Erythroid and megakaryocytic differentiation of K562 erythroleukemic cells by monochloramine.

The induction of leukemic cell differentiation is a hopeful therapeutic modality. We studied the effects of monochloramine (NH2Cl) on erythroleukemic K562 cell differentiation, and compared the effects observed with those of U0126 and staurosporine, which are known inducers of erythroid and megakaryocytic differentiation, respectively. CD235 (glycophorin) expression, a marker of erythroid differentiation, was significantly increased by NH2Cl and U0126, along with an increase in cd235 mRNA levels. Other erythroid markers such as γ-globin and CD71 (transferrin receptor) were also increased by NH2Cl and U0126. In contrast, CD61 (integrin ß3) and CD42b (GP1bα) expression, markers of megakaryocytic differentiation, was increased by staurosporine, but did not change significantly by NH2Cl and U0126. NH2Cl retarded cell proliferation without a marked loss of viability. When ERK phosphorylation (T202/Y204) and CD235 expression were compared using various chemicals, a strong negative correlation was observed (r = -0.76). Paradoxically, NH2Cl and staurosporine, but not U0126, induced large cells with multiple or lobulated nuclei, which was characteristic to megakaryocytes. NH2Cl increased the mRNA levels of gata1 and scl, decreased that of gata2, and did not change those of pu.1 and klf1. The changes observed in mRNA expression were different from those of U0126 or staurosporine. These results suggest that NH2Cl induces the bidirectional differentiation of K562. Oxidative stress may be effective in inducing leukemic cell differentiation.

Selective adsorption of bilirubin against albumin to oxidized single-wall carbon nanohorns.

Bilirubin adsorption capacities of single-wall carbon nanohorns (SWNHs) are investigated to develop an efficient adsorbent in plasma apheresis. Pristine, thermally oxidized and H2O2-oxidized SWNHs are examined and it is demonstrated that higher oxidization levels of the SWNHs enhance bilirubin adsorption capacity owing to increase in their dispersibility and formation of nanoscaled holes on the SWNH walls. Under co-existance of albumin molecules, the bilirubin adsorption capacity of the SWNHs increases with the oxidation level whereas the albumin adsorption capacity is kept small. Bilirubin is selectively adsorbed to the SWNHs, especially to the SWNHs with high oxidation levels, against albumin. This selectivity is maintained under high-concentrations of albumin in the near-clinical environment.

Phase I trial of combination chemotherapy with docetaxel, cisplatin and S-1 (TPS) in patients with locally advanced or recurrent/metastatic head and neck cancer.

BACKGROUND: we investigated the maximum tolerated dose (MTD) of combination therapy with docetaxel, cisplatin, and S-1 (TPS) in patients with locally advanced or recurrent/metastatic head and neck cancer (HNC). PATIENTS AND METHODS: treatment consisted of docetaxel (Taxotere) at doses of 50, 60, and 70 mg/m(2); cisplatin at 70 mg·m(2)/day on day 1; and S-1 twice daily on days 1-14 at doses of 40, 60, and 80 mg·m(2)/day, repeated every 3 or 4 weeks. RESULTS: forty patients were enrolled. MTD was not reached until level 4. Subjects at expanded dose were limited to patients with locally advanced disease. Two dose-limiting toxic effects (DLTs) were observed at dose level 5 (TPS: 70/70/80 mg·m(2)/day, every 3 weeks), namely one grade 3 infection and one grade 3 hyperbilirubinemia, establishing this as the MTD. Of 12 patients treated at dose level 6 (TPS: 70/70/60 mg·m(2)/day, every 3 weeks), 2 DLTs were seen. Six achieved a complete response and 22 a partial response, giving a response rate of 70%. CONCLUSIONS: TPS was well tolerated. The recommended phase II dose as induction chemotherapy for locally advanced HNC was determined as 70/70/60 mg·m(2)/day every 3 weeks. Antitumor activity was highly promising and warrants further investigation.

Properties of various carbon nanomaterial surfaces in bilirubin adsorption.

