Acute myeloid leukemia transforms the bone marrow niche into a leukemia-permissive microenvironment through exosome secretion.

Little is known about how leukemia cells alter the bone marrow (BM) niche to facilitate their own growth and evade chemotherapy. Here, we provide evidence that acute myeloid leukemia (AML) blasts remodel the BM niche into a leukemia growth-permissive and normal hematopoiesis-suppressive microenvironment through exosome secretion. Either engrafted AML cells or AML-derived exosomes increased mesenchymal stromal progenitors and blocked osteolineage development and bone formation in vivo. Preconditioning with AML-derived exosomes 'primed' the animals for accelerated AML growth. Conversely, disruption of exosome secretion in AML cells through targeting Rab27a, an important regulator involved in exosome release, significantly delayed leukemia development. In BM stromal cells, AML-derived exosomes induced the expression of DKK1, a suppressor of normal hematopoiesis and osteogenesis, thereby contributing to osteoblast loss. Conversely, treatment with a DKK1 inhibitor delayed AML progression and prolonged survival in AML-engrafted mice. In addition, AML-derived exosomes induced a broad downregulation of hematopoietic stem cell-supporting factors (for example, CXCL12, KITL and IGF1) in BM stromal cells and reduced their ability to support normal hematopoiesis. Altogether, this study uncovers novel features of AML pathogenesis and unveils how AML cells create a self-strengthening leukemic niche that promotes leukemic cell proliferation and survival, while suppressing normal hematopoiesis through exosome secretion.

Suppressive effects of 1,4-dihydroxy-2-naphthoic acid administration on bone resorption.

SUMMARY: The main component of the metabolic by-products of fermentation by Propionibacterium freudenreichii ET-3 is 1,4-dihydroxy-2-naphthoic acid (DHNA), which has a naphthoquinone skeleton, as in vitamin K2. This study showed that DHNA improved bone mass reduction with osteoporosis model mice caused by FK506. INTRODUCTION: Growth of the intestinal bacterium Lactobacillus bifidus is specifically facilitated by DHNA. The present study used osteoporosis model mice to investigate the effects of DHNA on bone remodeling. METHODS: FK506, an immunosuppressant, was used to prepare osteoporosis model mice. Thirty mice were divided into three groups: FK group, FK+DHNA group, and control group. In the FK group, FK506 was administered to induce bone mass reduction. In the FK-DHNA group, FK506 and DHNA were administered concurrently to observe improvements in bone mass reduction. To ascertain systemic and local effects of DHNA, we investigated systemic pathological changes in colon, kidney function and cytokine dynamics, and morphological and organic changes in bone and osteoclast dynamics as assessed by culture experiments. RESULTS: Compared to the FK group without DHNA, colon damage and kidney dysfunction were milder for FK+DHNA group, and production of inflammatory cytokines (interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha) was more suppressed. Furthermore, compared to the group without DHNA, histological analyses and radiography showed that bone resorption was suppressed for the DHNA group. Culture experiments using osteoclasts from murine bone marrow showed osteoclast suppression for the DHNA group compared to the group without DHNA. CONCLUSION: These results show that DHNA has some effects for improving bone mass reduction caused by FK506.

Molecular and biological characterization of gtf regulation-associated genes in Streptococcus mutans.

INTRODUCTION: Surface protein antigen (PAc) and glucosyltransferases (GTF) are major adhesive molecules of Streptococcus mutans, though the mechanism of their regulation has not been fully elucidated. METHODS: To investigate the regulation mechanism, we determined a nucleotide sequence in the upstream region of the pac locus in S. mutans and identified two open reading frames (ORF), designated as orf1 and orf2. Each ORF was inactivated and functional analyses were performed. RESULTS: Western blot analyses revealed that the expression level of PAc was unaffected, while that of cell-associated GTF was diminished in both mutant strains. Furthermore, they showed higher hydrophobicity levels and an impaired sucrose-dependent adherence to smooth surfaces. RNA dot blot analysis demonstrated that transcriptions of the gtfB and the gtfC genes, which encode GTF-I and GTF-SI, respectively, were downregulated, while that of pac was comparable to the wild-type strain. In addition, the GTF activities of the mutant strains were significantly lower than those of the wild-type, though a greater amount of total glucan produced by the mutants was noted in culture supernatants. CONCLUSION: These findings suggest that orf1 and orf2 are associated with positive regulation of the gtfB and gtfC genes.

