-fed tolerant mice, and were involved in the development of oral tolerance.

The chemoluminescence response of human polymorphonuclear leukocytes to Escherichia coli O and K antigens.

The interactions between Escherichia coli O or K antigens and polymorphonuclear leukocyte function were studied. Five types of O antigen and three types of K antigen were extracted from E. coli. These included O1, O6, O75 and K1 antigens from pyelonephritopathogenic strains, O44 and K74 antigens from an enteropathogenic strain and O14 and K7 antigen from a standard strain. The antigens all reacted specifically to their specific antisera and no cross-reactions were observed. The O1 or O44 antigen stimulated a significantly greater chemoluminescence response in polymorphonuclear leukocytes obtained from normal volunteers than O75, O6 or O14 antigen. In addition, the K1 or K74 antigen stimulated polymorphonuclear leukocytes significantly more than K7 antigen. These results suggest that pyelonephritopathogenic or enteropathogenic E. coli may produce severe tissue damage as a result of the response to their O or K antigens, as well as via adhesive agents such as pyelonephritopathogenic P-pili or the enteroadhesive factor, and exotoxins such as hemolysin or verotoxin.