Relationship between the Peripheral Lymphocyte Response to Mycophenolic Acid in vitro and the Level of ATP in Peripheral CD4+ Lymphocytes before and after Renal Transplantation.

OBJECTIVE: The lymphocyte immunosuppressant sensitivity test has been used to predict the pharmacodynamics of immunosuppressive drugs for the purpose of preventing acute rejection and infection after renal transplantation. On the other hand, measuring the ATP levels in peripheral CD4+ lymphocytes is also able to monitor the risks of rejection and infection in transplant recipients. In the present study, we examined the relationship between the mycophenolic acid pharmacodynamics and the ATP levels in peripheral lymphocytes before and after renal transplantation. METHODS: We examined both the pharmacological efficacy of mycophenolic acid and the lymphocyte ATP levels before and 2, 4 and 6 weeks after the operation in 20 renal transplant recipients. The drug's pharmacological efficacy was evaluated by the 50% inhibitory concentration of the drug against the in vitro proliferation of peripheral blood lymphocytes activated by T cell mitogen. The ATP levels in peripheral CD4+ lymphocytes were measured by the Immuknow assay kit. The relationships between the mycophenolic acid pharmacodynamics and ATP levels in peripheral lymphocytes were examined in these recipients. RESULTS: The immunosuppressive effects of mycophenolic acid against mitogen-activated lymphocyte proliferation were significantly and positively correlated with the lymphocyte ATP levels, but only at 6 weeks after transplantation. The relationship was not significant before or at 2 or 4 weeks after the operation. CONCLUSION: Our present data raised the possibility that evaluating the pharmacological efficacy of mycophenolic acid pre-transplantation and monitoring the ATP level 6 weeks after transplantation can predict the risk of rejection and/or infection in renal transplant recipients.

Correlation between the pharmacological efficacy of cyclosporine and tacrolimus as evaluated by the lymphocyte immunosuppressant sensitivity test (LIST) and the MTT assay procedure in patients before and after renal transplantation.

OBJECTIVES: Cyclosporine and tacrolimus are calcineurin inhibitors that are used to prevent acute rejection in renal transplant recipients. The lymphocyte immunosuppressant sensitivity test (LIST) can predict the pharmacological efficacy of these immunosuppressive agents for renal transplant recipients. There is a correlation between cyclosporine and tacrolimus pharmacological efficacy as evaluated by LIST by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay procedure prior to renal transplantation. However, the LIST can also evaluate patients before and after the transplantation. MATERIALS AND METHODS: The present study examined the relationship between cyclosporine and tacrolimus pharmacological efficacy by LIST using the MTT assay in 16 renal transplant recipients at 1, 3 and 12 months after transplantation, as well as before the operation. RESULTS: The relationship of cyclosporine and tacrolimus pharmacological efficacy gave a significant Kendall and Spearman's coefficient correlation in these transplant recipients by the LIST using the MTT assay procedure immediately prior to renal transplantation (rk = 0.711, rs = 0.877, p < 0.01). Furthermore, correlations between the cyclosporine and tacrolimus IC50 values were also observed with a significant Kendall and Spearman's coefficient correlation at 1 and 12 months after transplantation (rk1month = 0.65, rs1month = 0.829, p < 0.01, and k12month = 0.433, rs12month = 0.603, p < 0.01, respectively). However, no statistically significant relationship was observed between the pharmacological efficacies of the calcineurin inhibitors at 3 months after transplantation (rk3month = 0.117, rs3month = 0.1, p > 0.05). CONCLUSIONS: Both cyclosporine and tacrolimus exhibit pharmacological efficacy by the inhibition of calcineurin. However, the correlation between cyclosporine and tacrolimus pharmacological efficacies may be altered, due to immunosuppressive therapy or clinical events at 3 months after renal transplantation.

Cyclosporine pharmacological efficacy estimated by lymphocyte immunosuppressant sensitivity test before and after renal transplantation.

