Efficient production of fungal spores by the combination of reduction of nitrogen source content and embedding of hydrophobic polymer in an agar plate.

Fungal sporulation is affected by many environmental factors, for example, we previously observed that embedding of a hydrophobic polymer net in an agar plate medium significantly accelerates spore formation of some fungi. Here, it was found that the fungal spore formation depended on the surface hydrophobicity of cultivation vessels used for the plate cultivation. In a polypropylene (PP) vessel, six fungal strains produced spores of 1.5 to 514.8 times of those growing in a glass vessel. The contact of vegetative hyphae on the surface of the vessels might trigger the fungal spore formation. Moreover, the spore formation was synergistically accelerated by the reduction of nitrogen source content in an agar plate medium and by the contact to hydrophobic polymers. The synergistic effect depended on the surface area of the hydrophobic polymer. Thus, the combination of the reduction of nitrogen source and the embedding of hydrophobic polymer is expected as a novel and effective procedure for production of fungal spores which are useful for the inoculum in fermentation industry and biocontrol agent in agriculture.

Short-Term Crossover Study on the Effect of Orthogonalized Deep-Grooved Posterior Artificial Teeth on the Masticatory Efficiency of Complete Denture Wearers.

BACKGROUND: Mastication has been regarded to play an important role in achieving quality daily life for the elderly. On the occlusal surface of posterior artificial teeth, parallel grooves of 1 mm depth and 1 mm width with an inter-groove distance of 2 mm have been found to significantly improve masticatory efficiency on a mechanical simulator. MATERIALS AND METHODS: In the present study, the effect of the grooved design on masticatory efficiency in edentulous subjects using a short-term crossover trial was evaluated. Six edentulous participants, 1 male and 5 females, were assigned into two groups. One received duplicated complete dentures with grooved molars first, and the other received duplicated complete dentures with conventional molars first. The design of the teeth was crossover after evaluating masticatory efficiency. Raw carrot, raw lettuce and mixed foodstuffs were used to evaluate masticatory efficiency. Subjects were instructed to masticate 20 strokes and arbitrary strokes until they felt like swallowing. RESULTS: The number of masticatory strokes taken was significantly smaller using the grooved design (p<.05). Masticatory efficiency analysis revealed that the grooved design achieved significantly higher masticatory efficiency at 20 masticatory strokes for raw carrot and mixed foodstuffs (p<.05). CONCLUSION: Results show that compared with conventional lingualized occlusion, the grooved design accomplishes higher masticatory efficiency in vivo. Considering the distinction of this design that it can be applied to any existing denture occlusion, the design might help improve the dietary life of the elderly.

A new occlusal surface design for artificial posterior teeth to achieve high masticatory performance.

PURPOSE: The low masticatory efficiency of denture prostheses impairs the ability of wearers to consume high-fiber foods. Hence, artificial teeth with high masticatory efficiency are required. This study aimed to establish an occlusal surface design for posterior artificial teeth in denture prostheses that is compatible with the existing artificial teeth arrangement and that has high masticatory efficiency for the comminution of raw vegetables. METHODS: A masticatory simulator for occluding complete dentures was used to evaluate the masticatory efficiency of four occlusal surface designs, i.e., with parallel grooves occluding at right angles to the opposing teeth, groove depths of 1 and 0.5mm, and inter-groove distances of 1, 2, and 3mm. Raw carrots, rice, raw lettuce, chicken breasts, and peanuts were used as test foods to evaluate food comminution. RESULTS: Grooved occlusal surface designs with a 1-mm groove depth and a 2- or 3-mm inter-groove distance demonstrated significantly greater masticatory efficiency than the conventional occlusal form (p<0.05). CONCLUSIONS: The superiority of grooved designs over the conventional design was particularly evident for lettuce and raw carrots in this study, both of which are considered difficult foods to chew with complete dentures.

Persistence of recipient human leucocyte antigen (HLA) antibodies and production of donor HLA antibodies following reduced intensity allogeneic haematopoietic stem cell transplantation.

