Impact of prestorage leucoreduction of autologous whole blood on length of hospital stay with a subgroup analysis in bilateral hip arthroplasty.

BACKGROUND: Although prestorage leucoreduction (LR) of blood components for transfusion has gained favour around the world, evidence of its beneficial clinical effects is ambiguous. STUDY DESIGN AND METHODS: To reveal whether leucocytes and/or platelets in transfused blood are related to transfusion-related adverse effects, a prospective randomized crossover study was performed on patients who donated autologous blood prior to elective surgery. Among 1487 primary enrolees, a total of 192 patients undergoing two-stage, bilateral total hip arthroplasty were randomized to receive autologous blood that was either prestorage leucoreduced, or not, for the first procedure. For the second procedure, each patient was crossed over to receive alternatively processed autologous blood. Length of hospital stay served as a primary end-point, with perioperative infectious/thrombotic complications, pre- and postoperative laboratory values, and body temperature serving as secondary endpoints. RESULTS: No significant differences emerged between prestorage LR and non-LR cohorts in length of hospital stay, as well as perioperative infectious/thrombotic complications, postoperative body temperature and duration of fever. Postoperative laboratory values including white blood cell counts and C-reactive protein levels had no significant differences. CONCLUSION: This study could not prove any superiority of prestorage LR over non-LR for autologous whole blood among patients who underwent total hip arthroplasty.

Polypeptide N-acetylgalactosaminyl transferase 3 independently predicts high-grade tumours and poor prognosis in patients with renal cell carcinomas.

BACKGROUND: The polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) family of enzymes regulates the initial steps of mucin-type O-glycosylation. N-acetylgalactosaminyltransferases might show novel patterns of GalNAc-T glycosylation on tumour-derived proteins, which could influence cancer biology, but its mechanisms are unclear. We investigated the association of GalNAc-T3 and -T6 expressions with clinicopathological features and prognoses of patients with renal cell carcinomas (RCCs). METHODS: Expressions of GalNAc-T3/6 and cell-adhesion molecules were analysed immunohistochemically in 254 paraffin-embedded tumour samples of patients with RCC. RESULTS: Of 138 GalNAc-T3+ cases, 46 revealed significant co-expression with GalNAc-T6. N-acetylgalactosaminyltransferases-3+ expression showed a close relationship to poor clinical performance and large tumour size, or pathologically high Fuhrman's grading, and presence of vascular invasion and necrosis. The GalNAc-T3-positivity potentially suppressed adhesive effects with a significantly low ß-catenin expression. Univariate and multivariate analyses showed the GalNAc-T3+ group, but not the GalNAc-T6+ group, to have significantly worse survival rates. CONCLUSION: N-acetylgalactosaminyltransferases-3 expression independently predicts high-grade tumour and poor prognosis in patients with RCC, and may offer a therapeutic target against RCC.

Significance of pre-storage leucoreduction for autologous blood.

BACKGROUND AND OBJECTIVES: To reveal the associations between cytokines in blood and transfusion-related adverse events, we studied whether pre-storage leucoreduction of autologous blood could reduce the degree of inflammatory responses, infection rates, or the duration of hospitalizations. MATERIALS AND METHODS: Patients scheduled to donate autologous blood for elective orthopaedic surgery were assigned to receive either leucoreduced (LR) or non-leucoreduced (N-LR) autologous blood based on their date of birth. Levels of cytokines in the autologous blood, values for C-reactive protein, complete blood count and body temperature of the patients, as well as adverse clinical events, were evaluated periodically. RESULTS: Four hundred patients entered this study (LR group: 196, N-LR group: 204). The production of cytokines, excluding interleukin 1beta (IL-1beta), was suppressed for the LR group. However, for unknown reasons, IL-1beta actually increased during storage for the LR group. There were no differences between the two groups in the length of hospital stay, postoperative C-reactive protein changes, leucocyte count, or body temperature, and no clinical problems associated with blood transfusion were observed in either group. CONCLUSION: Pre-storage leucoreduction for autologous blood may be effective to suppress cytokine accumulation. However, clinical benefits such as prevention of febrile non-haemolytic reactions could not be demonstrated.

Molecular basis of the Dark Agouti rat drug oxidation polymorphism: importance of CYP2D1 and CYP2D2.