OBJECTIVE: Lymphocyte immunosuppressant sensitivity test (LIST) is useful for predicting the pharmacological efficacy of immunosuppressive agents. In this study, the pharmacological efficacy of cyclosporine was estimated by LIST before and after renal transplantation. METHODS: Lymphocyte immunosuppressant sensitivity test was performed by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay before and at 1, 3, and 12 months after transplantation in 19 consecutive renal transplant recipients. RESULTS: There was wide intersubject variability in cyclosporine IC50 before transplantation [Mean (SD) of 593.9 (1067.6) ng/mL]. This variability worsened 1 month after transplantation [525.7 (1532.7) ng/mL] but decreased at 3 months (193.5 (347.9) ng/mL) and 12 months (75.4 (95.4) ng/mL). In this small study, observed differences in IC50 values for the individual subjects at various time intervals was not associated with the occurrence of rejection, graft loss, and infection episodes. CONCLUSION: Lymphocyte sensitivity to cyclosporine assessed by the LIST assay showed a high level of inter-subject variability particularly before and 1 month after transplantation. The observed difference in IC50 values was not associated with clinical outcome in this small study.

Heparan sulfate levels in mucopolysaccharidoses and mucolipidoses.

Glycosaminoglycans are accumulated in both mucopolysaccharidoses (MPS) and mucolipidoses (ML). MPS I, II, III and VII and ML II and ML III patients cannot properly degrade heparan sulphate (HS). In spite of the importance of HS storage in the metabolic pathway in these diseases, blood and urine HS levels have not been determined systematically using a simple and economical method. Using a new ELISA method using anti-HS antibodies, HS concentrations in blood and urine were determined in MPS and ML II and ML III patients. HS concentrations were determined in 156 plasma samples from MPS I (n = 23), MPS II (n = 26), MPS III (n = 24), MPS IV (n = 62), MPS VI (n = 5), MPS VII (n = 5), ML II (n = 8) and ML III (n = 3), and 205 urine samples from MPS I (n = 33), MPS II (n = 33), MPS III (n = 30), MPS IV (n = 82), MPS VI (n = 7), MPS VII (n = 9), ML II (n = 8) and ML III (n = 3). The ELISA method used monoclonal antibodies against HS. MPS I, II, III and VII and ML II and III patients had significant elevation in plasma HS, compared to the age-matched controls (p < 0.0001). Eighty-three out of 89 (93.3%) of individual values in the above MPS types and ML were above the mean +2SD of the controls. In urine samples, 75% of individual values in patients with those types were above the mean +2SD of the controls. In contrast to the previous understanding of the HS metabolic pathway, plasma HS levels in all five MPS VI and 15% of MPS IV patients were elevated above the mean +2SD of the controls. These findings suggest that HS concentration determined by ELISA, especially in plasma, could be a helpful marker for detection of the most severe MPS I, II, III, VI and VII and ML II, distinguishing them from normal populations.

Keratan sulphate levels in mucopolysaccharidoses and mucolipidoses.

The mucopolysaccharidoses (MPS) is characterized by accumulation of glycosaminoglycans (GAGs), and mucolipidosis (ML) by accumulation of GAGs and sphingolipids. Each type of MPS accumulates specific GAGs. The lysosomal enzymes N-acetylgalactosamine-6-sulphate sulphatase and beta-galactosidase involve the stepwise degradation of keratan sulphate (KS). Deficiency of these enzymes results in elevation of KS levels in the body fluids and in tissues, leading to MPS IV disease. In this study, we evaluated blood and urine KS levels in types of MPS and ML other than MPS IV. Eighty-five plasma samples came from MPS I (n = 18), MPS II (n = 28), MPS III (n = 20), MPS VI (n = 3), MPS VII (n = 5) and ML (n = 11) patients while 127 urine samples came from MPS I (n = 34), MPS II (n = 34), MPS III (n = 32), MPS VI (n = 7), MPS VII (n = 9) and ML (n = 11) patients. KS levels were determined using the ELISA method. Plasma KS levels varied with age in both control and patient populations. In all age groups, the mean values of plasma KS in MPS and ML patients were significantly higher than those in the age-matched controls. Plasma KS values in four newborn patients were above the mean + 2SD of the age-matched controls (mean, 41 ng/ml). Overall, 85.9% of individual values in non-type IV MPS and ML patients were above the mean + 2SD of the age-matched controls. For urine KS levels, 24.4% of individual values in patients were above the mean + 2SD of the age-matched controls. In conclusion, KS in blood is elevated in each type of non-type IV MPS examined, in contrast to the conventional understanding. This finding suggests that measurement of KS level provides a new diagnostic biomarker in a wide variety of mucopolysaccharidoses and mucolipidoses in addition to MPS IV.