Periodontitis-induced lipid peroxidation in rat descending aorta is involved in the initiation of atherosclerosis.

BACKGROUND AND OBJECTIVE: Periodontitis is a risk factor for the development of atherosclerosis. Recent studies indicate that oxidative mechanisms, including lipid peroxidation, are involved not only in periodontitis but also in atherosclerosis. Lipid peroxidation may play an important role in the pathogenesis of atherosclerosis, particularly during its earliest stages. The purpose of this study was to investigate the relationship between lipid peroxidation induced by periodontitis and the initiation of atherosclerosis. MATERIAL AND METHODS: Sixteen rats were randomly divided into two groups of eight rats each. Periodontitis was ligature-induced for 4 wk in the experimental group, whereas the control group was left untreated. After the experimental period, the mandibular first molar regions were resected and then subjected to histological analysis and measurement of hexanoyl-lysine expression as an indicator of lipid peroxidation. Descending aorta was used for measuring the levels of hexanoyl-lysine, reactive oxygen species and lipid deposits, and for real-time polymerase chain reaction microarray analysis. The level of hexanoyl-lysine was also measured in serum. RESULTS: In the experimental group, the levels of hexanoyl-lysine in periodontal tissue and serum increased. Only aorta samples in the experimental group showed lipid accumulation, with increased expression of hexanoyl-lysine, reactive oxygen species and oxidative stress-related genes (including nitric oxide synthases 2 and 3), whereas the superoxide dismutase 1 gene level was down-regulated. CONCLUSION: In a ligature-induced periodontitis rat model, increased lipid peroxidation was found in serum and aorta as well as in periodontal tissue. Atherosclerosis-related gene expression and histological changes were also stimulated. Periodontitis-induced lipid peroxidation in the aorta may be involved in the early stage of atherosclerosis.

Oesophageal squamous cell carcinoma may develop within a background of accumulating DNA methylation in normal and dysplastic mucosa.

BACKGROUND: Oesophageal squamous cell carcinoma (OSCC) often arises from preceding dysplastic lesions in the oesophageal epithelium. However, the molecular changes occurring in premalignant lesions are not well understood. An epigenetic change is an example of OSCC that may occur within the epithelium. AIM: To investigate the methylation status of multiple promoters in cancer-derived DNA, as well as in the background epithelium of OSCC, including dysplastic lesions and non-neoplastic mucosa. The normal epithelium from patients without cancer was also examined. The findings were correlated with the mutational status of p53. PATIENTS AND METHODS: 56 patients with advanced OSCC, 21 patients with intraepithelial neoplasia (IEN), 56 patients with a background of non-neoplastic epithelium, adjacent to the OSCC, and 42 normal control epithelia from healthy volunteers were studied. The promoter methylation status of SFRP1, SFRP2, DCC, APC, p16(INK4a), p14(ARF), MINT1, MINT2, MINT31, CACNA1G, COX2, DAPK, hMLH1 and MGMT was examined by methylation-specific single polymerase chain reaction or combined bisulphite restriction analysis. The mutation of p53 by direct sequencing was assessed. RESULTS: DNA methylation was observed in OSCC and in its background epithelium. The frequency of CpG island methylation increased from a baseline level in the background non-neoplastic epithelium, through IEN, to advanced OSCC. However, mutations in p53 were almost exclusively observed in IEN and OSCC. More extensive DNA methylation was seen in the neoplastic lesions (OSCC or IEN) having a p53 mutation than in those with wild-type p53. CONCLUSION: DNA methylation is present at low levels in the non-neoplastic oesophageal epithelium and appears to contribute to the progression of the dysplasia-carcinoma sequence in OSCC carcinogenesis.

