Ligation-mediated amplification for effective rapid determination of viral RNA sequences (RDV)

BACKGROUND: Emerging infectious diseases pose a significant risk to public health. Methods for rapid detection of pathogens are needed to effectively treat these diseases. Recently, we developed new methods for the rapid determination of viral RNA sequences, RDV ver1.0 and ver2.0. We demonstrated that these methods were able to simultaneously detect cDNA fragments of many different viruses without using sequence specific primers. However, some species of viruses, including the Yokose virus (YOKV), a flavivirus, could not be detected using the conventional procedures. OBJECTIVE: The RDV method was further modified to reduce the candidate PCR primer sets. STUDY DESIGN: Primer sets were reduced to 256 sets in the improved RDV ver3.0, and theoretically, all viral cDNA fragments ligated by two kinds of adaptors after digestion by two restriction enzymes could be amplified in the PCR step for direct sequencing. RESULTS: We succeeded in obtaining 118 YOKV cDNA fragments of the 141 sequence fragments. The cDNA fragments covered diverse range of viral genome. CONCLUSION: We were able to reduce the combinations of PCR primer sets used in the RDV method. This RDV method ver3.0 has a potential to detect viral cDNA fragments of both known and unknown RNA viruses rapidly and conveniently.     

Peculiar axonal debris with subsequent astrocytic response (foamy spheroid body)

Summary Foamy spheroid bodies (FSBs) are described, as newly identified pathological structures occurring in human brain. FSBs favoured the substantia nigra pars reticulata (SNPR) and/or globus pallidus (GP) in degenerative conditions especially postencephalitic parkinsonism, progressive supranuclear palsy, pallido-nigro-luysial atrophy and multiple system atrophy. No FSBs were observed anywhere in the presence of substantia nigra pars compacta (SNPC) degeneration, such as occurs in idiopathic Parkinson's disease, or luysio-pallidal system degeneration, such as found in dentato-rubro-pallidoluysial atrophy or Joseph's disease. FSBs were also occasionally identified in the substantia nigra (SN) and/or GP of aged persons. In addition to SN and GP lesions, FSBs were seen in diffuse axonal lesions of long fibre tracts (the corpus callosum, the superior cerebellar peduncle) after non-missile head injuries, and in peri-infarct lesions. Under the light microscope, FSBs appear as slightly eosinophilic, foamy and nearly round objects with vague outlines, measuring approximately 10–50 m in diameter. Some FSBs contain coarse, eosinophilic clusters at their periphery. FSB stained black when stained by the Gallyas silver method. Some FSBs were immunohistochemically positive for synaptophysin and 68 kDa neurofilament. Glial fibrillary acidic proteinpositive fibres were observed alongside and/or inside some FSBs. Electron microscopically, FSBs were found to consist of collections of neuritic debris containing a variety of dense bodies and a small number of both mitochondria and neurofilaments. Some such collections were surrounded by astrocytic processes. These findings strongly suggest that FSBs are collections of small axonal debris destined for removal by astrocytes in due course. A variety of factors (degeneration of the SNPR and/or the GP, injury, infarction, ageing) seemed to be responsible for the histogenesis of FSBs.     

High levels of autoantibodies against drug-metabolizing enzymes in SLA/LP-positive AIH-1 sera

Autoimmune hepatitis type 1 (AIH-1) is characterized by the detection of smooth muscle autoantibodies, antinuclear antibodies and antineutrophil cytoplasmic autoantibodies, and AIH-2 is characterized by the presence of autoantibodies against LKM, which contain drug-metabolizing enzymes. In this study, we measured the levels of drug-metabolizing enzymes in AIH-1 patients (ANA-positive). We exhaustively investigated the level of autoantibodies against major CYPs and UDP-glucuronosyltransferases of typical phase II drug-metabolizing enzymes, a transporter (MDR1), and NADPH-cytochrome P450 reductase in 4 patients with AIH-1 and 6 controls, as a case report. Two (Patients 3 and 4) of the AIH patients exhibited high levels of autoantibodies, while two (Patients 1 and 2) of the patients and the controls did not. The levels of autoantibodies against CYP2C19, CYP2D6, CYP2E1, UGT1A6 and human liver microsomes in Patients 3 and 4 sera were over 2(3) times the levels in Patient 1, Patient 2 and the control sera. Meanwhile, the levels of autoantibodies against CYP1A2, CYP2A6, CYP2C9, UGT2B7, MDR1 and NADPH-cytochrome P450 reductase were 2-2(2) higher in Patients 3 and 4 than in the other subjects. We found that the pattern of elevation in the Patient 3 serum was not parallel with that in Patient 4. Thus, we found high levels of autoantibodies against drug-metabolizing enzymes in AIH-1 patients.     