The effects of reduced intensity conditioning (RIC) on human leucocyte antigen (HLA)-alloimmunization and platelet transfusion refractoriness (PTR) following allogeneic haematopoietic stem cell transplantation (Allo-HSCT) are unknown. We studied HLA-alloantibodies in a cohort of 16 patients (eight HLA-alloimmunized with pre-transplant histories of PTR and eight non-alloimmunized controls) undergoing Allo-HSCT using fludarabine/cyclophosphamide-based RIC. Pre- and post-transplant serum samples were analysed for HLA-antibodies and compared to myeloid, T-cell and bone marrow plasma cell chimaerism. Among alloimmunized patients, the duration that HLA-antibodies persisted post-transplant correlated strongly with pre-transplant HLA-antibody mean fluorescence intensity (MFI) and PRA levels (Spearman's rank correlation = 0·954 (P = 0·0048) and 0·865 (P = 0·0083) respectively). Pre-transplant MFI >10,000 was associated with post-transplant HLA antibody persistence >100 d (P = 0·029). HLA-antibodies persisted ≥100 d in 3/8 patients despite recipient chimaerism being undetectable in all lympho-haematopoietic lineages including plasma cells. Post-transplant de-novo HLA-antibodies developed in three control patients with two developing PTR; the donors for two of these patients demonstrated pre-existing HLA-antibodies of equivalent specificity to those in the patient, confirming donor origin. These data show HLA-antibodies may persist for prolonged periods following RIC. Further study is needed to determine the incidence of post-transplant PTR as a consequence of donor-derived HLA alloimmunization before recommendations on donor HLA-antibody screening can be made.

Escape of leukemia blasts from HLA-specific CTL pressure in a recipient of HLA one locus-mismatched bone marrow transplantation.

A case of leukemia escape from an HLA-specific cytotoxic T lymphocyte (CTL) response in a recipient of bone marrow transplantation is presented. Only the expression of HLA-B51, which was a mismatched HLA locus in the graft-versus-host direction, was down-regulated in post-transplant leukemia blasts compared with that in pre-transplant blasts. All CTL clones, that were isolated from the recipient's blood when acute graft-versus-host disease developed, recognized the mismatched B(∗)51:01 molecule in a peptide-dependent manner. The pre-transplant leukemia blasts were lysed by CTL clones, whereas the post-transplant leukemia blasts were not lysed by any CTL clones. The IFN-γ ELISPOT assay revealed that B(∗)51:01-reactive T lymphocytes accounted for the majority of the total alloreactive T lymphocytes in the blood just before leukemia relapse. These data suggest that immune escape of leukemia blasts from CTL pressure toward a certain HLA molecule can lead to clinical relapse after bone marrow transplantation.

Dexamethasone palmitate successfully attenuates hemophagocytic syndrome after allogeneic stem cell transplantation: macrophage-targeted steroid therapy.

Hemophagocytic syndrome (HPS) induced by uncontrolled macrophage activation and subsequent graft failure is a frequent and prominent complication after allogeneic stem cell transplantation (allo-SCT), a cause of severe morbidity and death, and a therapeutic challenge. Liposome-incorporated dexamethasone, dexamethasone palmitate (DP), shows greater efficacy against macrophages as compared to dexamethasone sodium phosphate (DSP). Based on our findings that DP achieves significantly larger decrease than DSP on the viability of primary human macrophages compared in vitro, we tested the effects of DP in patients with HPS. A decrease in number of macrophages in the bone marrow and prevention of engraftment failure were observed in all patients without any severe complications. In conclusion, these data provide a rationale for testing DP as a first-line treatment for patients with HPS after allo-SCT.

CTL clones isolated from an HLA-Cw-mismatched bone marrow transplant recipient with acute graft-versus-host disease.

