A 3.0-kb deletion including an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene in an individual with the Bm phenotype.

We developed a sequence-specific primer PCR (SSP-PCR) for detection of a 5.8-kb deletion (B(m) 5.8) involving an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene. Using this SSP-PCR, we performed genetic analysis of 382 individuals with Bm or ABm. The 5.8-kb deletion was found in 380 individuals, and disruption of the GATA motif in the regulatory element was found in one individual. Furthermore, a novel 3.0-kb deletion involving the element (B(m) 3.0) was demonstrated in the remaining individual. Comparisons of single-nucleotide polymorphisms and microsatellites in intron 1 between B(m) 5.8 and B(m) 3.0 suggested that these deletions occurred independently.

Blood group B gene is barely expressed in in vitro erythroid culture of Bm-derived CD34+ cells without an erythroid cell-specific regulatory element.

BACKGROUND AND OBJECTIVES: Previously, a weak phenotype Am or Bm was assumed to be caused by a reduction of A or B gene expression in bone marrow cells, but not in mucus-secreting cells. However, ABO expression has not been examined in erythroid progenitor cells of Am or Bm individuals. MATERIALS AND METHODS: We carried out in vitro erythroid differentiation of CD34(+) cells from peripheral blood of a Bm individual harbouring a 3.0-kb deletion including an erythroid cell-specific regulatory element, named the +5.8-kb site, in intron 1 of the human ABO blood group gene. RESULTS: During the in vitro differentiation of CD34(+) cells from this Bm individual into erythroid cells, B-antigens were not detectable on the cultured cells by flow cytometric analysis, and allele-specific RT-PCR consistently detected the transcripts from the O allele, but not from the B allele. Moreover, chromatin immunoprecipitation assay demonstrated that both RUNX1 and GATA-2 or GATA-1 were bound to the +5.8-kb site in cultured erythroid cells expressing ABO. CONCLUSION: It is likely that the +5.8-kb site enhances transcription from the ABO promoter in erythroid cells through binding of RUNX1 and GATA-2 or GATA-1.

Cytogenetic and clonal culture evaluation after response to low dose Ara-C in myelodysplastic syndromes.

Cytogenetic and bone marrow culture studies were performed sequentially in 13 patients with myelodysplastic syndromes (MDS) who responded to low dose cytosine arabinoside (Ara-C) treatment (complete in nine and partial in four patients). Of nine patients with initial clonal karyotypic abnormalities, six recovered a normal karyotype after attaining a response to treatment, but the other three patients retained partial or total karyotypic abnormalities. A new clonal karyotypic abnormality appeared after treatment in one patient. Eight patients showed normal colony growth of both granulocyte-macrophage colony-forming units and erythroid burst-forming units after treatment, but five were still defective. There was a clear difference in the duration of response to treatment between these two groups. Consolidation treatment was not effective in patients with persistent karyotypic abnormalities or defective colony formation. Although the number of patients studied is small, these results suggest that hemopoiesis in patients with MDS following a response to treatment with low dose Ara-C is heterogeneous. Consolidation chemotherapy is recommended to ensure and prolong the response in patients showing normalization of both cytogenetic and bone marrow culture results.

Disseminated coccidioidomycosis.

We report a case of disseminated coccidioidomycosis in a 39-year-old Japanese male whose illness developed after returning from a trip to an endemic area. He showed positive coccidioidin skin reaction throughout the entire course of his illness. The primary lesion in the lung subsequently spread to the bone. While the patient was on treatment with 5-FC, he made another trip to the same endemic area. After this episode, he developed pulmonary symptoms and cutaneous nodules on his wrist. The possibility of reinfection with Coccidioides immitis is discussed. Electron microscopy of the cutaneous nodules revealed that the spherules examined maintained their structural integrity in the granulomatous lesion, suggesting the high viability of the organism. Host-parasitic interaction in coccidioidomycosis is discussed.

[Pharmacokinetics of cefuzonam in peripheral venous serum, uterine arterial serum and intrapelvic female organs].

Concentrations of cefuzonam (CZON) in peripheral venous serum, uterine arterial serum and intrapelvic female organs of 29 women undergone simple total hysterectomy were determined by bioassay, using the cylinder-plate diffusion method. With an intravenous injection of CZON 1 g, the concentration at time 0 (Cp0) of peripheral venous serum and uterine arterial serum were 148.1 micrograms/ml and 155.4 micrograms/ml, respectively. Biological half-lives (T 1/2) of CZON were 1.07 hours in peripheral venous serum and 1.02 hours in uterine arterial serum. Concentrations in peripheral arterial serum were higher than 1.0 micrograms/ml at 4 hours after injection and remained at higher levels than minimal inhibitory concentrations necessary for most Escherichia coli strain for at least 4 hours. Concentrations of CZON in female organs were kept as high as peripheral venous serum, and ratios of CZON concentrations in uterine tube and endometrium to that in peripheral venous serum were 0.74 +/- 0.34 and 0.44 +/- 0.25, respectively. Since CZON is characterized by potent antibacterial activity and broad spectrum, it should be effective for infectious diseases of the female uro-genital tract.

