Efficacy of azithromycin in preventing lethal graft-versus-host disease.

Acute graft-versus-host disease (GVHD) following allogeneic bone marrow transplantation (BMT) is initiated by donor T lymphocytes that recognize histocompatibility antigens presented by recipient dendritic cells (DCs). Current approaches to reduce GVHD are focused on suppressing donor T lymphocyte responses to alloantigens. However, these strategies may be inadequate in the setting of allogeneic transplants (particularly histoincompatible transplants), may increase the risk of tumour relapse and are associated with high rates of opportunistic infections. We hypothesized that inhibition of recipient DCs might suppress GVHD. We recently demonstrated in vitro that azithromycin, a macrolide antibiotic, also acts as a nuclear factor (NF)-κB inhibitor of murine DCs and inhibits their maturation and functions, including allogeneic responses. We investigated whether azithromycin could prevent alloreactions in a murine histoincompatibility model. Oral administration of azithromycin to recipient mice for 5 days during major-histoincompatible BMT suppressed lethal GVHD significantly, whereas ex-vivo lymphocyte function was not affected by the drug. These data suggest that azithromycin has potential as a novel prophylactic drug for lethal GVHD.

The effect of azithromycin on the maturation and function of murine bone marrow-derived dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells capable of initiating primary/adaptive immune responses and tolerance. DC functions are regulated by their state of maturation. However, the molecular pathways leading to DC development and maturation remain poorly understood. We attempted to determine whether inhibition of nuclear factor kappa B (NF-κB), which is one of the pivotal pathways underlying these processes, could induce immunophenotypic and functional changes in lipopolysaccharide-induced mature DCs derived from murine bone marrow. A comparative in vitro study of five clinically used drugs that are known to inhibit NF-κB demonstrated that azithromycin, a macrolide antibiotic, significantly inhibited expression of co-stimulatory molecules (CD40 and CD86) and major histocompatibility complex (MHC) class II by DCs. It also reduced Toll-like receptor 4 expression, interleukin-12 production and the allostimulatory capacity of DCs. These data suggest that azithromycin, as not only an NF-κB inhibitor but also an antibiotic, has potential as a novel drug for manipulation of allogeneic responses.

Expression of ADAMTS4 in Ewing's sarcoma.

Ewing's sarcoma (EWS) is a malignant bone tumor that frequently occurs in teenagers. Genetic mutations which cause EWS have been investigated, and the most frequent one proved to be a fusion gene between EWS gene of chromosome 22 and the FLI1 gene of chromosome 11. However, a limited numbers of useful biological markers for diagnosis of EWS are available. In this study, we identified ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs) as a possible tumor marker for EWS using the retrovirus-mediated signal sequence trap method. ADAMTS4 is a secreted protein of 837 amino acids with a predicted molecular mass of 98-100 kDa. It is a member of metalloprotease family, is expressed mainly in cartilage and brain, and regulates the degradation of aggrecans. ADAMTS4 has been suggested to be involved in arthritic diseases and gliomas. Herein, we show that ADAMTS4 mRNA was expressed in all primary EWS samples and all EWS-derived cell lines examined, while its expression was detected only in small subpopulations of other solid tumors. Furthermore, ADAMTS4 expression was found to be regulated by EWS-FLI1 fusion gene-dependent manner. We also demonstrated that ADAMTS4 protein was highly expressed in tumor samples of the patients with EWS by using immunohistochemistry. These results suggest that ADAMTS4 is a novel tumor marker for EWS.

Optimal positive airway pressure predicts oral appliance response to sleep apnoea.

