HLA polymorphisms and risk of red blood cell alloimmunisation in polytransfused patients with sickle cell anaemia.

BACKGROUND: Red blood cell (RBC) alloimmunisation is an event that may occur due to factors such as numerous blood transfusions, age, gender and genetic factors such as human leukocyte antigen (HLA). AIMS/OBJECTIVES: The aim of the present study was to investigate the possibility of alloimmunisation to red blood cell group antigens associated with the HLA of individuals and to relate alloimmunisation to risk factors. METHODS: A total of 172 polytransfused patients with sickle cell anaemia (SCA) (44 alloimmunised, 128 non-alloimmunised) participated in this study. Blood group genotyping was performed by the DNA microarray method and HLA genotyping by polymerase chain reaction - specific sequence of oligonucleotides. RESULTS: The number of transfusions received directly influenced the incidence of alloimmunisation, and the most common alloantibodies were against Rh (48·8%) and Kell (17%) systems. The HLA-C*06 and HLA-DQB1*03 variants were significantly higher in alloimmunised patients. The HLA-DRB1*04 and HLA-DRB1*11 were more often found in individuals who developed the alloantibodies anti-Fya and anti-K, respectively. CONCLUSION: This study suggests that polytransfused patients with SCA possessing the HLA-DQB1*03 and HLA-C*06 allele variants are more susceptible to alloimmunisation. In addition, HLA-DRB1*04 and HLA-DRB1*11 alleles were seen to be associated with the production of anti-Fya and anti-K antibodies, respectively.

Developing seed cryobank strategies for Tabebuia heptaphylla (Bignoniaceae), a hardwood tree of the Brazilian South Atlantic Forest.

The conservation of Tabebuia heptaphylla, an economically significant, endangered tree of the South Atlantic Forest is confined to arboreta. Although its seeds are orthodox, they do not withstand long-term storage in conventional seed banks, motivating the development of cryopreservation for this species. Seeds within the moisture content (MC) range of 7.5 percent (0.08 g water g dry mass) to 8.4 percent (0.09 g water g dry mass) germinated after storage in liquid nitrogen (LN). Storage duration (15 min to 26 weeks) and rewarming regime (slow and rapid) did not significantly influence germination, which ranged between 54-67 percent. As no additional cryoprotective treatments were required, the protocol is time-, cost- and technically-efficient. Because transport of seeds in LN is problematic for safety, logistic and technical reasons, the feasibility of implementing germplasm transfer using T. heptaphylla seeds recovered from cryobanks was also tested. Viability was not negatively affected in seeds that had been rewarmed, recovered and maintained at room temperature for 2 weeks, allowing safe germplasm transfer in the unfrozen state. The vigor of seedlings from cryopreserved seeds, which was evaluated 90 days after transfer to soil was not influenced by LN storage compared to the controls.

A multidisciplinary treatment strategy that includes high-dose chemotherapy for metastatic retinoblastoma without CNS involvement.

The prognosis of patients with metastatic retinoblastoma is poor with conventional chemotherapy and radiation. Since retinoblastoma is highly chemosensitive, dose-escalation of chemotherapeutic agents with stem cell support should be promising. We report our experience with high-dose chemotherapy (HDC) and autologous stem cell transplantation (SCT) in patients with metastatic retinoblastoma. Five patients with metastatic retinoblastoma underwent HDC with autologous SCT following conventional chemotherapy and local radiation therapy. Stem cells (bone marrow in four and peripheral blood stem cells in one) were collected after marrow involvement was cleared. Melphalan was a key drug in all patients, and was administered in combination with other agents such as cisplatin, cyclophosphamide, carboplatin or thiotepa. Three patients are currently alive disease-free at 113, 107 and 38 months, respectively, from the time of SCT. They had no central nervous system (CNS) involvement. The two patients who died of disease had CNS involvement. No long-term sequelae of HDC have been noted. Our treatment strategy using HDC appears to be effective for treating metastatic retinoblastoma without CNS involvement.

Dimerumic acid as an antioxidant of the mold, Monascus anka.