Properties of various carbon nanomaterials in bilirubin adsorption have been studied to develop a new adsorbent in the plasma apheresis. Carbon nanomaterials we used as adsorbents are single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), and single-walled carbon nanohorns (SWNHs). The adsorbent was mixed with a bilirubin solution, and the adsorption capacity was obtained by measuring the concentrations of residual bilirubin in the solution after the adsorption process. We found that the bundled MWCNTs exhibit the largest capacity in the saturated amount of adsorbed bilirubin among the examined materials, and that the oxidized SWNHs exhibit the fastest rate in the early stage of the adsorption. We also found that the amount of adsorbed bilirubin increases with an increase in the dispersibility of the adsorbent.

Refractory response to growth factors impairs liver regeneration after hepatectomy in patients with viral hepatitis.

BACKGROUND/AIMS: Liver regeneration after surgical resection is important. The present study was designed to understand the effect of background liver damage on the rate of liver tissue regeneration after hepatectomy and the mechanism of any defective regeneration. METHODOLOGY: The subjects were 40 patients who underwent liver resection. They comprised 22 patients with chronic viral hepatitis-hepatocellular carcinoma (liver damage group) and 18 patients with hepatic metastases from colorectal cancer (normal liver group). Liver regeneration was evaluated by histopathological and immunohistochemical examination of the surgically resected tissue and by CT-scanning of the regenerated liver mass. The resected liver specimens were stained for c-met, gp-130 and nuclear factor-kappaB (NF-kappaB) proteins. RESULTS: Liver regeneration was significantly less in the liver-damage group than in the normal-liver group. Histopathological examination showed marked inflammatory cell infiltration in the liver-damage group. Expression of c-met, but not gp-130, was significantly higher on parenchymal cells of the liver-damage group than the normal-liver group. NF-kappaB expression in parenchymal liver cells was significantly higher than in non-parenchymal cells of the normal-liver group. In the liver-damage group, liver regeneration correlated negatively with the staining intensity of NF-kappaB protein in non-parenchymal cells. These findings suggest that non-parenchymal cells are constitutively activated in the damaged liver, probably explaining the refractoriness of hepatocytes to cytokine-induced proliferation after hepatectomy, in spite of increased receptor (c-met) expression. CONCLUSIONS: The refractory response of injured hepatocytes to cytokines may explain the impaired postoperative liver regeneration in patients with damaged liver.

Fabrication of carbon nanotube sheets and their bilirubin adsorption capacity.

Bilirubin adsorption on carbon nanotube surfaces has been studied to develop a new adsorbent in the plasma apheresis. Powder-like carbon nanotubes were first examined under various adsorption conditions such as temperatures and initial concentrations of bilirubin solutions. The adsorption capacity was measured from the residual concentrations of bilirubin in the solution after the adsorption process using a visible absorption spectroscopy. We found that multi-walled carbon nanotubes (MWCNTs) exhibit greater adsorption capacity for bilirubin molecules than that of single-walled carbon nanotubes (SWCNTs). To guarantee the safety of the adsorbents, we fabricated carbon nanotube sheets in which leakage of CNTs to the plasma is suppressed. Since SWCNTs are more suitable for robust sheets, a complex sheet consisting of SWCNTs as the scaffolds and MWCNTs as the efficient adsorbents. CNT/polyaniline complex sheets were also fabricated. Bilirubin adsorption capacity of CNTs has been found to be much larger than that of the conventional materials because of their large surface areas and large adsorption capability for polycyclic compound molecules due to their surface structure similar to graphite.

Site-specific gene transfer with high efficiency onto a carbon nanotube-loaded electrode.

A transfection array, which is specifically developed for use in high-throughput analyses of genome functions by the over-expression or suppression of genes on a chip, is expected to become an important method for post-genome research. High efficiency of gene expression or suppression is indispensable for high-throughput analyses because the adherent cell number on a single spot decreases as the density of spots increases in the transfection array. We have studied an electro-stimulated pore formation on the cell membrane for gene delivery. Fine pores should be formed on the cell membrane to increase the efficiency of gene transfection without cell damage. Herein, we examined the electrode carrying chemically functionalized carbon nanotubes (CNTs) on the surface. The CNTs were loaded on a gold electrode with a self-assembled monolayer membrane by electrostatic interaction. Adsorbed plasmid DNA was transfected with higher efficiency into adherent cells on the CNT-loaded electrode than on an electrode without CNTs. This result may be due to the strong but fine field emission formed from the tips of the CNTs.