Patients with less severe obstructive sleep apnoea (OSA) are usually prescribed oral appliances and/or smaller optimal nasal continuous positive airway pressure (P(nCPAP)) in nCPAP therapy. We hypothesised that OSA patients with greater P(nCPAP) would not respond favourably to oral appliances. Oral appliances were inserted in nCPAP users after washing-out the nCPAP effect. Follow-up polysomnography was undertaken with the adjusted oral appliance in place. The predictability of P(nCPAP) was evaluated with receiver-operating characteristic (ROC) curves. The median baseline apnoea/hypopnoea index (AHI) was reduced with the oral appliance from 36 to 12 events.h(-1) in 35 patients. When responders were defined as patients showing a follow-up AHI of <5 events.h(-1) with >50% reduction in baseline AHI, the area under the ROC curve for P(nCPAP) was 0.76. The best cut-off value of P(nCPAP) turned out to be 10.5 cmH(2)O with a high negative predictive value (0.93) and a low negative likelihood ratio (0.18). OSA patients with a P(nCPAP) of >10.5 cmH( 2)O are unlikely to respond to oral appliance therapy. This prediction is clinically helpful to both OSA patients and medical personnel in discussing oral appliances as a temporary substitute and/or alternative for nCPAP.

Haemodynamic changes in the liver under balloon occlusion of a portal vein branch: evaluation with single-level dynamic computed tomography during hepatic arteriography.

AIM: To assess haemodynamic changes in the liver under temporary occlusion of an intrahepatic portal vein. MATERIALS AND METHODS: Between February 2000 and October 2004, 16 patients with hepatobiliary disease underwent single-level dynamic computed tomography during hepatic arteriography (SLD-CTHA) under temporary balloon occlusion of an intrahepatic portal vein. All patients needed percutaneous transhepatic portography for therapy of their disease. SLD-CTHA was undertaken to clarify the time-attenuation curve influenced by portal vein occlusion, and it was performed continuously over a period of 30s. The difference in absolute attenuation of the liver parenchyma in segments with occluded and non-occluded portal vein branches was determined by means of the CT number, and the difference in absolute attenuation of the occluded and non-occluded portal veins themselves was also evaluated. RESULTS: SLD-CTHA demonstrated a demarcated hyperattenuation area in the corresponding distribution of the occluded portal vein branch. The attenuation of the liver parenchyma supplied by the occluded portal vein was significantly higher than that in the non-occluded area (p<0.01). The balloon-occluded portal branch enhancement in 15 of 16 cases (94%) appears due to arterio-portal communications. Failure to evaluate a remaining case for portal branch enhancement was due to absence of a visualized portal branch in the section. CONCLUSION: Under temporary occlusion of an intrahepatic portal vein, hepatic angiography produced enhancement of the occluded portal branches and their corresponding parenchymal distribution; this finding is considered consistent with the presence of arterio-portal communications.

Cryptic insertion and translocation or nondividing leukemic cells disclosed by FISH analysis in infant acute leukemia with discrepant molecular and cytogenetic findings.

Of 51 infants with acute leukemia, 13 (25%) had contradictory findings on 11q23/MLL rearrangements that were analyzed by cytogenetic and Southern blot methods: seven had rearranged MLL and normal karyotype, four had rearranged MLL and abnormal karyotype with no 11q23 translocation, and two had germline MLL and 11q23 translocations. Fluorescent in situ hybridization (FISH) analysis using an MLL probe that was performed to elucidate the discrepancy disclosed the presence of normal dividing cells and nondividing leukemic cells in the same bone marrow in five patients, and cryptic insertion or translocation in another five. Subsequent FISH and reverse transcription-polymerase chain reaction analysis identified the MLL-AF10, MLL-AF4, or MLL-AF1q fusions that were produced by the cryptic rearrangements in four of the five patients. In the remaining three patients, the breakpoint of 11q23 translocation was located distal to the MLL locus in one, and the discrepancy was unresolved in two. Thus, FISH should complement cytogenetic analysis when cytogenetic and molecular genetic findings are contradictory in infant leukemia, and when infant leukemia does not show 11q23 translocations or other specific translocations including t(7;12), t(1;22), etc that are recurrently found in infant leukemia.

Mutations/deletions of the WT1 gene, loss of heterozygosity on chromosome arms 11p and 11q, chromosome ploidy and histology in Wilms' tumors in Japan.