Analysis of Host Species Specificity of Magnaporthe grisea Toward Foxtail Millet Using a Genetic Cross Between Isolates from Wheat and Foxtail Millet.

ABSTRACT Host species specificity of Magnaporthe grisea toward foxtail millet was analyzed using F(1) cultures derived from a cross between a Triticum isolate (pathogenic on wheat) and a Setaria isolate (pathogenic on foxtail millet). On foxtail millet cvs. Beni-awa and Oke-awa, avirulent and virulent cultures segregated in a 1:1 ratio, suggesting that a single locus is involved in the specificity. This locus was designated as Pfm1. On cv. Ki-awa, two loci were involved and one of them was Pfm1. The other locus was designated as Pfm2. Interestingly, Pfm1 was not involved in the pathogenic specificity on cv. Kariwano-zairai. These results suggest that there is no "master gene" that determines the pathogenic specificity on all foxtail millet cultivars and that the species specificity of M. grisea toward foxtail millet is governed by cultivar-dependent genetic mechanisms that are similar to gene-for-gene interactions controlling race-cultivar specificity.

The combination of ionizing radiation and expression of a wild type p53 gene via recombinant adenovirus induced a prominent tumour suppressing effect in human oral squamous cell carcinoma.

Human oral squamous cell carcinoma (SCC) cell lines HSC4 and SAS were infected with wild type p53 (wt-p53)-encoding adenovirus (AxCAip53) and subsequently irradiated to investigate the effectiveness of p53 gene therapy in combination with radiation therapy for treating oral SCC. Western blot analysis using anti-p53 monoclonal antibody showed that a large amount of mutant p53 protein was accumulated in HSC4 cells, while no detectable p53 protein was observed in SAS cells. The induction of p53 expression by AxCAip53 infection was clearly observed in both HSC4 and SAS cells. A clonogenic cell survival assay demonstrated that AxCAip53 infection alone, or X-irradiation alone, significantly inhibited the growth of cancer cells, but that combined treatment was most effective, even in mutant p53-accumulated HSC4 cells. Flow cytometric analysis showed that the apoptotic pathway was induced in virus treated and radiation treated cells. Taken together, these findings suggest that the combination of p53 gene therapy and radiation therapy has a possibility to effectively treat oral SCC defective in p53 function.

Distribution of emm genotypes and superantigen genes of Streptococcus pyogenes isolated in Japan, 1994-9.

The purpose of this study was to examine characteristic profiles of Streptococcus pyogenes clinical isolates isolated in Japan during 1994-9. Genotyping of the M protein (emm typing) revealed that emm types 12 and 28 were the most common among 316 isolates. Most of the emm12 isolates were isolated from mucosa, while emm58 and emm89 were from skin. Moreover, the emm3 isolates were dominant in invasive infections. The distribution of 6 superantigen genes showed that all isolates harboured the mf gene and many had the speG gene. Invasive isolates were shown to have the ssa gene at a higher rate (76%) than noninvasive (37%). The distribution of superantigens was significantly different between emm types, but not between isolation sites. These results suggest that the distribution of emm types is related to isolation site, whereas superantigen distribution is related to clinical features of S. pyogenes infections.

Correlation of immunologic status with high-resolution CT and distributions of pulmonary tuberculosis.