We previously reported that the mold Monascus anka, traditionally used for fermentation of food, showed antioxidant and hepatoprotective actions against chemically induced liver injuries. In the present study, the antioxidant component of M. anka was isolated and identified. The antioxidant was elucidated to be dimerumic acid. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical was significantly scavenged by the antioxidant whereas hydroxyl radical and superoxide anion were moderately scavenged. When the antioxidant (12 mg/kg) was given to mice prior to carbon tetrachloride (CCl(4), 20 microl/kg, ip) treatment, the CCl(4)-induced liver toxicity in mice seen in an elevation of serum aspartate aminotransferase and alanine aminotransferase activities was depressed, suggesting the hepatoprotective action of the antioxidant. The liver microsomal glutathione S-transferase activity, which is known to be activated by oxidative stress or active metabolites, was increased by CCl(4) treatment and the increase was also depressed by pretreatment with the mold antioxidant. Thus these data confirmed that the dimerumic acid isolated from M. anka is the potential antioxidant and protective against CCl(4)-induced liver injury.

Increased accumulation of N-isopropyl-(I-123)p-iodoamphetamine in bronchial carcinoid tumor.

Radionuclide imaging with N-isopropyl-(I-123)p-iodoamphetamine ([123I]IMP) was performed in a patient with recurrent bronchial carcinoid tumor. Increased accumulation of [123I]IMP was observed in the known lesions of the brain and neck on both tomographic and planar images. Scintigraphy also revealed unknown metastatic lesions of the paraaortic lymph nodes, which were later confirmed on x-ray CT. Iodine-123-IMP may have a potential role in evaluating carcinoid tumors.

Tracheal relaxing effects and beta2 adrenoceptor selectivity of S1319, a novel sponge-derived bronchodilator agent, in isolated guinea-pig tissues.

1. S1319 (4-hydroxy-7-[1-(1-hydroxy-2-methylamino)ethyl]-1, 3-benzothiazol-2(3H)-one acetate), a novel non-catecholamine beta-adrenoceptor agonist, has been compared with isoprenaline, salbutamol and formoterol for activity in vitro on a range of beta-adrenoceptor containing preparations from guinea-pig. 2. S1319, like isoprenaline, salbutamol and formoterol, relaxed preparations of guinea-pig trachea (contracted by histamine) in a concentration-dependent manner. The relaxing activity of S1319 appeared to be more potent than that of isoprenaline and salbutamol, and similar to that of formoterol (pD2 values of 10.58+/-0.03 vs 7. 60+/-0.01, 7.50+/-0.01 and 10.52+/-0.04, respectively), and was blocked by the beta2-adrenoceptor selective antagonist (ICI 118,551). The intrinsic activity of S1319 was close to 1.0. 3. In the beta1-adrenoceptor containing preparations, guinea-pig right and left atria, a monophasic inotropic response of S1319 was observed. The pD2 value of S1319 for left atrial and right atrial inotropism was 6.70+/-0.15 and 7.81+/-0.01, respectively. 4. The selectivity ratio (trachea/left atrial inotropism) of S1319, formoterol, salbutamol and isoprenaline was 8523, 284, 4.8 and 0.45, respectively. The relative selectivity ratio of S1319 was 18743, 1858 and 30 times greater than that of isoprenaline, salbutamol and formoterol, respectively. 5. Relaxant responses of guinea-pig trachea to S1319 declined rapidly when the agonist was washed from the tissues, with complete recovery within 30 min. The duration of action of S1319 was similar to that of isoprenaline and less than that of salbutamol and formoterol. 6. In summary, S1319, a sponge-derived beta-adrenoceptor agonist, is a potent and selective beta2-adrenoceptor agonist with a short-duration of action in isolated guinea-pig tracheas.

In vivo administration of interleukin-6 in murine allogeneic bone marrow chimeras: early and delayed enhancement of hematopoiesis accompanied with split tolerance but not with graft-versus-host disease.

