Salvage allogeneic hematopoietic SCT for primary graft failure in children.

Primary graft failure (pGF) is associated with considerable morbidity and mortality. Salvage hematopoietic SCT (HSCT) can rescue pGF patients; however, the optimal preconditioning regimen and stem cell source are yet to be determined, particularly in children. In this study, we retrospectively analyzed 102 pediatric patients who received salvage allogeneic HSCT for pGF. Salvage HSCT from matched or one-Ag-mismatched related donors (rMM01) provided superior OS compared with that from two- or three-Ags-mismatched related donors (rMM23) or cord blood transplantation (CBT). CBT showed a trend toward a slightly lower engraftment rate and late engraftment achievement compared with rMM23; however, the OS rate was similar between the two groups (47.6±7.7% for rMM23 and 45.7±8.6% for CBT, at 1 year after salvage HSCT). Multivariate analysis showed that preconditioning regimens with fludarabine or irradiation were associated with a higher engraftment rate and those with alkylating agents were associated with better OS. In conclusion, our results showed that rMM01 was the most suitable donor for salvage HSCT for pediatric pGF, and that CBT was an equally important option compared with rMM23 for patients without rMM01.

Long-term outcome of childhood aplastic anemia patients who underwent allogeneic hematopoietic SCT from an HLA-matched sibling donor in Japan.

We report long-term outcomes of 329 childhood severe aplastic anemia (SAA) patients who underwent hematopoietic SCT (HSCT) from an HLA-matched sibling donor in the Japanese Hematopoietic Cell Transplantation Registry. OS and EFS at 10 years were as high as 89.7+/-1.7% and 85.5+/-2.0%, respectively. Five cases of late malignancies (LM) were identified (malignant peripheral nerve sheath tumor, thyroid carcinoma, colon carcinoma, MDS and hepatoblastoma). Cumulative incidence of LM was 0.8% at 10 years and 2.5% at 20 years, respectively, which was lower than that in previous reports. This low incidence is in keeping with the low occurrence of skin cancer in Japanese population and of acute GVHD in our study group. Radiation-containing conditioning was not significantly associated with the incidence of LM after HSCT probably because of absolute low patient number who developed LM in our series. In terms of LM development after HSCT, low-dose TBI in HSCT for SAA to avoid graft rejection, which is commonly used in Japan, might be tolerable in the Japanese population because of its low incidence.

TBI and melphalan followed by allogeneic hematopoietic SCT in children with advanced hematological malignancies.

We evaluated the efficacy and safety of the conditioning regimen that consisted of TBI and melphalan (L-PAM), followed by hematopoietic SCT (HSCT) in 23 children with advanced hematological malignancies. The median age at HSCT was 9 (range, 2-15) years. The underlying diseases were ALL in 16 patients (5 in CR2, 3 in CR3, 6 in relapse (RP) and 2 in induction failure (IF)), AML in 4 patients (3 in RP and 1 in IF) and non-Hodgkin's lymphoma in 3 patients (1 in CR3, 1 in CR4 and 1 in RP). The stem cell sources were BM for 19 patients and cord blood for 4 patients. All patients received the conditioning regimen that consisted of TBI 12 or 13.2 Gy and L-PAM 210 mg/m(2). In all, 22 patients engrafted on the median of day 16 (range, 10-23). The regimen was well tolerated and common regimen-related toxicities (RRTs) included grade II stomatitis and grade I hepatic toxicity. The cumulative incidences of RP and TRM were 47.6 and 21.5%, respectively. At a median follow-up of 24.4 months, the probability of disease-free survival was 41.0%. The regimen may provide sufficient anti-leukemic effect without increased RRT for advanced pediatric hematological malignancies.

Low-dose MTX for the treatment of acute and chronic graft-versus-host disease in children.

We report the results of a retrospective analysis in 27 pediatric patients who received low-dose MTX as the second-line treatment for steroid-refractory or -dependent acute and chronic GVHD. Between July 2000 and May 2006, 10 patients with aGVHD and 17 with cGVHD were treated with MTX at a dose of 3-10 mg/m(2) weekly. Seven of ten patients (70%) with aGVHD responded well to MTX, thus resulting in the achievement of either a complete response (CR) or a partial response (PR). The dose of prednisone could be reduced to equal to or lower than 1 mg/kg in the responding patients at the end of MTX therapy. The median number of MTX administrations was five (range, 1-7). Ten (58.8%) of seventeen patients with cGVHD achieved CR or PR. The dose of prednisone could be reduced to lower than 0.4 mg/kg in 16 of 17 patients and seven patients could discontinue prednisone. The median duration of MTX administration was 18 months (range, 1-68). The toxicities of grade III to IV occurred in only six patients presenting cytopenias or elevated levels of serum transaminases. Low-dose MTX was tolerable and effective for the steroid-refractory or -dependent GVHD in reducing the dose of steroid without increasing the risk of opportunistic infection.