The Dark Agouti rat has been proposed as a model for the human debrisoquine 4-hydroxylase polymorphism. Earlier studies suggested that the poor metabolizer phenotype in the Dark Agouti rat is caused by the absence of the expression of CYP2D1 mRNA. Although CYP2D1 is the major enzyme catalyzing debrisoquine 4-hydroxylation, other reports have indicated the involvement of a CYP2D, purified from rat hepatic microsomes and presumed to be CYP2D2, which also exhibits this activity. The levels of CYP2D1 and CYP2D2 mRNAs were markedly lower in Dark Agouti as compared to Sprague Dawley rats. Using a baculovirus expression system, recombinant CYP2D1 and CYP2D2 from Spodoptera frugiperda insect cells were examined and were found to both forms catalize debrisoquine 4-hydroxylase activity. These results suggest that reduced debrisoquine 4-hydroxylase activity in the Dark Agouti rat is caused by the low level expression not only of CYP2D1, but also of CYP2D2. Interestingly, bunitrolol 4-hydroxylation was catalyzed by recombinant CYP2D2, while CYP2D1 was inactive toward this substrate. Thus, the low bunitrolol 4-hydroxylation in Dark Agouti rats was caused by the low level of CYP2D2 expression in this rat strain.

Duane syndrome and congenital upper-limb anomalies. A familial occurrence.

We report a family in which five members in three generations have been afflicted with Duane syndrome. Four of the five members also have congenital hypoplasia of the thenar eminence. One also was afflicted with Hirschsprung disease and another was born deaf. A sixth member, who does not have Duane syndrome, is afflicted with a more extensive malformation of the upper extremities and unilateral deafness. We present a discussion of Duane syndrome and its association with other congenital anomalies. Although some members of this family presented in this report show features that are similar to the Holt-Oram syndrome, Wildervanck's syndrome, and others reported in the literature, there are several unique features about this family that we thought were worth reporting.

Regio- and stereoselectivity in propranolol metabolism by dog liver microsomes and the expressed dog CYP2D15.

We have studied the regio- and stereoselectivity of ring-hydroxylation and N-desisopropylation of S(-)- and R(+)-propranolol, using dog liver microsomes and the expressed dog CYP2D15 in insect cells. In dog liver microsomes, 4-hydroxylation was the preferred pathway in S(-)-propranolol oxidation, while N-desisopropylation was the preferred pathway in R(+)-propranolol oxidation. S(-)-Propranolol was preferred over R(+)-propranolol as substrate for 4- and 5-hydroxylations, while R(+)-propranolol was the preferred substrate for N-desisopropylation at higher substrate concentrations. The expressed CYP2D15 had high catalytic activities toward 4-, 5-hydroxylation, as well as N-desisopropylation of both enantiomers. At the substrate concentrations used, 4-hydroxylation was the preferred pathway for the metabolism of both enantiomers, and S(-)-propranolol was the preferred substrate over R(+)-propranolol for all three monooxygenations catalyzed by CYP2D15. Anti-CYP2D15 peptide antibody strongly inhibited 4- and 5-hydroxylation of both enantiomers in dog liver microsomes, while it did not inhibit their N-desisopropylation. These findings suggest that CYP2D15 is highly responsible for the stereoselective 4- and 5-hydroxylations of propranolol in dog liver microsomes.

Expression and characterization of dog CYP2D15 using baculovirus expression system.

Dog CYP2D15 was expressed in Sf9 cells with a recombinant baculovirus. Infection of Sf9 insect cells with a recombinant dog CYP2D15-virus resulted in the expression of a protein which cross-reacted with a polyclonal antibody against a dog CYP2D15-specific peptide. The difference spectrum of CO-complex of reduced P450 of the infected cell microsomes had a maximal absorbance at 449 nm. The specific content of P450 was calculated to be 0.56 nmol/mg of Sf9 cell microsomal protein. Although the expressed dog CYP2D15 showed high catalytic activity for the hydroxylations of bunitrolol and imipramine at low substrate concentration (10 microM), the catalytic activity for that of debrisoquine (50 microM) was extremely low as compared with that of CYP2D from other species. Dog liver microsomes also showed bunitrolol and imipramine hydroxylase activities, but not debrisoquine hydroxylase activity at the same substrate concentrations. In addition, the expressed CYP2D showed high catalytic activity for imipramine N-demethylation. Thus, our study reveals that the expressed dog CYP2D15 engages in high catalytic activity and has a unique substrate specificity from other CYP2D subfamilies. Western blot analysis suggested that the dog CYP2D15 contents were less than 4% of the total liver P450 content, assuming that 100% of expressed CYP2D15 incorporated heme.