Interleukin-6 (IL-6) induced increased, leukocyte and platelet counts on around day 20 when it was administered into [BALB/c-->C3H/He] bone marrow chimeras from day 1 to day 12. Increased leukocyte counts and hemoglobin (Hb) levels were also observed at around day 60 and from day 41 to 80, respectively. On the other hand, hematopoietic recovery in [C3H/He-->C3H/He] bone marrow chimeras injected with IL-6 was different from that in [BALB/c-->C3H/He] bone marrow chimeras, showing no delayed and long-lasting increase in Hb levels but showing an early and transient increase in Hb levels and platelet counts. Sera from [BALB/c-->C3H/He] bone marrow chimeras injected with IL-6 showed predominant productions of IL-3 and/or IL-4. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that stem cell factor (SCF) mRNA expression was increased in bone marrow or spleen cells from [BALB/c-->C3H/He] bone marrow chimeras injected with IL-6 on day 36. Furthermore, we analyzed influence of IL-6 on graft-versus-host disease (GVHD) in [BALB/c-->C3H/He] bone marrow chimeras injected with IL-6. Decreased survival days and body weights were not observed when compared with the control. Histopathological changes of the liver due to GVHD were also not obvious. However, alloreactive mixed lymphocyte reactions (MLRs) were readily detected although cytotoxic T cells were not generated. Since H-2 typing showed that donor-type chimerism was predominantly observed, it was suggested that split tolerance might be induced by IL-6 administration. Increased IL-2 levels were not detected in sera from [BALB/c-->C3H/He] bone marrow chimeras injected with IL-6 whereas IL-4 was detected in the same sera, indicating that type 2 helper T (TH2) cells appeared to be predominantly generated. These results suggest that IL-3/IL-4 and SCF appeared to synergistically support delayed effects on hematopoiesis in [BALB/c-->C3H/He] bone marrow chimeras injected with IL-6 although early effects appeared to be mediated mainly by IL-6 directly or indirectly. Furthermore, IL-6 could induce split tolerance in [BALB/c-->C3H/He] bone marrow chimeras via a preferable activation of TH2 type cells without inducing severe GVHD.

Structural composition of central pulmonary arteries. Growth potential after surgical shunts.

Failure to achieve adequate pulmonary artery growth in patients with cyanotic congenital heart disease is a major obstacle to surgical correction. To assess whether differences in structural composition of central pulmonary arteries influence their growth potential after surgically created shunts, we obtained full-thickness biopsy specimens from the hilar pulmonary arteries of eight patients with pulmonary atresia or tetralogy of Fallot undergoing modified Blalock-Taussig shunts under 1 year of age. Tissue was processed for electron microscopic studies and a morphometric assessment was made of the volume proportions of smooth muscle, collagen, ground substance, and elastin. Initial pulmonary artery size was determined angiographically during the diagnostic cardiac catheterization. Pulmonary artery size was determined by cross-sectional echocardiograms 7 to 27 months later (mean 19 months). Pulmonary artery growth did not correlate with the interval between examinations but did correlate with the volume proportion of elastin (r = 0.73, standard error of the estimate = 1.45, p less than 0.05). Thus the structural composition of pulmonary arteries may influence their potential for growth after surgical shunts. In particular, an inadequate proportion of elastin may be a hindrance to growth.

Analysis of left ventricular motion after mitral valve replacement with a technique of preservation of all chordae tendineae. Comparison with conventional mitral valve replacement or mitral valve repair.

The postoperative regional left ventricular motion of 22 patients with a diagnosis of mitral regurgitation, and who underwent mitral valve replacement with preservation of chordae tendineae, were retrospectively analyzed by cineangiography in the early postoperative period and by multiple-gated cardiac blood pool scintigraphy in the mid-to-late postoperative period. The operation consisted of the division of the anterior leaflet into anterior and posterior segments, the shifting and reattachment of the divided segments to the mitral ring of the respective commissural areas, and the use of a low-profile bileaflet prosthetic valve. Control groups consisted of 28 patients with mitral regurgitation who underwent mitral valve replacement with a conventional technique and 16 patients who underwent mitral valve repair. Compared with the conventional mitral valve replacement group, the radial shortening of the left ventricle of the chordae-preserved mitral valve replacement group was greater at the apical septal, inferoapical, anterobasal, and anterolateral portions, whereas the radial shortening of the repair group was greater than that of the chordae-preserved group only at the inferolateral portion. The ejection fraction of the whole left ventricle was statistically greater in the chordae-preserved group, and also regional ejection fraction of the chordae-preserved mitral valve replacement group was greater at the apical septal, inferoapical, inferolateral, anterobasal, and anterolateral portions than that of the conventional mitral valve replacement group at these portions. On the other hand, the postoperative regional and global motion was identical to that of the mitral valve repair group except at the inferolateral portion. The result of this study supports a concept that maintenance of continuity between the mitral anulus and the papillary muscle has a beneficial effect on postoperative left ventricular performance.