Incidence rates of Wilms' tumor (WT) markedly differ in East Asian and Caucasian children. In the present study, we examined WT1 deletions/mutations and loss of heterozygosity (LOH) on 11p and 11q in a large number of WTs and compared our findings with those from 4 series of Caucasian WTs. Incidence rates of the subtle WT1 mutation in 3 of the 5 series of sporadic and unilateral WTs including ours were 4.3-6.2% and similar. However, gross homozygous WT1 deletion was more frequent in our series than in some others. In addition, our series tended to show a higher incidence of LOH limited to 11p13 and a lower incidence of LOH including 11p15 than the Caucasian one. These findings indicate some genetic differences in WT between the 2 regions. One of the 4 Caucasian series reported a correlation of germinal WT1 mutation with the predominantly stromal histology. The present study not only confirms the correlation of germinal WT1 deletion/mutation with predominant stromal histology but also establishes a correlation with somatic WT1 deletion/mutations with predominant stromal histology. While WTs with WT1 abnormalities usually showed pseudodiploidy and predominant stromal histology, those without WT1 abnormalities showed various chromosome numbers and histologic subtypes.

Myelodysplastic syndrome in childhood: a retrospective study of 189 patients in Japan.

We report a retrospective analysis of children with myelodysplastic syndrome (MDS) diagnosed between 1990 and 1997 in Japan. In total, 189 patients were enrolled: 122 cases of primary MDS (26 RA, 18 RAEB, 25 RAEBt, 53 CMML/JMML), 24 cases with constitutional predisposition to MDS, and 43 cases of therapy-related MDS (t-MDS). The frequency of pediatric MDS was estimated to be 7.7% of all leukemias. Cytogenetic abnormalities were observed in 41% of primary MDS and 90% of t-MDS cases. The 4-year survival rate, estimated by Kaplan-Meier analysis, for primary RA was 78.9%, while other types of MDS and JMML had rates lower than 40%, and t-MDS showed an even more unfavorable prognosis. In primary MDS, the survival rate of patients with cytogenetic abnormalities was significantly lower. Among prognostic variables by IPSS, only the cytogenetic pattern was useful for predicting outcome in childhood MDS. There was no apparent advantage to chemotherapy for RA, and the survival rate in patients with primary RA, JMML, or t-MDS receiving stem cell transplantation was significantly higher. More precise designs of our diagnostic and classification systems, as well as therapeutic trials in large-scale prospective studies, are necessary for further improvements in MDS outcome.

A pivotal role of Rho GTPase in the regulation of morphology and function of dendritic cells.

Dendritic cell (DC) is the most potent activator of CD4+ T cells and has unique dendrites and veils. To explore the function of Rho in DC, exoenzyme C3 from Clostridium botulinum was used as a specific inhibitor of Rho. Treatment of DC with C3 (DC/C3) resulted in profound morphological changes by losing dendrites and emerging of shrunk membrane processes that were in parallel with marked reduction of polymerized actin in the marginal area. Inactivation of Rho-associated coiled coil-containing kinase (p160ROCK) by a specific ROCK inhibitor Y-27632 also led to disappearance of dendrites of DC with retaining large membrane expansions. In scanning electron microscopy, untreated DCs interacted with CD4+ T cells more efficiently than DC/C3. Conjugate formation assay showed that the number of DCs associated with CD4+ T cells was 2-fold higher in untreated DCs than that of DC/C3. Alloantigen-presenting capacity of DC/C3 was significantly suppressed in a dose-dependent manner. Because C3 treatment did not affect the surface expression of HLA, costimulatory, and adhesion molecules of DC, we examined cytokine production of DC and naive CD4+ T cells to further elucidate the inhibitory mechanism of MLR. Unexpectedly, DC/C3 increased IL-12 production after LPS stimulation. Naive CD4+ T cells cocultured with DC/C3 produced the increased percentage of IFN-gamma-producing cells, whereas the percentage of IL-2-producing T cells was decreased. These results demonstrate that Rho GTPase in DC controls both characteristic shape and immunogenic capacity.

Differential responsiveness of cord and adult blood monocytes to hepatocyte growth factor.