Quantitative study of local distribution of noncholesterol sterols and cholesterol in gallstones.

Quantitative analysis of the local distribution of four noncholesterol sterols, 24-methylene cholesterol, campesterol, stigmasterol, and beta-sitosterol, and of the local distribution of cholesterol in gallstones was performed by mass spectrometry, with D6-cholesterol as an internal standard. The role played by trace amounts of these four noncholesterol sterols in the formation of gallstones was investigated by comparing the amounts of these sterols in different parts of gallstones. It was found that the amounts of the noncholesterol sterols in the inside part were significant greater than the amounts in the outside part of various structural types of gallstones. However, the distribution of the cholesterol did not show such variation. The amounts of noncholesterol sterols distributed locally suggested that these sterols play a role in the formation of gallstones.

Pharmacological approaches to reduce perioperative transfusion requirements in the aged.

Although iron deficiency is undoubtedly the commonest cause of anaemia even in elderly people, the aetiology is not always clear owing to various underlying diseases. Correction of anaemia is sometimes needed before surgery. The use of drugs that may influence blood coagulation, such as aspirin (acetylsalicylic acid), should be checked. Perioperative allogenic blood transfusion can often be avoided by the use of autologous blood and improved surgical techniques. Autologous blood donations are preferable in cases of planned surgery. Epoetin (recombinant human erythropoietin) in combination with iron supplementation facilitates the donation of autologous blood, even in elderly patients. Another method of avoiding allogenic blood transfusion is the collection and reuse of the blood a patient sheds in operations. During and/or after surgery, many haemostatic agents are available. Moreover, recent developments in gene engineering have enabled the utilisation of recombinant cytokines and coagulation factors. Further work remains to be done to define the proper use of these agents.

Pseudorabies virus (PRV) early protein 0 activates PRV gene transcription in combination with the immediate-early protein IE180 and enhances the infectivity of PRV genomic DNA.

Pseudorabies virus (PRV) early protein 0 (EP0) functions as a transactivator of the viral gene promoters. In transient expression assays employing chloramphenicol acetyl transferase (CAT) reporter constructs, EP0 and the immediate-early protein IE180 act in an additive manner to activate transcription from the thymidine kinase (TK) and glycoprotein G (gG) gene promoters. EP0 enhanced the synthesis of infectious virus in cotransfection experiments with the EP0-expression plasmid and PRV genomic DNA. EP0 was detected by Western blot analysis in the purified virions. These results may indicate that EP0 in the virions acts as an important transactivator to express the immediate-early gene efficiently in the first stage of infection, and IE180 and EP0 expressed after the infection cooperatively activate the early and late gene expression in the later stage of infection.

Inhibition of pseudorabies virus replication by a dominant-negative mutant of early protein 0 expressed in a tetracycline-regulated system.

Pseudorabies virus (PRV) early protein 0 (EP0) consisting of 410 amino acids is a transactivator of viral genes. A mutant consisting of amino acids 1-113 exhibits dominant-negative properties. In order to assess the antiviral potential of the EP0 mutant, Vero cells were transformed with the EP0 mutant gene expressed in a tetracycline-regulated system. The transformed cell lines showed marked resistance to PRV infection when expression of the EP0 mutant gene was induced. In the transformed cell line infected with PRV, synthesis of the immediate-early protein (IE180) and of EP0 was inhibited, whereas the levels of IE and EP0 messenger RNA (mRNA) were not decreased, as compared with those of the control cell line. The present results suggest that the EP0 mutant may not alter the efficiency of the viral gene transcription but rather translation efficiency of the viral mRNA.

Degradation and epimerization kinetics of moxalactam in aqueous solution.

The kinetics of epimerization and degradation of moxalactam in aqueous solution was investigated by HPLC. The pH-rate profiles of the degradation and epimerization were determined separately over the pH range of 1.0-11.5 at 37 degrees C and constant ionic strength 0.5. The degradation and simultaneous epimerization were followed by measuring both of the residual R- and S-epimers of moxalactam and were found to follow pseudo-first-order kinetics. The degradation was subjected to hydrogen ion and hydroxide ion catalyses and influenced by the dissociation of the side chain phenolic group. The epimerization rates were influenced significantly in the acidic region by the dissociation of the side chain carboxylic acid group and in the basic region by hydroxide ion catalysis. The pH-degradation rate profile of moxalactam showed a minimum degradation rate constant between pH 4.0 and 6.0. The pH-epimerization rate profiles of moxalactam showed minimum epimerization rate constants at pH 7.0. The epimerization rate constants of the R- and S-epimers were not very different.