Reconstructing the history of 14C discharges from Sellafield: part 1--atmospheric discharges.

14C specific activities, above ambient background levels, were determined in individual treerings (corresponding to the years 1950-1999) sectioned from an oak tree that was felled in autumn 1999, from a location 1.5 km east of the Sellafield nuclear fuel reprocessing plant in Cumbria. north-west England. The data were used to produce a new, improved, reconstruction of Sellafield's annual atmospheric 14C discharges between 1951 and 1999, using the most reliable discharge data set (1994-1999) as the primary basis for the determination of a new calibration factor that relates excess 14C activity in individual tree rings to the annual discharge during the corresponding year. The results indicate that the current British Nuclear Fuels plc (BNFL) estimate of total 14C discharges to the atmosphere prior to 1978 is significantly overestimated, while the current estimate of total 14C discharges after 1978 is very similar to that determined in this study. In this study, the total activity of 14C discharged to the atmosphere from Sellafield between 1951 and 1999 is estimated to be 259+/-63 TBq (at 2 std. dev.). The BNFL current estimate is 360TBq.

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.

Evaluation of accumulated mucopolysaccharides in the brain of patients with mucopolysaccharidoses by (1)H-magnetic resonance spectroscopy before and after bone marrow transplantation.

In seven patients with mucopolysaccharidoses (1 Hurler, 1 Hurler-Scheie, 4 Hunter, 1 Sly), cranial (1)H-magnetic resonance spectroscopy was performed to evaluate the accumulation of mucopolysaccharides and biochemical changes in the CNS in vivo before and after bone marrow transplantation (BMT). In two of seven patients, (1)H-magnetic resonance spectroscopy was performed before and after BMT. Nuclear magnetic resonance spectra of dermatan sulfate and chondroitin sulfate-C and magnetic resonance spectroscopy of chondroitin sulfate-C and urine from patients with mucopolysaccharidoses showed resonance higher than the chemical shift of myoinositol in the brain (3.7 ppm). The resonance was considered to contain signals from mucopolysaccharide molecules. The resonance was measured as presumptive mucopolysaccharides (pMPS). In white matter lesions detected by magnetic resonance imaging, pMPS/creatine ratios and choline/creatine ratios were consistently higher than control ratios. In white matter without lesions, choline/creatine ratios were higher than control ratios. Patients with higher developmental quotient or intelligence quotient tended to show higher N:-acetylaspartate/creatine ratios and lower pMPS/creatine ratios in basal ganglia. After BMT, the pMPS/creatine ratio in white matter lesions of patient 3, with Hunter syndrome, was slightly decreased, but in none of the patients was the ratio ever below the control ratios, even 7 y after BMT. In white matter without lesions, the pMPS/creatine ratio in patient 3 was decreased to the control ratios after BMT, but although the choline/creatine ratios were gradually decreased, they remained higher than the control ratio, 2 y after BMT. These results suggest that evaluation of pMPS, choline, and N:-acetylaspartate by (1)H-magnetic resonance spectroscopy is an important technique that may provide useful biochemical information in vivo on the neurologic process and the efficacy of BMT in patients with mucopolysaccharidoses.

Mixed connective tissue disease following interstitial cystitis.

A 64-year-old woman complained of severe infrapubic pain and pollakisuria with nausea, vomiting and diarrhea, but with normal urinalysis since 1987. The clinical diagnosis of interstitial cystitis (IC) was made, and cystectomy was performed in 1996. The bladder taken was markedly shrunken with a capacity of 50 ml, and showed bleeding on the mucosal surface. Histological findings of the bladder showed ulcer formation in the mucous membrane, and marked infiltration of mononuclear cells, edema and fibrosis in the submucosal tissue. She had noticed exudative erythema, swelling and sclerosis on the bilateral fingers and dorsal aspects of the hands since 1993, and Raynaud's phenomenon and morning stiffness of the fingers from November, 1998. Laboratory data showed positive anti-nuclear antibody (titer: 1: 2,560) and anti-U1 RNP antibody (titer: 69.5 by ELISA). A diagnosis of mixed connective tissue disease following IC was made.