Monocytes as antigen-presenting cells play an important role in host defence. There are several cytokines affecting monocyte function. We demonstrate that both adult and cord blood monocytes constitutively express hepatocyte growth factor (HGF) receptor, MET. HGF significantly down-regulated MET expression of adult blood monocytes, compared with cord blood monocytes, when cultured either in RPMI-1640 containing 10% FBS or serum-free medium. Surface levels of MET correlated with c-met mRNA levels both in adult and cord blood when cultured. MET expression was down-regulated by treating with actinomycin D or cycloheximide. HGF stimulated DNA synthesis of adult monocytes, but not cord blood. HGF enhanced antigen-presenting capacity of adult blood monocytes but not cord blood monocytes. HGF up-regulated HLA class I expression in adult monocytes but not in cord blood monocytes. The current results suggest that the failure of cord blood monocytes to respond to HGF may be responsible, in large part, for their functional immaturity.

Successful unrelated BMT in a patient with Kostmann syndrome complicated by pre-transplant pulmonary 'bacterial' abscesses.

Kostmann syndrome, severe congenital neutropenia, is often associated with life-threatening bacterial infections. A 5-year-old girl with Kostmann syndrome developed pulmonary abscesses. She was refractory to granulocyte colony-stimulating factor and antibiotics. She underwent unrelated HLA-matched BMT. Myeloablative conditioning consisted of 12-Gy TBI with lung shielding, antithymocyte globulin, etoposide, and cyclophosphamide. After successful engraftment, the pulmonary abscesses resolved by day 75 post-transplant. Although the option of transplantation is not established in the setting of unrelated HLA-matched BMT in Kostmann syndrome, this case may provide useful information. Furthermore, pre-transplant pulmonary bacterial abscesses may not be a contraindication for BMT in some patients with Kostmann syndrome.

Polio vaccine virus-associated meningoencephalitis in an infant with transient hypogammaglobulinemia.

The case of an infant with transient hypogammaglobulinemia who developed meningoencephalitis, retinitis and sensorineural hearing loss is presented. The neurovirulent variant of the Sabin type 2 oral poliovirus vaccine was detected in cerebrospinal fluid and stool.

Frequent increase of DNA copy number in the 2q24 chromosomal region and its association with a poor clinical outcome in hepatoblastoma: cytogenetic and comparative genomic hybridization analysis.

In a cytogenetic and comparative genomic hybridization (CGH) study of 38 hepatoblastomas, we found gain of 1q in 17 tumors (44.7%), that of 2 / 2q in 14 (36.8%), that of 20 / 20q in 9 (23.7%) and that of 8 / 8q in 8 (21.0%), loss of 4q in 4 (10.5%) and no DNA copy changes with normal karyotype or no mitotic cells in 11 (28.9%). Eleven tumors with 2 / 2q gain detected by CGH had a total chromosome 2 gain, a partial 2q gain, or a total chromosome 2 gain with an augmented partial 2q region; the common region for DNA copy gain was 2q24. Two-color fluorescence in situ hybridization (FISH) analyses using probes covering the centromere of chromosome 2 or HOXD13 (2q31) confirmed the CGH findings, and showed that the common region for gain in 2q was centromeric to HOXD13. Event-free survival (EFS) +/- standard error (SE) at 5 years was lowest in patients with 2q gain [37 +/- 15%], highest in those with no DNA copy changes [82 +/- 12%], and intermediate in those with DNA copy changes other than 2q gain [74 +/- 13%] (P = 0.0549). Multivariate analysis showed that 2q gain was an independent factor predicting a poor outcome. These findings suggest the presence of a growth-promoting gene or an oncogene in the 2q24 chromosome band, and a tumor suppressor gene in terminal 4q, which have important roles in the development and progression of hepatoblastoma.

Plasma levels of natriuretic peptides in relation to doxorubicin-induced cardiotoxicity and cardiac function in children with cancer.