Improvement of hypertonus after treatment for sleep disturbances in three patients with severe brain damage.

Treatment for sleep disturbance was given to three patients with severe brain damage (a 14-year-old boy, an 8-year-old girl and a 9-year-old boy), and changes in their muscle tone were estimated using F-wave analysis. In all patients, F-wave analysis was performed in the ulnar nerve before and 2 weeks after treatment with flunitrazepam or melatonin. From 16 recordings of F-waves, the mean amplitude and latency, the ratio of F-wave amplitude to M-wave amplitude (F/M ratio), the mean F-wave conduction velocity and the F-wave occurrence were evaluated. All patients showed at least one significant decrement of mean F-wave amplitude, the F-wave occurrence and mean F/M ratio, which suggests a reduction in muscle tone after treatment for sleep disturbance. It is concluded that treatment for sleep disturbance occurring in brain damage is important in view of the improvement of increased muscle tone.

Muscle histopathology in spastic cerebral palsy.

We studied the histopathology of spastic muscles in patients with spastic cerebral palsy using specimens obtained from the gastrocnemius muscles during orthopedic operations. Though there was no disease-specific abnormality, we found changes in fiber type distribution, i.e., type-1 fiber predominance and type-2B fiber deficiency. These changes were not found in the patient with brain infarction at age 1 year 5 months. Variation in fiber size, especially in type-1 fibers, was also detected in older patients and at the more severely affected sides. We speculated that the influence on the spinal motor unit from upper motor cortex might be applicable to the muscle histological findings and the time of the onset of the brain damage might be important.

Dose evaluation based on 24Na activity in the human body at the JCO criticality accident in Tokai-mura.

24Na in the human body, activated by neutrons emitted at the JCO criticality accident, was observed for 62 subjects, where 148 subjects were measured by the whole body counter of JNC Tokai Works. The 148 subjects, including JCO employees and the contractors, residents neighboring the site and emergency service officers, were measured by the whole-body counter. The neutron-energy spectrum around the facility was calculated using neutron transport codes (ANISN and MCNP), and the relation between an amount of activated sodium in human body and neutron dose was evaluated from the calculated neutron energy spectrum and theoretical neutron capture probability by the human body. The maximum 24Na activity in the body was 7.7 kBq (83 Bq(24Na)/g(23Na)) and the relevant effective dose equivalent was 47 mSv.

Significance of platelet-reactive antibody screening for patients facing frequent platelet transfusions.

It is not clear whether platelet-reactive antibody screening is clinically significant for patients facing frequent platelet transfusions. On the basis of data from 96 patients who had been examined for platelet-reactive antibodies by the mixed passive hemagglutination method for a variety of reasons, we investigated the following three issues retrospectively: (1) the relationship between platelet-reactive antibodies and the occurrence of problems in platelet transfusions, such as refractoriness or nonhemolytic reactions; (2) the influence of a history of transfusion on the production of those antibodies; and (3) the effect of screening for those antibodies on the prompt administration of appropriate platelet components. More than half of the platelet transfusion-related problems were associated with platelet-reactive antibodies. For patients with a history of transfusion, the mean period before a clinical problem occurred with platelet transfusions was 9 days,compared with 66 days for those without such a history. Accordingly, during the period, patients with a history of transfusions received fewer units of platelets and had fewer donor exposures than did patients without such a history. On the other hand, most patients who had been screened in advance for those antibodies received appropriate platelet components without delay, whereas an average of 10 days was needed before those who had not been screened received compatible platelets. The patients who had not been screened were transfused with 68 units of random platelets on average during the period. When frequent platelet transfusions are anticipated, especially for patients with a history of transfusion, screening for platelet-reactive antibodies beforehand would be helpful for prompt administration of appropriate platelets, although problems, such as the cost of those platelets and the burden on donors, remain to be resolved.

Twenty-six-week carcinogenicity study of sulfamethoxazole in CB6F1-Tg-rasH2 mice.