Effects of long-term and reduced-dose hormone replacement therapy on endothelial function and intima-media thickness in postmenopausal women.

OBJECTIVE: Short-term estrogen therapy improves endothelial function in postmenopausal women. However, there are few reports on its long-term effects on endothelial function and carotid intima-media thickness. Further, we determined whether a reduced dosage of estrogen may maintain its beneficial effects. DESIGN: Eighteen postmenopausal women (53.7+/-1.1 years) who had been diagnosed as having osteoporosis were enrolled. Among them, 11 women were prescribed oral conjugated estrogen 0.625 mg and medroxyprogesterone acetate 2.5 mg per day, and 7 women were prescribed an oral calcium supplement as the control group. Each patient decided whether she would take hormone replacement therapy or a calcium supplement. We performed ultrasound measurement of endothelial function of the brachial artery and carotid intima-media thickness. Examinations were scheduled to be performed pre-therapy and after 3, 6, 12, 18, 24, and 36 months of therapy. RESULTS: After three years of therapy, 6 women in the hormone replacement therapy group agreed to take half the dose of oral conjugated estrogen. Improvement of flow-mediated dilatation was observed at 3 months and the improvement was preserved up to 36 months. A similar improvement was also observed while women were on hormone replacement therapy even at the reduced dosage. Intima-media thickness of the common carotid artery in the control group increased after 12 months, which was not observed in the hormone replacement therapy group. CONCLUSIONS: Our results indicate that even at half the dose of estrogen, hormone replacement therapy may improve endothelial function and prevent the progression of carotid intima-media thickening in postmenopausal women.     

Non-human primate behaviors as models for development of higher cognitive functions]

Non-human primate behaviors have a special value for the neurobiological study of the development of higher cognitive functions of humans, because of the near evolutional relation between two species. We surveyed results and futures of neurobiological studies of a retrieval task, a learning-set and a self-injurious behavior expressed by non-human primates. On the retrieval task that is related to the development of inhibitory control, it was revealed a hierarchical ordering of inhibitory control processes in which the distinct neuronal circuits were involved. On the learning-set that is related to the development of abstract thinking, neural circuits for the individual learning dramatically changed from an automatic process to a cognitive process depending on the learning-set formation. The self-injurious behavior is expressed during early normal development in humans, and no other animals but non-human primates express it without administration of drugs. For that behavior, probable change in interactions of multiple monoaminergic systems was suggested as its underlying causes. Further studies on development of higher cognitive functions using non-human primates could be required for understanding the nature of human cognition.     

Surgical treatment of neuroblastoma with micrometastasis

BACKGROUND/PURPOSE: The aim of this study was to define the role of surgery in neuroblastoma with micrometastasis, which is detectable only by the polymerase chain reaction (PCR) method. METHODS: Fifty samples (peripheral blood 9, bone marrow 41) were harvested sequentially from 27 neuroblastoma patients, and the micrometastases were examined using the previously described single-step PCR method. The results were reviewed with the clinical courses. RESULTS: Radical surgery was performed in 9 patients with bone marrow micrometastasis. Event-free survival was obtained in 2 patients with stage IV disease (25.0%) for a follow-up period of 2 to 6 years in this group. Both patients received intraoperative radiation and subsequent autologous bone marrow transplantation (ABMT) using purged marrow. Radical surgery was performed in 18 patients without micrometastasis, and 6 of 9 advanced patients (66.7%) survived without the disease including 4 patients who received unpurged stem cell transplantation. CONCLUSIONS: Persistent micrometastasis in bone marrow should be considered predictive as a poor prognostic factor in neuroblastoma. Intensive local control with surgery and radiation is important for the patients with micrometastasis and should be followed by ABMT using purged marrow. Unpurged marrow may be safely used if the single-step PCR detects no micrometastasis.     