Mediastinal emphysema and bilateral pneumothoraces with chronic GVHD in patients after allogeneic stem cell transplantation.

It is difficult to treat lung complications caused by chronic graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-SCT). We retrospectively analyzed the characteristics of five patients with mediastinal emphysema (ME) and bilateral pneumothoraces (BP) caused by chronic lung GVHD after allo-SCT. Four of these patients had undergone unrelated SCT, and three had had HLA-identical unrelated donors. All patients received total body irradiation (TBI) during conditioning. Immunosuppressive agents were administered as GHVD prophylaxis, but two patients developed acute GVHD and all the five developed chronic GVHD. The onset of lung complications was 99-1915 days (median, 202 days) after SCT. The onset of ME and BP was 6-48 days (median, 23 days) after the onset of lung complications. Immunosuppressive agents were initially beneficial on the lung complications, but the patients later showed no response to therapy, and all died from respiratory failure 7-195 days (median, 28 days) after the development of ME and BP. The results suggest that these complications progress rapidly, are resistant to treatment, and have a poor prognosis. It is therefore important to start prophylaxis and treatment as early as possible.

Cytokine gene expression by concanavalin A-stimulated peripheral mononuclear cells after bone marrow transplantation: an indicator of immunological abnormality due to chronic graft-versus-host disease.

The response of IFN-gamma, IL-2 and IL-5 mRNA expression to the stimulation of concanavalin A (Con A) in peripheral blood mononuclear cells (PBMC) after bone marrow transplantation (BMT) was analyzed using reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the recovery of T cell function. The subjects were 23 patients undergoing allogeneic BMT, 1 syngeneic BMT, 1 autologous BMT and 2 normal individuals. IFN-gamma mRNA expression increased after Con A stimulation in 6 patients who had limited chronic graft versus host disease (GVHD), 14 patients who did not have chronic GVHD, each one patient receiving syngeneic and autologous BMT and 2 normal individuals. On the other hand, IFN-gamma mRNA expression was not increased by Con A stimulation in 4 patients who had extensive chronic GVHD. Also, the concentration of IFN-gamma in cultured medium in a patient with extensive chronic GVHD was not detectable. A similar low response of IL-2 and IL-5 mRNA expression to Con A was observed in these patients with extensive chronic GVHD. These findings indicate that the cytokine productive capacity of T cell (IFN-gamma and IL-2 could be produced by type 1 T helper (Th1) cells and IL-5 could be produced by type 2 T helper (Th2) cells) was suppressed in patients who had extensive chronic GVHD, while that capacity was almost normal in patients without chronic GVHD and with limited chronic GVHD. Therefore, the analysis of cytokine gene response to Con A stimulation may provide useful information regarding immune reconstitution after BMT.

15-Deoxyspergualin controls cyclosporin- and steroid-resistant intestinal acute graft-versus-host disease after allogeneic bone marrow transplantation.

We report a case of cyclosporin- and methylprednisolone-resistant intestinal acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation which responded to a new immunosuppressant, 15-deoxyspergualin (DSG). Endoscopy showed lymphoid hyperplasia with CD3+, CD4+, CD8- lymphocyte infiltration into the submucosa of the jejunum and colon. DSG effectively suppressed this intestinal acute GVHD.