[Susceptibility testing of clinical strains of Legionella spp. isolated in Japan].

We determined antimicrobial susceptibilities of 23 strains of Legionella spp. clinically isolated in Japan, between September 1994 through November 1999 by using Etest. The MICs of all isolates against macrorides, fluoroquinolones and rifampicin showed 1 microgram/ml or less. Especially rifampicin showed the most potent activity, of which the MICs of all isolates showed 0.064 microgram/ml or less. Although we could not find resistant strains against any antibiotics tested in this study, we think it is important to follow up the susceptibilities of clinical isolates of Legionella.

Bone marrow transplantation for the treatment of X-linked adrenoleukodystrophy.

The effects of bone marrow transplantation (BMT) in X-linked childhood adrenoleukodystrophy (ALD) are described in four Japanese patients. Two older patients, 10-year-old boys with IQ 60 and difficulty in school, had favourable results. IQ levels and MRI findings were generally maintained after BMT. One patient showed improvement of gait disturbance. They have both attended ordinary schools after BMT, although a learning disorder persists. On the other hand, two other younger patients with a rapid course and indeterminate IQ at BMT showed deterioration of neurological functions. Indication for BMT seems to be a maintained IQ level, preferably higher than 80, since it seems to be difficult to normalize IQ level after BMT. Younger patients have higher risk of developing a rapidly progressive form of the disease. Identification of presymptomatic boys, and serial and careful follow-up by neuropsychological and neuroradiological studies, are essential prerequisites to successful BMT in X-ALD.

The mouse N-acetylgalactosamine-6-sulfate sulfatase (Galns) gene: cDNA isolation, genomic characterization, chromosomal assignment and analysis of the 5'-flanking region.

Deficiency of lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) leads to mucopolysaccharidosis IV A (MPS IV A), for which there is no definitive treatment so far. Although a number of mutations of the GALNS gene of MPS IV A patients have been described, pathogenesis of the disorder still remains elusive. In order to facilitate in vivo studies using model animals for MPS IV A, we isolated and performed molecular characterization of the mouse homolog of human GALNS. The 2.3-kb cDNA contains a 1560-bp open reading frame encoding 520 amino acid residues. The coding region has 84% similarity to the human GALNS cDNA at amino acid level. The mouse Galns gene was mapped by interspecific backcross analysis to the distal region of chromosome 8 where it co-segregates with Aprt. Northern blot analysis showed a wide expression of a single-copy gene, being higher especially in liver and kidney. The Galns gene was isolated from S129vJ genomic library and its genomic organization was characterized. The mouse Galns gene was about 50-kb long and organized into 14 exons and 13 introns. All intron-exon splice junctions conformed to the GT/AG consensus sequence except exon 8/intron 8 junction. Primer extension shows multiple transcription initiation sites between -44 and -75 although major transcription initiation site was observed at -90 bp from the ATG codon. The 5'-flanking region lacks canonical TATA and CAAT box sequences, but is G+C rich with 10 GC boxes (potential Sp1 binding sites), characteristic of a housekeeping gene promoter.

Ataxia telangiectasia associated with B-cell lymphoma: the effect of a half-dose of the drugs administered according to the acute lymphoblastic leukemia standard risk protocol.

Ataxia telangiectasia (A-T) is a rare autosomal recessive disorder characterized by cerebellar ataxia, oculocutaneous telangiectasia, and variable degrees of humoral and cellular immunodeficiency. Affected individuals are known to exhibit a high incidence of lymphoma and leukemia. Because of increased chemosensitivity, the treatment of A-T patients with malignancies requires extremely careful planning and caution with respect to the use of chemotherapy. The authors report on a 12-year-old boy with A-T who developed B-cell lymphoma. He received a half-dose of the drugs administered according to the acute lymphoblastic leukemia (ALL) protocol issued by our children's cancer study group (9104 Standard Risk Protocol, Tokai Pediatric Oncology Study Group). As a result, he continues to be in complete remission and free of treatment complications 32 months after the diagnosis of B-cell lymphoma.