Sulfamethoxazole (SMX), a hormone-mediated rodent-specific nongenotoxic carcinogen, was administered to CB6F1 mice carrying a human prototype c-Ha-ras gene (Tg-rasH2) at doses of 0, 25, 100 or 400 mg/kg/day and to the wild-type mice at a dose of 400 mg/kg/day in feed for 26 weeks to evaluate the carcinogenicity and to validate the Tg-rasH2 model. N-Methyl-N-nitrosourea was administered at an intraperitoneal dose of 75 mg/kg to Tg-rasH2 as a positive control and the experimental system was confirmed to be valid. Histopathological examination revealed adenomas of the lung and Harderian gland and hemangiosarcoma of the spleen at low frequencies in the Tg-rasH2 treated with SMX; however, no statistically significant differences were observed either in the onset or prevalence rates of these neoplasms compared with that in the control group. Between the wild-type mice and Tg-rasH2, the onset rate and prevalence of the neoplasms were not significantly different, but the neoplasms tended to be more frequent in Tg-rasH2 mice showing a sensitivity to tumorigenicity. Follicular epithelial cell hyperplasia was observed in the thyroid gland in the groups of Tg-rasH2 given 100 mg/kg SMX or more, but no neoplastic lesion was observed. SMX was judged to be negative for carcinogenic potential in Tg-rasH2 in the present study.

Decrease in hepatic CYP2C11 mRNA and increase in heme oxygenase activity after intracerebroventricular injection of bacterial endotoxin.

We previously reported (Arch. Toxcol. 1998, 72, 492-498) that the differential decrease in the levels of hepatic cytochrome P450 (CYP) isozymes in rats was observed 24 hr after intracerebroventricular (i.c.v.) injection of bacterial lipopolysaccharide (LPS) at the dose ineffective (0.1 microgram) when injected intraperitoneally (i.p.). Among CYP isozymes we examined, the male specific CYP isozyme, CYP2C11 was most severely affected by i.c.v. injection of LPS. In this study, we examined the gene expression of CYP2C11, the total P450 contents, the CYP2C11-dependent activity of imipramine N-demethylase (IMND) and protein of CYP2C11 10 hr after i.c.v. or i.p. injections of LPS. Intracerebroventricular injection of LPS significantly decreased the level of CYP2C11 mRNA (to 63% of saline i.c.v. control), the total P450 contents (to 70% of saline i.c.v. control), the IMND activity (to 74% of saline i.c.v. control), but not protein of CYP2C11 in rat liver. In contrast, i.p. injection of LPS at the same dose as i.c.v. did not significantly affect these parameters. Since CYP is a heme protein, we also measured the activity of heme oxygenase (HO) using the same rat liver microsomes. The HO activity was increased to 166% by i.c.v. injection of LPS and 135% by i.p. injection of LPS compared to corresponding saline control. It is suggested that i.c.v. injection of LPS down-regulates the expression of CYP2C11 at transcriptional level and that both the decrease in CYP2C11 mRNA and the increase in heme degradation may be involved in the decreased level of protein and activity of CYP2C11 by i.c.v. injection of LPS in rat liver.

Strain differences in age-associated change in testosterone 6ß-hydroxylation in Wistar and Dark Agouti rats.

This study examines strain differences in testosterone (T)-hydroxylations between Wistar and Dark Agouti (DA) rats of both genders. The DA rat, an animal model, is a poor metabolizer of such drugs as debrisoquine, which are metabolized by cytochrome P450 (CYP) 2D. T-16α-, 2α-hydroxylations, which are linked to CYP2C11, were catalyzed at similar rates by the microsomes of both strains. In contrast, the liver microsomes from mature male DA rats catalyzed T-6ß-hydroxylation, the CYP3A mediated activity, at higher rates (∼ 2-fold) than Wistar rat liver microsomes did. There was no difference between immature male DA and Wistar rats for T-6ß-hydroxylation, indicating that the activity in male DA rat increases with maturation. Polyclonal antibodies raised against rat liver microsomal CYP3A2 and a CYP3A inhibitor, troleandomycin (TAO), effectively inhibited T-6ß-hydroxylation by liver microsomes from both strains of rats. The level of T-6ß- hydroxylation activity correlated well with the amount of CYP3A protein in the microsomes in mature as well as in immature male and female Wistar and DA rats. Northern blot analysis repeatedly indicated that the cellular contents of CYP3A2 mRNA are slightly (∼ 20%) higher in the liver of mature DA rats than in that of mature Wistar rats. These results indicate that the increased levels of CYP3A are responsible for the increased T-6ß-hydroxylation activity and protein in DA rat.