Successful treatment of meningoencephalitis caused by methicillin-resistant Staphylococcus aureus with intrathecal vancomycin in an allogeneic peripheral blood stem cell transplant recipient.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common infectious pathogen during stem cell trans-plantation. We report a case of meningoencephalitis with multiple abscess formation caused by MRSA, which occurred in a 4-year-old boy soon after allogeneic peripheral blood stem cell transplantation. We successfully cured the infection with a combination of intravenous and intrathecal vancomycin.

The important balance between cytokines derived from type 1 and type 2 helper T cells in the control of graft-versus-host disease.

We have investigated cytokine mRNA expression in the peripheral blood mononuclear cells of 20 patients who received allogeneic hematopoietic stem cell transplants to assess the cytokine network after transplantation. IL-4 mRNA expression decreased in five of five (100%) patients with > or = grade III (severe) acute GVHD and increased in 10 of 22 (45%) patients without severe GVHD. In contrast, IL-12 mRNA expression increased in two of two (100%) patients with severe GVHD, but increased in only six of 18 (33%) patients without severe GVHD. Furthermore, IL-10 and/or IL-13 mRNA expression increased in 19 of 22 (86%) patients without severe GVHD, but increased in only one of three (33%) patients with severe GVHD. In patients with allogeneic PBSCT who had severe acute GVHD, the cytokine mRNA expression in patients with allogeneic PBSCT, who had no severe GVHD, showed a similar pattern to that in patients with allogeneic BMT. IL-4 mRNA expression increased in three of five (60%) patients and IL-10 and/or IL-13 mRNA expression increased in five of five (100%) patients. In contrast, IL-12 mRNA expression increased in only one of three (33%) patients. Serum IL-4 concentration in allogeneic PBSCT patients in the early engraftment phase was relatively high, while serum IL-12 concentration was low. These findings suggest that severe GVHD may be related to the cytokine imbalance between type 1 helper T (Th1) cells and type 2 helper T (Th2) cells.

Prophylaxis of superficial bladder cancer with instillation of adriamycin or mitomycin C.

A multicenter trial for postoperative prophylaxis of superficial Ta-T1, G1-G2 bladder cancer was performed. Intravesical instillation using either 20-30 mg adriamycin or 20 mg mitomycin C per dose was carried out for 4 weeks or 2 years. Patients without instillation served as controls. A total of 259 patients was considered eligible for the evaluation. The instillation group showed a better disease free survival rate than the control group. Better prophylactic effects of instillation therapy were observed when one of following factors was present: multiple tumors, large tumors, T1 and G2 bladder cancer. The total dose of drug instilled seemed to correlate with the effects, but there were no differences between adriamycin and mitomycin C. The side effects were minimal and temporary.

Krypton-81m single photon emission tomography and the collateral circulation in carotid occlusion: the role of the circle of Willis and leptomeningeal anastomosis.

Cerebral perfusion images were investigated in patients with carotid artery occlusion, using single photon emission computed tomography with the infusion of krypton-81m into the internal, common carotid and vertebral arteries. The contribution of the circle of Willis and cerebral cortical anastomoses to the maintenance of adequate blood supply into the involved hemisphere was analysed. It was concluded that the cerebral perfusion image is superior to angiography in evaluating collateral circulation, and in the case of carotid occlusion, the circle of Willis is important in preventing infarction in the territory of the perforating arteries, while the cerebral cortex mainly receives its blood supply through the cortical leptomeningeal anastomoses, illustrating the major role of the leptomeningeal anastomosis as a collateral channel.

Antitumor effect in cyclosporine A- or FK506-treated mice after syngeneic bone marrow transplantation.

We investigated the possibility for induction of graft-versus-tumor (GVT) effects in cyclosporine A (CsA)- or FK506 (FK)-treated DBA/2 mice after syngeneic bone marrow transplantation (BMT). For in vitro assays of spleen cells, the CsA-treated mice had more enhanced cytotoxic activity against YAC1 and P388, while the FK-treated animals had more against P815, YAC1, and P388. IL-4 mRNA expression was detected in spleen cells of the FK-treated mice and IL-6 mRNA expression was clearly detected in both the treated groups. Concerning GVT effects, FK had more pronounced immunostimulatory potential than CsA in this experimental setting using DBA/2 mice. In tumor-loading in vivo experiments, we could not show any antitumor effect on survival. However, this immunostimulation could be expected to eradicate the minimal residual disease after autologous BMT and autologous peripheral blood stem cell transplantation.