Developmental failure of the intra-articular ligaments in mice with absence of growth differentiation factor 5.

OBJECTIVE: To show the phenotypic characteristics of the knee joints in brachypodism mice (bp mice), which carry a functional null mutation of the growth differentiation factor 5 (GDF5) gene, we investigated the adult and embryonic bp mice. METHOD: Radiographic and macroscopic examinations of the knee joint of adult bp mice were performed. A histological examination of the knee joint of bp mice from E12.5 to E18.5 was also performed. RESULTS: Radiographic and macroscopic examinations of the adult bp mice showed anterior dislocation, hypoplastic condyles, and absence of the intra-articular ligaments. Safranin O staining of knee joints of the embryonic bp mice showed severe hypoplasty of the chondroepiphyses and intra-articular ligaments at E16.5. There was no difference in the number and location of 5-bromo-2'-deoxyuridine (BrdU)-positive cells between wild-type and bp mice through E12.5 to E14.5. A terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) study showed excessive cell death of mesenchymal cells of the future knee joint in bp mice at E12.5 and E13.5. CONCLUSION: bp mice exhibit developmental failure of the condyles and intra-articular ligament of the knee joints.

Development and characterization of human constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies.

Delta (Y), MB1 (X), and Z are the three catalytic beta-subunits located in the inner rings of the constitutive proteasome, an intracellular multicatalytic complex responsible for the generation of peptides presented by human leukocyte antigen (HLA) class I antigens to T cells. When cells are incubated with interferon-gamma, delta (Y), MB1 (X), and Z are replaced by LMP2, LMP7, and LMP10, respectively, leading to the expression of immunoproteasome which generates peptides with increased affinity for HLA class I antigens. The characterization of the expression of constitutive proteasome and immunoproteasome subunits in cells, normal tissues, and malignant lesions has been hampered by the lack or limited availability of constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies (mAbs), which are suitable for immunohistochemical staining. To overcome this limitation, we generated human delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10-specific mAb-secreting hybridomas from BALB/c mice immunized with peptides and recombinant fusion proteins. The mAbs SY-5, SJJ-3, NB-1, SY-1, HB-2, and TO-7 were shown to be specific for delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10, respectively, as they react specifically with the corresponding molecules when tested with a human B lymphoid LG2 cell lysate in Western blotting and with the peptide derived from each molecule in enzyme-linked immunosorbent assay. The reactivity of the six mAbs with the corresponding intracellular antigens resulted in intracellular staining when the mAbs were tested with microwave-treated and saponin-permeabilized cells in indirect immunofluorescence and with formalin-fixed, paraffin-embedded tissue sections in immunohistochemical reactions. These results suggest that the constitutive proteasome and immunoproteasome subunit-specific mAbs we have developed are useful probes to characterize the expression of proteasome subunits in normal tissues and in pathological lesions.

Effect of specific antigen stimulation on intraepithelial lymphocyte migration to small intestinal mucosa.

Migration of intraepithelial lymphocytes (IELs) into intestinal epithelium is not yet well understood. We established an IEL-cell line from ovalbumin (OVA) 23-3 transgenic (Tg) mice and investigated the effect of antigen stimulation on the dynamic process of IEL migration into small intestinal mucosa. The cell line was a T cell receptor (TCR) alphabeta(+) CD4(+) CD8(-) phenotype, expressing alphaEbeta7 integrin in 90% of cells. Under intravital microscopy, the lined IELs adhered selectively to the microvessels of the intestinal villus tip of the Tg mice. The accumulation of IELs was significantly inhibited by an antibody against beta7-integrin and MAdCAM-1. When IELs were stimulated with OVA, the accumulation was attenuated compared to that of resting cells, with decreased expression of alphaEbeta7 integrin. In Tg mice fed with OVA, the number of IELs which migrated in the villus mucosa was significantly smaller than in the non-fed controls. The preferential migratory capacity of IELs to villus mucosa may be altered by specific antigen stimulations.