Organically-bound 3H concentration in rice around atomic energy facilities.

Rice samples collected around atomic energy facilities in the Tokai area were analyzed for organically-bound 3H (OBT). Measurement results were compared with those for samples from control areas in Japan and the People's Republic of China. OBT concentrations of rice in the Tokai area were found to be 4 times higher than the control area as maximum. Although this relatively high 3H concentration shows the effect of 3H release from the facilities into the environment, the concentration level is comparable with those in the rice sample from a northern interior region of China. The OBT concentration of rice sample from the general environment in interior China was 4.5 times higher than the control area in Japan. The radiation dose from the increasing 3H level is negligible in comparison with that from natural radiation.

Mutation analysis in the iduronate-2-sulphatase gene in 43 Japanese patients with mucopolysaccharidosis type II (Hunter disease).

Our series of studies on Hunter disease in Japanese patients showed allelic heterogeneity of IDS gene mutations, genotype/phenotype correlation and racial differences in distribution of mutations. Twenty-five different small mutations have been characterized. Small mutations in the Japanese population are widely distributed through the IDS gene, although some mutations were unevenly concentrated on exon 5 (28%) and on exon 9 (24%). Mutations were seen at the same codon 468 in exon 9 in 5 patients. These findings are in good agreement with data on other ethnic groups. Two unique mutations linked to a severe phenotype were apparently associated with aberrant splicings; one was a point mutation within exon 3 (P86L), partially activating a cryptic splice acceptor site at 28 bp downstream from the mutation site within exon 3 and producing a 44-base truncated mRNA, and the other was a point mutation at the consensus sequence of the splice donor site of intron 2, causing exon 2 skipping.

[Clinical efficacy of post-TUR prophylactic chemotherapy for superficial bladder cancer--the result of co-operative prospective randomized trial].

We have performed a prospective randomized clinical trial of post-operative prophylactic therapy for superficial bladder cancer since October in 1991. The criteria were as follows; age < or = 80 y.o., Ta/T1, TCC G1/G2, without CIS lesion and resectable cancer by TUR. The therapeutic arm was divided into three as follows: arm A intracystic instillation with Epirubicin; arm B: oral administration with 5-FU; arm C: combination of arm A and arm B. The number of registered patients were 20 in arm A, 18 in arm B, and 18 in arm C. The patient characteristics in every group were not significantly different. The prophylactic efficacy of arm A was superior to that in the other two groups.

[Clinical investigation of prostatic cancer patients who underwent radical prostatectomy: analysis of patients in the Tokai Urological Cancer Registry].

Between 1989 and 1991, 815 cases of prostatic carcinoma were registered in the Tokai Urological Cancer Registry. We investigated the clinical features and prognosis of 69 patients who underwent radical prostatectomy. The median age and clinical stage, were 65.9 +/- 4.7 years and A in 7 patients, 66.3 +/- 5.8 years and B in 31, 68.2 +/- 2.7 years and C in 26, 63.4 +/- 13.5 years and N(+) in 5. Neoadjuvant therapy consisting of endocrine therapy and chemoendocrine therapy was administered to 28 and 14 patients, respectively. In comparison to the clinical stage, pathologically, 4 cases (9.5%) showed downstaging or overstaging, 15 (35.7%) understaging and 23 (54.8%) accurate staging in the neoadjuvant therapy group, and, respectively, 2 (7.4%), 12 (44.4%) and 13 (48.2%) in the non-neoadjuvant therapy group. There was a significant difference in the pretreatment prostatic antigen (PA) value between pT0 and pT3 (p < 0.05), between pT0 and pN(+) (p < 0.01) and between pT2 and pN(+) (p < 0.05). In addition, the pretreatment PA value in the recurrence group was higher than that in the non-recurrence group in both neoadjuvant and non-neoadjuvant groups, with a significant difference (p < 0.01) in the neoadjuvant group. Cancer recurrence was noticed in 2 patients given non-neoadjuvant therapy and 3 given neoadjuvant therapy. There was no significant difference in the non-recurrence rate with the pathological stage either with or without neoadjuvant therapy. However, the non-recurrence rate in the neoadjuvant therapy group was higher than that in the non-neoadjuvant therapy group.