HLA-Cw disparity in a donor increases the risk of acute graft-vs-host disease (GVHD) after bone marrow transplantation. Acute GVHD is mediated by donor CTLs. However, mismatched HLA-Cw-specific CTLs generated in posttransplant recipients who developed acute GVHD have not been characterized in detail. In this study, CTL clones isolated from a recipient at the onset of acute GVHD who was transplanted from an HLA-A, -B, and -DRB1-matched, HLA-Cw-mismatched (recipient, Cw*0303/Cw*0702; donor, Cw*0801/Cw*0702), unrelated donor were characterized. The seven isolated CTLs, including CD4(+), CD8(+), and CD4(+)CD8(+) T lymphocytes, lysed recipient cells, HLA-Cw*0303-transfected 721.211 cells, and HLA-Cw*0303-transfected donor cells, but not untransfected 721.211 cells or donor cells. Thus, all CTLs recognized the mismatched Cw*0303 molecule as an alloantigen. The sequences of Cw*0303 and Cw*0801 differ by 16 aas. Stimulation of CTLs by COS cells transfected with Cw*0303 cDNA constructs demonstrated that Cw*0303 mutants in which individual amino acids constituting peptide-binding pockets were substituted with the corresponding Cw*0801 amino acids significantly decreased IFN-gamma production by all CTLs, whereas Cw*0303 mutants bearing Cw*0801 amino acids outside the positions constituting peptide-binding pockets stimulated all CTLs to the same degree as the wild-type Cw*0303 construct. These data suggest that all CTLs recognized the Cw molecule in a peptide-dependent manner. ELISPOT revealed that Cw*0303-reactive T cells accounted for one-half of the total of alloreactive T cells in the blood during GVHD. Taken together, non-self Cw-specific CTL clones with a variety of phenotypes and peptide specificities can be generated in posttransplant recipients with acute GVHD.

Potential role of a mismatched HLA-specific CTL clone developed pre-transplant in graft rejection following cord blood transplantation.

Graft rejection is a serious complication in cord blood transplantation (CBT), but little is known about the mechanism of rejection. To investigate the potential role of T lymphocytes in graft rejection, we isolated a CD8(+) cytotoxic T lymphocyte (CTL) clone of recipient origin from blood obtained from a patient with graft rejection after CBT from an HLA-mismatched unrelated donor. The isolated CTL clone specifically recognized an HLA-B( *)1501 molecule as an alloantigen, which was expressed in donor cells but not in recipient cells. The results of a microchimerism analysis specific for HLA-B( *)1501 and a polymerase chain reaction assay specific for the T cell receptor on DNA from pretransplant peripheral blood mononuclear cells revealed that the patient was exposed to HLA-B( *)1501 prior to CBT, and that the CTL clone was in the patient's blood prior to transplantation. The present study demonstrates a potential role for pretransplant CTL in graft rejection following CBT.

Decreased risk of acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation in patients with the 5,10-methylenetetrahydrofolate reductase 677TT genotype.

Polymorphism in 5,10-methylenetetrahydrofolate reductase (MTHFR), a central enzyme in folate metabolism, has been shown to affect the sensitivity of patients to folate-based drugs such as methotrexate. In this study, we investigated whether a common single nucleotide polymorphism at position 677 in the donor or recipient's MTHFR gene affects the risk for acute graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (HSCT) from HLA-identical sibling donors when the recipient receives prophylactic treatment with methotrexate for GVHD. MTHFR genotypes were determined in 159 recipients with a hematological disease and their donors using polymerase chain reaction-restriction fragment length polymorphism analysis of genomic DNA. The 677TT genotype, which encodes an enzyme with approximately 30% of the activity of the wild-type (677CC), was observed in 13% of patients and in 8% of normal donors. Multivariate analyses demonstrated a significant association between 677TT genotype in patients and a lower incidence of grade I-IV acute GVHD (relative risk, 0.35; 95% confidence interval, 0.13-0.95; P = 0.040). There was no association between the incidence of acute GVHD and the donor MTHFR genotypes. These results suggest that greater immunosuppression by methotrexate due to low MTHFR enzyme activity decreases the risk of acute GVHD in recipients of allogeneic HSCT.

Long-term outcome after bone marrow transplantation for aplastic anemia using cyclophosphamide and total lymphoid irradiation as conditioning regimen.