[Pharmacokinetics of aztreonam in peripheral venous serum, uterine arterial serum and intrapelvic female organs].

The concentrations of aztreonam (SQ 26,776, AZT, Squibb) in peripheral venous serum, uterine arterial serum and intrapelvic female organs were determined by bioassay, using the cylinder-plate diffusion method, in 27 women with simple total hysterectomy. With an intravenous injection of AZT 1 g, the maximum levels of peripheral venous serum and uterine arterial serum were 76.39 micrograms/ml and 76.38 micrograms/ml, respectively. Also, the biological half-life (T1/2) was 1.56 hours in peripheral venous serum and 1.54 hours in uterine arterial serum. The concentration in uterine arterial serum was more than 1.8 micrograms/ml at 8 hours after injection and maintained at a high level than the minimal inhibitory concentration necessary for most Gram-negative bacteria for at least 8 hours. The concentrations of AZT in female genital organs were kept higher than the minimal inhibitory concentration against E. coli at 4 hours after injection, and the ratios of the concentrations in uterine tube and endometrium to that in peripheral venous serum were 0.40 +/- 0.18 and 0.30 +/- 0.20, respectively. Since AZT is characterized by more potent antibacterial activity against Gram-negative bacteria and the minimal side effects, intravenous administration of AZT at 1 g or 2 g per day may be an adequate dose for infections of the female urogenital tract.

[Clinical evaluation of cefbuperazone in severe infections complicated with hematological disorders].

The efficacy and the safety of an antibiotic in cephamycin group, cefbuperazone (CBPZ), were investigated in 93 patients with severe infections complicated with hematological disorders. The efficacy evaluation was made in 85 cases with underlying hematological disorders including 49 cases (57.6%) of leukemia and 18 cases (21.2%) of malignant lymphoma. The overall efficacy rate was 50.6% of the 85 evaluable cases. The clinical efficacy rate for sepsis and suspected sepsis was 53.4%. The most frequently used group of antibiotics for combination therapy was aminoglycosides in 37 cases, in which an efficacy rate of 62.2%, a higher rate than the efficacy rate of 48.5% for all the combination therapy cases, was obtained. In 16 cases in which penicillins were used as combination drug, the efficacy rate obtained was low, 31.3%. Efficacy rates obtained for cases with different neutrophil counts at the start of therapies were as follows: 52.2% in 23 cases with neutrophil counts below 100/mm3, 46.2% in 13 cases with neutrophil counts between 100 and 499/mm3 and 51.3% in 39 cases with neutrophil counts equal to or above 500/mm3, thus no significant differences in efficacy rates were observed for patients with different neutrophil counts. These results appear to suggest that CBPZ, alone or in combination with other antibiotic such as aminoglycosides, may be quite useful in the treatment of severe infections in patients with hematological disorders.

[Successful treatment with high dose chemotherapy following peripheral blood stem cell transplantation for relapsed Burkitt's lymphoma with central nervous system involvement].

A 19-year-old man complaining of right upper abdominal pain underwent surgery for the removal of a testicular tumor in October 1997. He was given a diagnosis of Burkitt's lymphoma and was in clinical stage II B. The patient went into completed remission with VABCOP-M combination chemotherapy, but relapsed with involvement of the central nervous system (CNS). He was treated with ICE, then CHASE together with total cranial irradiation and simultaneous intrathecal MTX and cytosine arabinoside through Ommaya reservoir until a second remission was achieved. Afterward, the patient was given high-dose chemotherapy and total body irradiation followed by an autologous peripheral blood stem cell transplant (auto-PBSCT), and maintained complete remission. Though the prognosis for Burkitt's lymphoma with CNS involvement is considered to be poor, high-dose chemotherapy with PBSCT was a successful treatment for relapsed Burkitt's lymphoma in our patient.

A unique monoclonal antibody mNI-11 rapidly enhances spread formation in human umbilical vein endothelial cells.