Clonotypic analysis of T cell reconstitution after haematopoietic stem cell transplantation (HSCT) in patients with severe combined immunodeficiency.

Haematopoietic stem cell transplantation (HSCT) is performed for treatment of a broad spectrum of illnesses. Reconstitution of an intact immune system is crucial after transplantation to avoid infectious complications, and above all, the establishment of T cell receptor (TCR) diversity is the most important goal in the procedure. Until recently, little has been known of the mechanism of T cell reconstitution in the very early period after HSCT. In this study, we analysed TCR repertoires sequentially in four patients with severe combined immunodeficiency (SCID) before and after HSCT. In all patients, the TCR repertoires were extremely abnormal before HSCT, whereas after transplantation there was progressive improvement in TCR diversity, based on analysis of the TCR Vbeta repertoire and CDR3 size distributions. Somewhat unexpectedly, there was a significant but transient expansion of TCR diversity 1 month after transplantation in all cases. Clonotypic analysis of TCRs performed in one case showed that many T cell clones shared identical CDR3 sequences at 1 month and that the shared fraction decreased progressively. These results indicate that early expansion of TCR diversity may reflect transient expansion of pre-existing mature T cells from the donor blood, independent of de novo T cell maturation through the thymus.

Assessment of room temperature influence on finger blood flow response induced by short-term grasping of vibrating handle.

OBJECTIVE: To investigate the influence of room temperature on finger blood flow (FBF) change in healthy subjects exposed to short-term grasping of a vibrating handle under different room temperatures. METHODS: FBF was measured using a blood flowmeter in six male subjects on the dorsum of the middle phalanx of third finger in both hands once at the end of every minute for an equal duration of 5 min at pre-exposure, during exposure to grasping of vibrating handle with sinusoidal vibration and after exposure. Vibration was generated with a frequency of 125 Hz and an rms acceleration of 40 m/s(2). Measurements were conducted in four room temperatures of 15+/-1, 20+/-1, 25+/-1 and 30+/-1 degrees C. RESULTS: Compared with the baseline measurements in the exposed hand during grasping of vibrating handle most significant increase in FBF was observed at 15+/-1 degrees C (P<0.001) and least at 30+/-1 degrees C (P<0.05), and after vibration least significant FBF was found at 25+/-1 degrees C (P<0.05). In case of the unexposed hand significant increase in FBF was exhibited at 20+/-1 degrees C (P<0.01) and 30+/-1 degrees C (P<0.01) during vibration, and only at 15+/-1 degrees C (P<0.05) after vibration. CONCLUSIONS: Response in FBF due to grasping of vibrating handle was of different patterns from the baseline measurement under different room temperature conditions in both exposed and unexposed hands and it was influenced by room temperature. Overall, the influence was greater at lower test room temperature, inducing more significant increase in FBF.

Effect of room temperature on percentage finger systolic blood pressure response to finger cooling.

Percentage finger systolic blood pressure (%FSBP) in response to finger cooling is used to assess vascular components of the hand-arm vibration syndrome and the measurement method is under discussion for standardization. It has been suggested that measurement circumstances including room temperature may affect %FSBP. We investigated the effect of room temperature on %FSBP response to finger cooling in healthy subjects. Six healthy male subjects who were medical students volunteered for the study. Multi-channel plethysmograph was used for simultaneous multi-finger FSBP measurements. The examination room was kept at 21 +/- 1 degrees C and 25 +/- 1 degrees C, and the subjects were randomly assigned. Percentage finger systolic blood pressures for the index, middle, ring and little fingers at 15 degrees C and 10 degrees C cuff-water temperatures were calculated. Four-way analysis of variance was performed to determine the independent effect of subject, room temperature, finger and cuff-water temperature factors on %FSBP. The room temperature as an independent factor affecting %FSBP was statistically significant (P < 0.01). From the results, it can be concluded that %FSBP response to finger cooling in healthy subjects may be affected by room temperature. Therefore, room temperature is expected to be controlled when assessing peripheral vascular components of the upper extremities using %FSBP response to finger cooling.

Unrelated donor bone marrow transplantation for 100 pediatric patients: a single institute's experience.