Improved shoulder contour following forequarter amputation with an osteomyocutaneous free flap from the amputated extremity: two cases.

To cover a large soft-tissue defect and to reconstruct the shoulder contour after forequarter amputation, we used an osteomyocutaneous free flap incorporating an elbow joint from the amputated extremity in two patients. These flaps were well vascularised and reliable. They provided excellent coverage of large soft-tissue defects and they maintained shoulder contours. This procedure is useful for reconstruction after extended forequarter amputation and chest wall resection.

Teratogenic mechanisms of longitudinal deficiency and cleft hand.

In order to better understand the teratogenic mechanisms of congenital defects of the digits, we analyzed clinical cases and induced similar types of congenital hand anomalies in rat fetuses by oral administration of busulfan. In clinical cases, radial and ulnar deficiencies had common characteristic features. We induced radial and ulnar deficiencies in rat fetuses with the same drug. Radial and ulnar deficiencies induced in rats have similar clinical manifestations and these anomalies might be caused by the same teratogenic mechanism. Then, the formation of the digital rays was examined histologically. The results of histological examination suggested that these deficiencies were not caused by localized damage of the limb bud. They also suggested that the cause of missing digits in longitudinal deficiency is closely related to a deficit of mesenchymal cells in the limb bud. Cleft hand is considered to be one of the types of longitudinal deficiency. However, several investigators have suggested that the abnormal induction of finger rays in the process of formation of fingers induced central polydactyly, osseous syndactyly and also cleft hand. X-rays of the clinical cases and skeletal changes of the anomalies induced in rats appear to demonstrate that cleft hand formation proceeds from osseous syndactyly and central polydactyly. The results of our experimental study show that the critical periods of central polydactyly, osseous syndactyly and cleft hand are the same. They also suggest that central polydactyly, syndactyly and cleft hand might be induced when the same teratogenic factor acts on embryos at the same developmental stage in the human being. Because they have a similar causation, cleft hand, syndactyly and central polydactyly should be classified into the same entity, that is, abnormal induction of digital rays. Based on these clinical and experimental studies, we modified the Swanson classification. In our modified classification, typical cleft hand, syndactyly and polydactyly are included in the same category of abnormal induction of digital rays as the fourth new category.

Serological and genetic characterisation of a unique strain of adenovirus involved in an outbreak of epidemic keratoconjunctivitis.

AIMS: To characterise a novel strain of adenovirus (Ad) type Ad8 (genome type Ad8I) involved in an epidemic keratoconjunctivitis (EKC) outbreak in Hiroshima city using serological testing and sequence analysis of the fibre and hexon gene. METHODS: A neutralisation test (NT) was performed in microtitre plates containing a confluent monolayer of A549 cells using 100 tissue culture infectious doses of virus and type specific antisera. The haemagglutination inhibition test was also carried out in microtitre plates with rat erythrocytes using four haemagglutination units of virus and twofold dilutions of serum. The fibre gene was sequenced by generating overlapping polymerase chain reaction products or by direct sequencing of genomic DNA. Primer selection was based on alignment of the fibre genes of human adenovirus serotypes Ad8, Ad19, Ad37, Ad9, and Ad15 available from Gene Bank. RESULTS: The virus strain was specifically neutralised by anti-Ad8 antibodies, although there was a major crossreaction with anti-Ad9 antibodies. Haemagglutination was equally inhibited by anti-Ad8 and anti-Ad9 antibodies. The predicted amino acid sequences of the hypervariable regions (HVRs) of the Ad8I hexon gene showed higher homology with Ad9 (83.3%) than with Ad8 (62.0%). However, the Ad8I fibre knob was more homologous to Ad8 (94.4%) than to Ad9 (91.6%). CONCLUSIONS: Ad8I is a unique strain of adenovirus because of its lower genomic homology with Ad8, major crossreactivity with Ad9 in NT, and mixed genetic organisation of HVRs of the hexon gene. These factors may have enabled the virus to circumvent acquired immunity, resulting in the outbreak.