Cytokine receptor gene expression in peripheral blood mononuclear cells during graft-versus-host disease after allogeneic bone marrow transplantation.

Interleukin-1 receptor type 1 (IL-1R), IL-2 receptor alpha subunit (IL-2R) and IL-6 receptor alpha subunit (IL-6R) mRNA expression in peripheral blood mononuclear cells (PBMC) in 17 patients who underwent allogeneic bone marrow transplantation (allo BMT) and 2 patients who underwent autologous transplantation were analyzed using a semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR). There were several exceptions in some cases and IL-1R expression was found to vary in a rather wide range, however, the expression of IL-2R and IL-6R mRNA tended to increase during the development of graft-versus-host disease (GVHD). In particular, IL-2R mRNA expression was increased in four patients with GVHD and graft failure. In contrast, IL-2R and IL-6R mRNA expression was not increased in autologous (auto) BMT and auto peripheral blood stem cell transplantation (PBSCT) patients. These findings suggest that IL-2R and maybe IL-6R mRNA expression in PBMC play an important role in the development of an allo response and GVHD. Therefore, the analysis of cytokine receptor mRNA expression in PBMC after allo BMT may provide important information concerning the immune response and the cytokine network system in marrow transplants.

Establishment and characterization of a new Ph1-positive ALL cell line (ALL/MIK) presenting bcr gene rearrangement on bcr-2 and ALL-type bcr/abl transcript: suggestion of in vitro differentiation to monocytoid lineage.

A new Ph1-positive acute lymphoblastic leukemia cell line, designated as ALL/MIK, has been developed from a patient with Ph1-positive acute leukemia. The ALL/MIK cells showed an immunophenotype of common ALL with rearranged JH and Jk genes. The ALL/MIK cells showed no M-bcr rearrangement using Southern blot analysis with either 3' or 5' M-bcr probes, but had the bcr gene rearrangement on bcr-2 within the first intron of the bcr gene. Consistent with this result, the reverse transcriptase-dependent polymerase chain reaction (RT-PCR) assay revealed that the ALL/MIK cells contained the transcript derived fusion of the first exon of bcr gene and the second exon of abl gene. Although the ALL/MIK cells were defined as early pre-B cells by immunophenotypical and genotypical analyses, they were capable of differentiating into monocytoid lineage by when cultured with TPA. Furthermore, another Ph1-positive ALL cell line, (TOM-1), was investigated for its ability to differentiate to monocytoid lineage. TOM-1 was also induced to monocytoid lineage by TPA. Thus, the present study suggested that the leukemic transformation in some Ph1-positive ALL may occur at the level of multipotential hematopoietic cells capable of differentiating towards lymphoid and myelo-monocytoid lineage.

A translocation t(8;14) and c-myc gene rearrangement associated with the histological transformation of B-cell acute lymphocytic leukemia (FAB-L2) into Burkitt's type (FAB-L3) leukemia.

We report a case of B-cell acute lymphocytic leukemia which showed histological transformation from an FAB-L2 into a Burkitt's type (FAB-L3). Both leukemias had identical immunoglobulin heavy-chain joining gene and kappa light-chain joining gene rearrangements, indicating the clonal identity of the two leukemias. A chromosomal analysis of leukemia cells on the onset indicated normal karyotype, whereas that of the transformed FAB-L3 showed t(8;14)(q24;q32). Furthermore, the proto-oncogene c-myc was in the germline configuration in the initial leukemia but in the rearranged configuration after transformation. Presence of t(8;14)(q24;q32) and the c-myc gene rearrangement after transformation suggested that the chromosomal translocation followed by the activation of the c-myc proto-oncogene might be involved in the Burkitt's type transformation of the FAB-L2 leukemic clone, but not in the leukemogenesis of the initial FAB-L2 leukemia.