In all, 100 unrelated donor bone marrow transplantations (UD-BMT) were performed in our institute between October 1993 and January 2003. Of 93 evaluable patients, 73 patients had hematological malignancy, 13 had nonmalignancy and seven had lymphoproliferative disease. The estimated 9-year event-free survival (EFS) rate was 57.1+/-5.5% in all patients. In the following analyses of the patients with hematological malignancy, the standard group had significantly better EFS than the high-risk group (61.5+/-7.0 vs 35.6+/-9.7%, P=0.02), and the EFS rate of the tacrolimus (FK-506)+methotrexate (MTX)+/-methylprednisolone prophylactic group for graft-versus-host disease was superior to that of the FK-506 without MTX group (75.7+/-8.0 vs 55.8+/-7.6%, P=0.02). When we compared the EFS rates of the FK506+MTX+/-methylprednisolone (mPSL) group and the HLA-matched related donor BMT group in our institute, these were almost similar (75.7+/-8.1 vs 68.4+/-9.3%). Therefore, UD-BMT using FK-506+MTX+/-mPSL is a safe and useful method for children with hematological malignancy who require allogeneic BMT.

A stereoselective synthesis of the C10-C31 (BCDEF ring) portion of pinnatoxin A.

[reaction: see text] An efficient synthesis of the C10-C31 (BCDEF ring) portion of pinnatoxin A has been achieved. The key step is a highly stereoselective construction of the dispiroketal (BCD ring) system employing an intramolecular hetero-Michael reaction of a reversibly formed hemiketal alkoxide through the use of LiOMe.

A specific ligand for beta(2)-glycoprotein I mediates autoantibody-dependent uptake of oxidized low density lipoprotein by macrophages.

beta(2)-Glycoprotein I (beta(2)-GPI) is a major antigen for antiphospholipid antibodies (Abs) present in patients with the antiphospholipid syndrome (APS). We previously reported that beta(2)-GPI specifically binds to oxidized low density lipoprotein (oxLDL), but not to native low density lipoprotein (LDL). In the present study, a ligand specific for beta(2)-GPI, oxLig-1, was purified from the extracted lipids of oxLDL. The structure of oxLig-1 was shown to be identical to that of synthesized 7-ketocholesteryl-9-carboxynonanoate by mass spectroscopy and nuclear magnetic resonance analyses. Both purified and synthesized oxLig-1 were recognized by beta(2)-GPI and subsequently by anti-beta(2)-GPI auto-Abs, either in enzyme-linked immunosorbent assay (ELISA) or in ligand blot analysis. Binding of liposomes containing oxLig-1 (oxLig-1-liposomes) to mouse macrophages, J774A.1 cells, was relatively low, as compared with that of phosphatidylserine (PS)-liposomes. In contrast, binding of oxLig-1-liposomes was enhanced more than 10-fold in the presence of both beta(2)-GPI and an anti-beta(2)-GPI auto-Ab (WB-CAL-1), derived from (NZW x BXSB) F1 mouse, an animal APS model. Anti-beta(2)-GPI auto-Abs derived from APS patients with episodes of arterial thrombosis were detected in ELISA, using a solid phase oxLig-1 complexed with beta(2)-GPI. We suggest that autoimmune atherogenesis linked to beta(2)-GPI interaction with oxLDL and Abs may be present in APS.

IgG anti-laminin-1 autoantibody and recurrent miscarriages.

PROBLEM: The present study assesses the clinical significance of anti-laminin-1 auto-antibodies (auto-Abs) in recurrent miscarriages. METHOD OF STUDY: A total of 207 recurrent aborters with a history of two or more consecutive first-trimester miscarriages were tested for the presence of anti-laminin-1 Abs, beta2-glycoprotein I-dependent anticardiolipin Abs, lupus anticoagulants, anti-DNA Abs, and anti-nuclear Abs, before they had conceived again. Recurrent aborters then were followed up during subsequent pregnancies and their outcomes were evaluated relative to their blood test results prior to pregnancy. RESULTS: Fifty-five (31.1%) women out of 177 recurrent aborters were positive for IgG anti-laminin-1 auto-Abs. The levels of IgG anti-laminin-1 auto-Abs in recurrent aborters were significantly higher than those in healthy pregnant women and in healthy non-pregnant women (P = 0.0043 and 0.0073, respectively). The live birth rate of subsequent pregnancies in IgG anti-laminin-1 auto-Abs-positive recurrent aborters was significantly lower than the IgG anti-laminin-1 auto-Abs-negative recurrent aborters (P = 0.0320). There were no specifically significant relationships observed between IgG anti-laminin-1 auto-Abs and other tested auto-Abs. CONCLUSION: IgG anti-laminin-1 auto-Abs are associated with recurrent miscarriages and the subsequent pregnancy outcome of recurrent aborters.

Protein translocation within chloroplast is similar in Euglena and higher plants.