We previously reported a novel monoclonal antibody (MAb), designated mNI-11, recognizing an adhesion-associated antigen distinct from any previously reported ones. In this article, this adhesion-associated antigen with a molecular weight of about 97 kDa was found to be strongly expressed on human umbilical vein endothelial cells (HUVECs) by fluorescence-activated cell sorter (FACS) analysis. Expression of this antigen on HUVECs was slightly increased in response to the exposure to tumor necrosis factor-alpha (TNF-alpha) or phorbol myristate acetate (PMA). As a biological function exerted by this antigen, it was of great interest that immobilized mNI-11 directly and rapidly enhanced the spread formation of HUVECs, whereas MAbs binding other adhesion-associated antigens such as mNI-58A (anti-CD11a), L130 (anti-CD18), L133.1 (anti-CD31), L178 (anti-CD44), L25.3 (anti-CD49d), or LB-2 (anti-CD54) did not carry such activity under the same conditions. The HUVECs spread formation enhanced by mNI-11 was completely blocked in the presence of a microfilament formation inhibitor, cytochalasin D (CyD), a Ca2+ calmodulin inhibitor, W-7, EDTA, and was partially blocked by a microtubule formation inhibitor, nocodazole, a protein kinase C (PKC) inhibitor, H-7, and a protein synthesis inhibitor, cycloheximide (CHX). However, a protein tyrosine kinase (PTK) inhibitor, genistein, did not affect the spread formation under the same conditions. Taken together, it was suggested that the spread formation of HUVECs enhanced by mNI-11 was mainly associated with the influx of Ca2+ and microfilament reorganization. In addition, the novel property associated with mNI-11 to enhance the spread formation of HUVECs was possibly mediated through its reaction against a unique epitope on HUVECs.

Granulopoiesis-supporting effects of marrow stromal cells in continuous culture from patients with primary myelofibrosis.

The granulopoiesis-supporting effects of marrow adherent cells from seven patients with primary myelofibrosis (PMF) were studied by a continuous allogeneic co-culture system in which the survival of donor granulocyte-macrophage colony-forming cells (CFU-GM) depends upon the supporting ability of adherent cells. Marrow adherent cells from all these seven patients were able to sustain the same number of CFU-GM as those from control subjects. Cytochemical studies showed that colonies grown from cells sustained on fibrous marrow adherent cell layers were predominantly neutrophilic, as were those in control cultures, although many eosinophil colonies grew from patients' bone marrow. These results indicate that marrow stromal cells from PMF patients function normally in their ability to support granulopoiesis.

Wolff-Parkinson-White syndrome: mechanocardiographic study on the mechanical consequences of ventricular pre-excitation.

To elucidate the mechanical consequences of ventricular pre-excitation in patients with the W,W syndrome, electrical and mechanical events in the ventricles during anomalous pathway conduction and normal atrioventricular conduction were examined mechanocardiographically in 11 cases of Group A and 19 cases of Group B, in whom anomalous pathway conduction was stopped by procaine amide, resulting in normalization of conduction. Eight healthy persons were employed as a control group. In the control group, procaine amide had no significant effect on the mechanocardiographic values. In the WPW syndrome, significant prolongation of the P-X, P-J, P-T, P-C, P-I, P-Ao, and P-II intervals was induced by the drug. From the results of statistical analyses of measured values, it would appear that mechanical events in the ventricles were accelerated by ventricular pre-excitation but the extent of acceleration of the former was less than the extent of prematurity of the latter. The anomalous ventricular pre-excitation occurred earlier in cases of Group B than in those of Group A, while initiation of ventricular contraction, atrioventricular valve closure, and aortic vlave opening were accelerated more in Group A. In one case of Group B, electrical phenomena could not be related to mechanical events.

Functional changes in marrow stromal cells in aplastic anaemia.

To determine whether or not abnormalities exist in the bone marrow stroma in aplastic anaemia, we analysed the ability of marrow stromal cells to support haemopoiesis using a long-term culture system. Marrow stromal cell layers from 3 of 9 patients with this disorder failed to maintain granulocyte-macrophage colony-forming cells in vitro. The stromal dysfunction was reversible in 1 patient who recovered after androgen therapy. The results of the present study add to the available evidence for a functional defect of marrow microenvironments in some cases of aplastic anaemia.

Granulocytic and stromal progenitors in the bone marrow of patients with primary myelofibrosis.

Quantitative and qualitative changes in granulocyte-macrophage (CFU-GM) and fibroblast colony-forming cells (CFU-F) were studied in 7 patients with primary myelofibrosis (MF). Marrow cells were collected from bone biopsy specimens after treatment with collagenase. The number of CFU-GM correlated with the amount of haemopoietic tissue noted in the bone marrow histology and ranged between 0-400/mg of bone. CFU-F were increased in 2 patients with moderate fibrosis. Circulating CFU-GM were increased in all patients studied (169-3749/ml of blood). There was no significant correlation between the number of CFU-GM in the bone marrow and that in the blood. Cytochemical studies showed a high incidence in eosinophil progenitors in the bone marrow and especially in the blood of patients with MF. These data suggest a functional abnormality of myeloid progenitors in this disease.