PURPOSE: To investigate the correlation of cluster of differentiation (CD)4 lymphocyte counts with high-resolution CT (HRCT) findings and distribution of pulmonary tuberculosis. MATERIAL AND METHODS: Thirty-seven bacteriologically proven pulmonary tuberculosis, clinically non-AIDS, patients underwent HRCT and CD4 lymphocyte counts in peripheral blood cells were obtained within 3 days after the CT examinations. Patients were categorized into four groups according to CD4 lymphocyte counts (A >800; B 800-500; C 500-200; D <200). HRCT findings analyzed were as follows: typical, atypical, and mixed findings of post-primary tuberculosis, common, uncommon, and mixed distribution, and number of lobes involved. RESULTS: CD4 lymphocyte counts correlated with the degree of the mixture of atypical CT findings (rho=0.565, p<0.001) and the degree of the mixture of uncommon distribution (rho=0.431, p<0.01). Number of involved lobes showed no statistically significant correlation (rho=0.209, p=0.21). CONCLUSION: In patients with low CD4 levels, atypical HRCT findings co-exist with typical findings, and uncommon sites are involved in addition to common sites.

Diagnostic and interventional ultrasound of the pediatric spine.

Ultrasound is useful as a diagnostic tool in the evaluation of the pediatric spine, and can also help guide procedures in the interventional radiology suite or the operating room. This pictorial exhibit will display examples of diagnostic and interventional uses of ultrasound with respect to the pediatric spine.

Fba, a novel fibronectin-binding protein from Streptococcus pyogenes, promotes bacterial entry into epithelial cells, and the fba gene is positively transcribed under the Mga regulator.

In infection by Streptococcus pyogenes, fibronectin (Fn)-binding proteins play important roles as adhesins and invasins. Here, we present a novel Fn-binding protein of S. pyogenes that exhibits a low similarity to other Fn-binding proteins reported. After searching the Oklahoma Streptococcal Genome Sequencing Database for open reading frames (ORFs) with an LPXTG motif, nine ORFs were found among those recognized as putative surface proteins, and one of them was designated as Fba. The fba gene was found in M types 1, 2, 4, 22, 28 and 49 of S. pyogenes, but not in other serotypes or groups of streptococci. Fba, a 37.8 kDa protein, possesses three or four proline-rich repeat domains and exhibits a high homology to FnBPA, the Fn-binding protein of Staphylococcus aureus. Recombinant Fba exhibited a strong binding ability to Fn. In addition, Fba-deficient mutants showed diminished invasive capabilities to HEp-2 cells and low mortality in mice following skin infection. The fba gene was located downstream of the mga regulon and analysis using an mga-inactivated mutant revealed that it was transcribed under the control of the Mga regulator. These results indicate that Fba is a novel protein and one of the important virulence factors of S. pyogenes.

[Surgical treatment of coarctation and interrupted aortic arch complex in infants].

One hundred forty-two consecutive neonates or early infants with coarctation and interrupted aortic arch complex who underwent biventricular repair at the Fukuoka Children's Hospital between January 1991 and December 2000 were reviewed. One-stage repair was performed in 33 patients (35%) with coarctation complex and in 41 patients (85%) with interrupted aortic arch complex. The overall mortality rate was 6.1% in one-stage repair and 6.6% in two-stage repair of coarctation complex patients and 9.8% in one-stage repair and 28.6% in two-stage repair of interrupted aortic arch complex patients. The recoarctation rate was 5.3% in coarctation complex and 2.1% in interrupted aortic arch complex. All patients with recoarctation underwent successful catheter intervention and required no reoperation. In conclusion, one-stage repair of interrupted aortic arch and coarctation complex with the anterior approach resulted in good outcomes. Then descending aorta cannulation through a median sternotomy combined with the cerebral perfusion technique enables complete avoidance of circulatory arrest and is a useful technique. However, a two-stage procedure can be useful in the patients whose condition has deteriorated substantially or in whom intracardiac anomalies are severe.

Early onset Pneumocystis carinii pneumonia after allogeneic peripheral blood stem cell transplantation.