BACKGROUND: Anthracyclines are effective anticancer drugs for childhood cancer with dose-limiting cardiotoxicity. Children who have received anthracyclines thus need periodical cardiac evaluation. The plasma levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have been shown to increase in proportion to severity of cardiac dysfunction. We examined whether plasma levels of ANP and BNP, in addition to echocardiographic evaluation, can be used as specific markers for doxorubicin-induced cardiotoxic effects in children. PROCEDURE: Consecutively, 34 patients (18 boys and 16 girls) who had previously received doxorubicin-containing chemotherapy were enrolled in this study. Plasma ANP and BNP were assayed simultaneously at the time of first cardiac function evaluation by echocardiography. RESULTS: Of the 34 patients, 8 (23.5%) had left ventricular dysfunction as assessed by echocardiography. Both ANP and BNP plasma levels in these patients were significantly elevated in comparison with healthy controls (P < 0.01) or patients with normal cardiac function (P < 0.05). It should be also noted that ANP and BNP levels were correlated significantly with cardiac systolic function, but not with diastolic function. CONCLUSIONS: These results suggest that plasma ANP and BNP levels could be markers for doxorubicin-induced cardiotoxicity in children. Measurement of natriuretic peptide levels during treatment may allow earlier-identification of individuals at risk for severe cardiac damage.

Functional immaturity of cord blood monocytes as detected by impaired response to hepatocyte growth factor.

BACKGROUND: Monocytes as antigen-presenting cells play an important role in host defense and transplantation. However, there are little reports on cord blood monocytes, and the role of monocytes in cord blood transplantation is largely unknown. METHODS AND RESULTS: There are several cytokines affecting monocyte function. These include interferon-gamma, interleukin-4, interleukin-10, granulocyte macrophage-colony stimulating factor and hepatocyte growth factor (HGF). We investigated the effect of these cytokines on antigen-presenting capacity (APC) of cord and adult blood monocytes. Using either mononuclear cells or purified CD4+ T cells as responder cells, HGF enhanced APC of adult monocytes most effectively among these cytokines. In contrast, cord blood monocytes failed to respond to HGF. As HLA, costimulatory and adhesion molecules may affect APC function, we examined these antigens of monocytes following HGF stimulation. The HGF upregulated integrin alpha5 subunit (CD49e) and intercellular adhesion molecule-1 (CD54) was expressed in adult blood monocytes, but not in cord blood. In kinetic studies, HGF downregulated c-met protein/HGF receptor expression of adult monocytes in lower concentrations and at shorter incubation time as compared with that of cord blood. CONCLUSIONS: The results suggest that impaired response of cord blood monocytes to HGF may be responsible, in large part, for their functional immaturity.

Transient renal tubular acidosis in a neonate following transplacental acetazolamide.

Renal tubular acidosis (RTA) was observed in a preterm boy shortly after birth. His mother had glaucoma and had been treated during pregnancy with oral acetazolamide, a carbonic anhydrase inhibitor. When RTA developed, acetazolamide was detected in his serum demonstrating transplacental acetazolamide passage.

Is the sleep initiating process affected by psychological factors?

The aim of this study was to investigate the effects of psychological factors on sleep initiating process. The Y-G personality test (Y-G), Lazarus-type stress coping inventory (SCI), Zung self-rating depression scale, General health questionnaire, and Sleep health questionnaire were administered to 418 subjects (mean age = 21.13 +/- 8.76 years), and the data were analyzed by multiple regression analysis (stepwise variable reduction method). Self-rating depression scale; Co-Em, Pla, Sel scale in SCI; C, N, O, Co scale in Y-G were significantly included in the final model. The results showed that psychological factors, such as depression, emotion-focused coping behaviour, prudent planning to solve problems, low self-control, small changes of feeling, nervousness, subjectiveness, and cooperativeness are associated with difficulty initiating sleep.

Successful immunization following cord blood transplantation in a child with Diamond-Blackfan anemia.

Cord blood transplantation (CBT) has been increasingly used to treat patients with hematological diseases, but active immunizations for patients have not been described. Patients certainly need immunizations following CBT, since transplanted cord blood is naive. The authors previously reported successful hematopoietic reconstitution following cord blood transplantation from an HLA-matched sibling in a transfusion-dependent child with Diamond-Blackfan anemia. No graft-versus-host disease, either acute or chronic, has been observed so far. Here, the authors report that immunological recovery of the patient has been rapid shortly after CBT and immunization has been done successfully. Vaccines (diphtheria, pertussis, tetanus, rubella, measles, and BCG) were administered during 22-34 months post-transplant. Seroconversion to these vaccines was excellent without significant adverse effects. These results indicate that both toxoid and live vaccines have been safely administered in the patient who underwent related cord blood transplantation.