We retrospectively studied 49 patients in a single institute to evaluate the long-term outcome of total lymphoid irradiation (TLI) conditioning for allogeneic stem cell transplantation (allo-SCT) to treat aplastic anemia (AA). Most of the patients had received transfusions and had undergone previous treatment, with 33 receiving related transplants and 16 receiving unrelated transplants. Conditioning consisted of cyclophosphamide (Cy; 200 mg/kg) plus TLI (750 cGy) for related transplantation and Cy plus total body irradiation (TBI; 500 cGy) and TLI (500 cGy) for unrelated transplantation. Antithymocyte globulin (ATG) was added for 6 of the unrelated transplantations. Graft-versus-host-disease (GVHD) prophylaxis consisted mainly of cyclosporine (CSA) and methotrexate (MTX). Graft failure developed in 2 patients (4.1%). With a median follow-up of 7 years, overall survival (OS) was 81% and was not statistically significantly different between the patients receiving related transplants and those receiving unrelated transplants. In multivariate analyses, a history of previous treatment with ATG was the sole factor associated with a worse survival rate, and the interval from diagnosis to treatment was not prognostic. The incidence of acute (grade II to IV) GVHD (aGVHD) was 23%, and that of chronic GVHD (cGVHD) was 29%. Female-to-male transplantation was the sole factor associated with chronic GVHD. B cell lymphoproliferative disorder developed only after the ATG-containing conditioning. No other secondary malignancies developed after long-term follow-up. Our findings suggest that TLI conditioning is feasible and effective for patients with AA.

Unrelated-donor bone marrow transplantation with a conditioning regimen including fludarabine, busulfan, and 4 Gy total body irradiation.

We investigated the feasibility of reduced-intensity conditioning with 4 Gy total body irradiation, fludarabine (30 mg/m2 for 6 days), and busulfan (4 mg/kg for 2 days) for bone marrow transplantation from a serologically HLA-matched unrelated donor. Seventeen adult patients (median age, 55 years; range, 27-67 years) with various hematologic malignancies (6 in remission, 11 not in remission) were treated. Successful engraftment was achieved in all patients at a median of day 18 (range, day 14-35) after transplantation, although subsequent secondary graft failure was observed in 2 patients. The cumulative incidence of acute graft-versus-host disease (GVHD) of grades II to IV at day 100 was 48%. With a median follow-up of 286 days (range, 56-687 days), the rates of 1-year overall survival, 100-day nonrelapse mortality, and 1-year nonrelapse mortality were 41%, 14%, and 46%, respectively. Eleven patients died, and the causes of death were relapse (n = 4), pulmonary complications (n = 4), acute GVHD (n = 2), and sepsis (n = 1). The remaining 6 patients (at transplantation, 2 were in remission, and 4 were not in remission) are currently still in remission. These results suggest that this regimen reduces the risk of graft failure, but further studies are needed to ameliorate transplantation-related toxicities, primarily GVHD and/or pulmonary complications.

Serologically HLA-DR-mismatched unrelated donors might provide a valuable alternative in allogeneic transplantation: experience from a single japanese institution.

To clarify the clinical significance of a serologic HLA-DR mismatch after unrelated-donor transplantation, we evaluated for hematologic malignancies 123 cases of unrelated bone marrow transplantation carried out in a single institution between 1995 and 2004. Of the patients in these cases, 12 were serologically mismatched at the single HLA-DR locus. Eighty-two patients who received HLA-matched transplantations were used as controls. Conditioning consisted of a conventional total body irradiation-based regimen or a fludarabine-based reduced-intensity regimen. Prophylaxis for graft-versus-host disease (GVHD) consisted of tacrolimus plus short-term methotrexate. Graft failure did not develop. With a median follow-up of 42 months (range, 11-99 months), rates of overall survival, nonrelapse mortality, and relapse at 4 years were 63%, 38%, and 0%, respectively, all of which were comparable with those after HLA-matched transplantation. The frequency of acute GVHD of grades II to IV was 75%, significantly higher than after HLA-matched transplantation (42%, P = .046), and there was a trend toward an increased incidence of acute GVHD of grades III to IV after serologically HLA-DR-mismatched unrelated transplantation (27% versus 10%, P = .093). Chronic GVHD developed in 4 of 11 evaluable patients, an incidence comparable with that after HLA-matched transplantation. In summary, serologically HLA-DR-mismatched unrelated transplantation is feasible and might be an acceptable alternative for the Japanese population, although the higher incidence of acute GVHD is notable.