Pneumocystis carinii (P. carinii) is one of the major opportunistic pathogens responsible for hematopoietic stem cell transplantation (HSCT)-related pneumonias. Although trimethoprim-sulfamethoxazol (TMP/SMX) prophylaxis has been shown to prevent almost all P. carinii infections, 1%-2% of patients may still experience this complication. P. carinii pneumonia (PCP) is usually a late complication in patients receiving TMP/SMX prophylaxis, with most cases occurring later than 2 months post-transplant. We report a patient who developed early onset PCP after allogeneic peripheral blood stem cell transplantation (PBSCT) from an HLA-identical sibling donor. On day 12, the patient complained of dyspnea and cough. A chest X ray showed infiltrates in right upper lobe with bilateral pleural effusion. By the findings of Grocott stain on bronchoalveolar lavage fluid obtained on day 14, he was diagnosed as having PCP. Intravenous TMP/SMX failed to improve the lesion. This is the earliest onset PCP in the literature after HSCT despite the prophylactic administration of TMP/SMX before transplant.

[Clinical study on renal trauma].

A total of 64 patients with renal trauma were treated at the Department of Urology, St. Marianna University School of Medicine, from January 1988 to December 1996. Patients were between 3 and 66 years old (mean 28.2); 56 (87.5%) of them were male and 8 (12.5%) were female. The main cause of injury was a traffic accident in 33 cases (51.6%). According to the Classification of Renal Injury by the Japanese Association for the Surgery of Trauma in Japan, there were 27 cases (42.2%) of Type I (subcapsular injury), 12 cases (18.8%) of Type II (superficial injury), 16 cases (25.0%) of Type III (Deep injury), 6 cases (9.4%) of Type IV (Pedicle injury). Surgical treatment was performed in 6 cases (9.3%). The major associated injuries were bone fracture in 33 cases (51.6%), lung injury in 20 cases (31.3%) and liver injury in 16 cases (25.0%). The literature is also reviewed briefly.

Granular lymphocytic leukemia derived from gamma delta T-cell expressing cytotoxic molecules.

We here present an extremely rare case of granular lymphocytic leukemia derived from gamma delta T-cell (gamma delta T-GLL). The blood picture at diagnosis was as follows; white cell count 25.7 x 10(9)/l containing 94% atypical lymphocytes with cytoplasmic granules, hemoglobin 11.8 g/dl and platelet count 124 x 10(9)/l. The atypical lymphocytes were positive for CD2, CD3, CD5, CD7, CD56 and TCR gamma delta, but negative for CD4, CD8, CD57, TCR alpha beta and B-cell antigens. The cytotoxic molecules, T-cell intracellular antigen-1 (TIA-1) and granzyme B, were positive by immunocytochemical analysis. Southern blot analysis showed rearrangement of T-cell receptor J gamma and C beta genes but germline configuration of the JH gene. Neither serum antibody against human T-cell leukemia virus type-I (HTLV-I) nor the integration of HTLV-I proviral DNA was detected. CT scan showed splenomegaly but no lymph node enlargement. A diagnosis of gamma delta T-GLL was made, and she has been followed up without any therapies for more than 4 years.

Radiotherapy combined with transcatheter arterial infusion of cisplatin versus oral fluoropyrimidine anticancer agent for locally advanced carcinoma of the uterine cervix: a prospective follow-up study.

We randomized patients with locally advanced cervical cancer to receive radiotherapy combined with transcatheter arterial infusion (TAI) of cisplatin or oral fluoropyrimidine anticancer agents, and compared the prognosis by a prospective follow-up study. Sixty patients were studied who completed their planned radiation therapy with chemotherapy at the Department of Obstetrics and Gynecology of Hiroshima University Hospital between January 1991 and December 1998. Patients were randomly assigned to receive (A) radiotherapy with TAI of 120 mg/body cisplatin twice a month at the interval of 4 weeks or (B) radiotherapy with 200 mg/day oral 5-FU or UFT every day. In both groups, radiotherapy is routinely 50 Gy of external beam irradiation to the whole pelvis and 18-20 Gy (point A dose) of intracavitary irradiation using a remote after loading system (RALS). Serious adverse reactions interfering with treatment did not appear in either group. The effective histologic response was 28/32 (87.5%) in group A and 25/28 (89.3%) in group B. The median follow-up period were 28.3 months and 25.4 months in group A and B, respectively. There was no significant difference in the overall survival and disease-free survival rates for all patients, clinical stage III and squamous cell carcinoma. We could not conclude that radiotherapy with TAI of cisplatin achieved superior therapeutic efficacy in locally advanced cervical cancer. To improve the therapeutic effects, it is important to establish a new cisplatin-containing chemoradiotherapy regimen.