Endoplasmic reticulum chaperone-specific monoclonal antibodies for flow cytometry and immunohistochemical staining.

Endoplasmic reticulum (ER) chaperones of the antigen processing machinery play a crucial role in HLA class I antigen complex assembly and antigen presentation. The characterization of the expression of these chaperones in normal tissues and malignant lesions has been hampered by the lack or limited availability of ER chaperone-specific monoclonal antibodies (mAb) that are suitable for immunohistochemical staining. To overcome this limitation, we have generated human calnexin, ERp57, calreticulin and tapasin-specific mAb-secreting hybridomas from BALB/c mice immunized with peptides and recombinant proteins. The mAb TO-5, TO-2, TO-11 and TO-3 were shown to be specific for calnexin, ERp57, calreticulin and tapasin, respectively, as they react specifically with the corresponding immunizing peptides in ELISA and with the corresponding proteins when tested with human lymphoid cell lysates in Western blotting. Furthermore, the reactivity of the four mAb with the corresponding intracellular antigens yielded intracellular staining when the mAb were tested with formalin-fixed, microwave-treated and saponin-permeabilized cells in indirect immunofluorescence and with formalin-fixed, paraffin-embedded tissue sections in the immunoperoxidase reaction. These results suggest that the ER chaperone-specific mAb we have developed are useful probes for characterizing the expression of ER chaperones of the antigen processing machinery in normal and pathological cells. This information will contribute to defining the effects of abnormalities in their expression on HLA class I antigen expression and function and on the interactions of target cells with the host's immune system.

Longitudinal study of auditory brainstem response in leigh syndrome.

To assess the utility of auditory brainstem response (ABR) in diagnosing brainstem changes in patients with Leigh syndrome (LS), we performed a longitudinal study of five patients with LS using both ABR and neuroimaging techniques (CT and MRI). The brainstem components of the initial ABRs we performed on the patients were abnormal in all five patients. In four of the patients, these abnormal findings preceded any clinical signs of brainstem impairment. Improvements in clinical findings were reflected in improvements in ABR findings in three patients. In one of these three patients, improvements in clinical findings were also reflected in improvements in MRI findings. In the other two patients, MRI findings showed no improvements, despite the improvements in clinical findings. In two of our patients, ABR clearly revealed functional improvements in the brainstem which were not revealed by MRI. Therefore, we conclude that ABR is an essential diagnostic technique for patients with LS.

Genetic characterisation of adenovirus type 8 isolated in Hiroshima city over a 15 year period.

AIMS: To investigate the genetic differences among the strains of adenovirus type 8 (Ad8) circulating in Hiroshima city, Japan, and to study their circulation pattern. METHODS: One hundred and twenty nine strains of adenovirus type 8 (Ad8) were isolated in Hiroshima City over a 15 year period (1983-97) from patients with keratoconjunctivitis, and analysed with six restriction enzymes-BamHI, HindIII, PstI, SacI, SalI, and SmaI-to investigate possible relations among the isolates and their genetic variability. Seven hypervariable regions of the hexon gene that carry the type specific epitope were also sequenced to investigate the variation among the genome types. RESULTS: Restriction endonuclease analyses yielded three known genome types (Ad8A, 13 samples; Ad8B, seven samples; and Ad8E, 35 samples) and a novel genome type (Ad8I, 74 samples). Ad8A, Ad8B, and Ad8E were closely related, with 96% homology, whereas Ad8I had only 71% homology. Ad8A, Ad8B, and Ad8E shared 91.8% and 96.4% homology with regard to their amino acid and nucleotide sequences, respectively, with the isolate 1127 (accession no X74663). However, when compared with Ad8A, Ad8B, Ad8E, and isolate 1127, Ad8I shared only 62.7% and 69.9% homology with regard to amino acid and nucleotide sequences, respectively. Ad8A, Ad8B, and Ad8E had a unique 31 amino acid deletion in the hypervariable region 1 of the hexon gene, whereas Ad8I had a 33 residue deletion. The Ad8E strain that circulated from 1984 to 1995 was stable among the study population. Ad8I was isolated from an outbreak of epidemic keratoconjunctivitis in 1995 and was also isolated from sporadic cases until 1997. CONCLUSIONS: These results confirmed that genetic variability occurs in Ad8 in the microenvironment and revealed the emergence of a new genome type (Ad8I).