It is currently thought that chloroplasts of higher plants were derived from endosymbiont oxygenic photosynthetic bacteria (primary endosymbiosis), while Euglena, a photosynthetic protista, gained chloroplasts by secondary endosymbiosis (i.e., incorporation of a photosynthetic eukaryote into heterotrophic eukaryotic host). To examine if the protein transport inside chloroplasts is similar between these organisms, we carried out heterologous protein import experiments with Euglena precursor proteins and spinach chloroplasts. The precursor of a 30-kDa subunit of the oxygen-evolving complex (OEC30) from the thylakoid lumen of Euglena chloroplasts contained the N-terminal signal, stroma targeting, and thylakoid transfer domains. Truncated preOEC30s lacking the N-terminal domain were post-translationally imported into spinach chloroplasts, transported into the thylakoid lumen, and processed to a mature protein. These results showed that protein translocations within chloroplasts in Euglena and higher plants are similar and supported the hypothesis that Euglena chloroplasts are derived from the ancestral Chlorophyta.

Proteolytic cleavage of beta(2)-glycoprotein I: reduction of antigenicity and the structural relationship.

Binding of beta(2)-glycoprotein I (beta(2)-GPI)-dependent anticardiolipin antibodies (aCL) derived from antiphospholipid syndrome (APS) is significantly reduced in aCL ELISA due to loss of the phospholipid (PL) binding property of beta(2)-GPI by plasmin treatment. In the present study, the treatment generated a nicked form of beta(2)-GPI and resulted in loss of antigenicity for the autoantibodies detected in ELISA, using an beta(2)-GPI directly adsorbed polyoxygenated carboxylated plate, the assay system of which was not related to PL binding. The nicked form bound to neither Cu(2+)-oxidized low-density lipoprotein (oxLDL) nor to beta(2)-GPI-specific lipid ligands isolated from oxLDL, the result being a complete loss of subsequent binding of anti-beta(2)-GPI autoantibodies. The conformational change in the nicked domain V was predicted from its intact structure determined by an X-ray analysis (implemented in Protein Data Bank: 1C1Z), molecular modeling and epitope mapping of a monoclonal anti-beta(2)-GPI antibody, i.e. Cof-18, which recognizes the related structure. The analysis revealed that novel hydrophobic and electrostatic interactions appeared in domain V after the cleavage, thereby affecting the PL binding of beta(2)-GPI. Such a conformational change may have important implications for exposure of cryptic epitopes located in the domains such as domain IV.

Expression and properties of recombinant HbA2 (alpha2delta2) and hybrids containing delta-beta sequences.

Hemoglobin A2 (alpha2delta2), which is present at low concentration (1-2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic. However, reports on its functional properties regarding O2 binding are conflicting. We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A2 and systematically studying its functional properties. The construct expressing HbA2 contains only alpha and delta genes so that the extensive purification required to isolate natural HbA2 is circumvented. Although natural hemoglobin A2 is expressed at low levels in vivo, the amount of recombinant alpha2delta2 expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the alpha + beta or the alpha + gamma genes, respectively, are present on the construct. Recombinant HbA2 is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA. However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin. Molecular modeling studies which attempt to understand these properties of HbA2 are described.

Determining the site of the primary cancer in patients with skeletal metastasis of unknown origin: a retrospective study.

BACKGROUND: When skeletal metastasis is the presenting problem and the primary site is occult, there is a need to identify the primary site as soon as possible. However, the search for the primary tumor is often time-consuming and difficult. The purpose of this study was to analyze the efficacy of particular diagnostic approaches and to devise an efficient and optimal diagnostic strategy. METHODS: Among 213 patients with skeletal metastasis treated between 1990 and 1996 were 64 in whom skeletal lesions were the first manifestation of malignancy. The authors retrospectively analyzed both the final diagnosis and the process by which it was made in these 64 cases. RESULTS: The primary cancer was identified antemortem in 56 (88%) of the 64 patients by examination and in 3 patients at autopsy. Lung carcinoma, the most frequently observed primary lesion, was identified in 23 patients. Other primary lesions were prostate carcinoma in 11 patients, breast carcinoma in 5, and hepatocellular carcinoma in 5. The primary malignancy was not determined in 5 patients. Thoracic and abdominal computed tomography (CT) scans were useful, especially in the diagnosis of patients with lung, hepatocellular, renal cell, and pancreatic carcinomas. Tumor markers were abnormally elevated in 73% of patients with carcinomas. CONCLUSIONS: Although thoracic and abdominal CT scans were useful, examination of the gastrointestinal tract and pelvic CT scan seldom revealed the primary lesion and therefore should not be performed as an initial routine study in the absence of abdominal symptoms. Tumor markers are useful in differentiating carcinoma from hematologic malignancy and primary bone tumor.