Growth of the pulmonary arteries after systemic-pulmonary shunt.

Pulmonary artery growth after a systemic-pulmonary shunt was angiographically evaluated in 19 out of 35 patients. The mean age of the subjects at the time of the initial operation was 18+/-18 months including 12 patients under a year old. The preoperative diagnosis was tetralogy of Fallot (TOF) in 10 patients, TOF plus pulmonary atresia in five and transposition of great arteries in four. A Blalock-Taussig shunt (BTS) operation was performed in 16 patients (15 classical and 1 modified) and a central shunt was performed in three patients as an initial operation. The preoperative pulmonary artery index (PAI) was 129+/-42 in all patients and there were no significant differences between patients under or over a year old (139+/-42 vs. 115+/-49). Postoperative angiography was performed 32+/-13 months after the surgery. Room air arterial O2 pressure increased significantly from 29+/-5 mmHg to 42+/-5 mmHg just after an initial palliative shunt operation. PAI change in patients under a year old was 214+/-73%, which was higher than 145+/-27% in patients over a year old after a palliative shunt operation. On the ipsilateral side, PAI change was almost the same between patients under and over a year old. On the contralateral side, PAI change in patients under a year old was 216+/-68%, which was significantly higher than the 116+/-21% in patients over one year old. There was a significant negative correlation (r=-0.65, p<0.05) between PAI change and arterial O2 pressure as measured just after a palliative shunt operation. In conclusion, a palliative shunt operation prior to a year old is desirable in order to produce sufficient and bilateral pulmonary artery growth.

[Clinical characterization of blaIMP positive gram-negative rods isolated cases].

We detected the metallo-beta-lactamase gene blaIMP positive strains of the gram-negative rods (GNR) isolated in Oita Medical University Hospital between 1993 and 1999 and studied the clinical characteristics of patients infected or colonized with blaIMP positive GNR. 25 strains (20 Pseudomonas aeruginosa and 5 Serratia marcescens) were detected and most of them were isolated from urinary samples after 1997. In the studies of antimicrobial susceptibility, some strains had sensitivity to aztreonum or imipenem although most of the strains showed multidrug resistance. When blaIMP positive GNR were isolated from patients, these strains were thought to have caused infection in 88% of the patients. About half of the patients were over 65 years old and had malignant diseases. Most of the patients had inserted urinary tract catheters, intratracheal tube or intravernous catheters. It was suggested that the insertion of the catheters were related to infection of blaIMP positive GNRs. Two patients were not treated with any antibiotics before the isolation of blaIMP positive GNRs although more than half of the patients were administered carbapenems and cephems. Most of strains were isolated in the same department and showed the same genotype by pulsed field gel electrophoresis.

Thrombotic thrombocytopenia purpura caused by piperacillin successfully treated with plasma infusion.

An 81-year-old man was admitted to the hospital with a fever and loss of appetite. After treatment with piperacillin sodium (PIPC), the patient exhibited thrombocytopenia, hemorrhagic colitis, and drug-induced skin eruption. On the fifth day after PIPC induction, he further experienced neurological abnormalities, such as disorientation and confusion, renal dysfunction, and microangiopathic hemolytic anemia (MAHA). The patient was diagnosed with thrombotic thrombocytopenic purpura (TTP) on the basis of thrombocytopenia, MAHA, renal dysfunction, fever, and neurological abnormalities. Infusion of fresh-frozen plasma was initiated for treatment. His condition improved markedly after this treatment. It is rare for TTP to be accompanied with hemorrhagic colitis and skin eruption. These symptoms were induced by PIPC and were successfully treated with plasma infusion.