The relationship between paroxysmal kinesigenic choreoathetosis and epilepsy.

PURPOSE: To clarify the relationship between paroxysmal kinesigenic choreoathetosis (PKC) and epilepsy, we investigated the clinical and electroencephalographic (EEG) findings of patients with familial PKC and epilepsy, as well as sporadic cases with both PKC and epilepsy. PATIENTS AND METHODS: Patients consisted of 12 familial cases from seven families and three sporadic cases. The period of follow-up ranged from 17 months to 33 years, 7 months (average: 16 years, 8 months). During the follow-up, a total of 163 EEGs (11 EEGs per subject) were studied, including interictal and ictal EEGs. RESULTS: Transient epileptic discharges were found in ten of the 15 patients (66.7 %) during the clinical course. As for focus, centro-midtemporal and frontal spikes were most often observed. The ictal EEG of an afebrile convulsion in one patient showed a partial seizure with secondary generalization which originated from the frontal area. CONCLUSIONS: It appears that patients who suffer from both PKC and epilepsy have a functional abnormality of the cerebral cortex, particularly in the perirolandic and frontal regions.

Radiological features of long bones in synovitis, acne, pustulosis, hyperostosis, osteitis syndrome and their correlation with pathological findings.

Abstract The purpose of this study was to demonstrate the radiological features of long bones in synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome and to correlate these with the clinical findings. Eleven long bone lesions in seven cases of SAPHO syndrome were examined. The patients ranged in age from 6 to 63 years, with a mean of 47 years. In all seven cases, radiography, (99m)technetium bone scintigraphy, CT scan, and magnetic resonance imaging (MRI) were performed. In six of the cases, bone biopsy and bone culture were carried out for 7 long bones. Seven of the involved lesions were from the shaft of the femur, one each was from the neck and the shaft of the humerus, and one was from the proximal tibia. These lesions showed radiologically hyperostosis, osteolysis, and bone infarction-like lesion. Osteolysis was occasionally accompanied by sclerotic change. Hyperostosis usually showed diaphyseal involvement, presenting low signal intensity on T1- and T2-weighted MR images. Histologically, these findings corresponded to massive bone necrosis, new bone formation, fibrosis, or a mixture of these associated with mild inflammatory cell infiltration. Osteolysis involved dyaphysis, metaphysis, or epiphysis associated with arthritis, and presented low signal intensity on T1-weighted images, nonhomogeneous signal intensity lower than fat on T2-weighted images, and high signal intensity on fat suppression images. These findings corresponded to fibrosis, granulation, and inflammatory cell infiltration with lymphocyte aggregation. Bone infarction-like lesion was observed in the shaft or neck of the femur and the humerus and accompanied by calcification and cystic change. Bone cultures were negative in all cases in which bone biopsy was performed. Although hyperostosis is thought to be a characteristic bone lesion in SAPHO syndrome, the long bone lesion can occasionally show not only hyperostosis but also osteolytsis and bone infarction-like lesions.

Quantitative analysis of mRNA expression of TIMPs in the periprosthetic interface tissue of loose hips by real-time PCR system.

Matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), have been reported to play a critical role in extracellular degradation around artificial hip joints. Although messenger ribonucleic acid (mRNA) expression patterns of several MMPs and TIMPs were reported, there is no report of quantitative mRNA analysis of TIMPs in periprosthetic tissues. In this study, mRNA expression of four different types of TIMPs in periprosthetic interface tissue of loose hips was analyzed by a quantitative polymerase chain reaction system. The mRNA expression level of TIMP-1, -2, and -3 in periprosthetic interface tissue was significantly higher than that in control. In contrast, the mRNA expression level of TIMP-4 in the periprosthetic interface tissue was lower. This study suggested that increased levels of TIMP-1, -2, and -3, and decreased levels of TIMP-4 may contribute to pathologic extracellular matrix degradation in combination with MMPs, thus leading to prosthetic loosening and osteolysis around artificial total hip joints.