Similarities between civilian gunshot wounds to the head and nongunshot head injuries

BACKGROUND: This investigation compared the cerebral pathophysiologic status of gunshot wounds to the head (GSWH) with that of severe head injury of other causes (non-GSWH). METHODS: Data were collected prospectively from 71 GSWH and 541 non-GSWH patients. The two groups had similar demographic characteristics and injury severities. Cerebral metabolic parameters for each patient were averaged for the entire period of monitoring. These per-patient averages were compared between GSWH and non-GSWH groups. RESULTS: Median intracranial pressure was 21.4 mm Hg in GSWH patients vs. 16.7 mm Hg in non-GSWH patients (p < 0.001). Mean arterial pressures were similar, but the higher intracranial pressure in GSWH patients produced a lower median cerebral perfusion pressure. Cerebral blood flow, cerebrovascular resistance, cerebral metabolic rate of oxygen, average jugular venous oxygen saturation, and number of jugular venous desaturations did not differ significantly between the groups. Three-month outcome was death in 43% of GSWH patients and 32% of non-GSWH patients, persistent vegetative state or severe disability in 33% and 32%, respectively, and moderate disability or good recovery in 24% and 36%, respectively. These outcomes were not significantly different (p = 0.11). CONCLUSION: GSWH patients suffer global cerebral metabolic disturbances that are at least as severe as those seen in non-GSWH patients with injuries of comparable severity. This selected population of GSWH patients may enjoy outcomes comparable to those of non-GSWH patients if they are treated by the same aggressive protocols.     

Quality of micturition in male patients with orthotopic neobladder replacement

We evaluated the quality of micturition in patients with orthotopic neobladder replacement. In the present study, 58 patients who had undergone orthotopic neobladder replacement following cystectomy were enrolled. The quality of micturition was evaluated by means of uroflowmetry, a self-administered questionnaire and a 24-h frequency–volume chart. The questionnaire consisted of 26 questions concerning storage (9 questions), evacuation of urine (9), patient's satisfaction with neobladder (3), limitations in daily life (3) and pain (2). In addition, 11 of the questions were followed immediately by a question concerning inconvenience to the patients. The mean follow-up period was 35 months (range 6–80). On uroflowmetry, the mean peak flow rate was 13.5 ml/s with a low average flow rate (mean: 5.7 ml/s), and the voiding time was considerably prolonged (mean: 70.2 s). According to the frequency–volume chart and questionnaire responses, although the frequency and tidal volume of micturition were normal, the majority of patients were conscious of retarded, intermittent, prolonged, and weak stream and of straining during voiding. Diurnal or nocturnal incontinence was noticed subjectively in as many as 69% of the patients. However, the degree of urine leakage was minimal. In spite of the fact that 66% of the patients replied that their micturition status was worse than before surgery and that 69% of the patients felt dissatisfied, the degree of inconvenience was low in every item and the limitations in daily life were minimal. Although micturition in patients with neobladder replacement appeared to be of lower quality than that in normal subjects and was considered by many to be unsatisfactory, most patients were able to adjust themselves to it without significant inconvenience.     

Expression of cyclooxygenase-2 in renal cell carcinoma: correlation with tumor cell proliferation, apoptosis, angiogenesis, expression of matrix metalloproteinase-2, and survival.

PURPOSE: Cyclooxygenase (COX)-2 plays an important role in tumor cell proliferation, resistance to apoptosis, angiogenesis, and invasion in various malignant tumors. However, the relationships between COX-2 expression and these biological processes, clinicopathological features, and survival rate in patients with renal cell carcinoma are not clear. EXPERIMENTAL DESIGN: Tumor sections surgically removed from 131 patients were examined for COX-2 expression by immunohistochemistry. We also examined Ki-67 labeling index, apoptotic index, microvessel density, and matrix metalloproteinase (MMP)-2 expression, and correlated COX-2 expression with various clinicopathological features and survival. RESULTS: Of 131 sections, 70 (53.4%) were positive for COX-2 expression. COX-2 expression was associated significantly with various clinicopathological features, and correlated with the Ki-67 labeling index, microvessel density, and MMP-2 expression (P < 0.01), but not with the apoptotic index (P = 0.054). COX-2 expression was also identified as an independent risk factor for large tumor size (>7 cm) in multivariate logistic regression model. COX proportional hazards analysis showed that distant metastasis and high T stage were independent prognostic factors [odds ratio (OR), 9.41; 95% confidence interval (CI), 2.16-41.11; P < 0.01 and OR, 5.19; 95% CI, 1.02-26.54; P = 0.048, respectively), whereas COX-2 expression was not (OR, 1.46; 95% CI, 0.24-9.00; P = 0.68). CONCLUSION: COX-2 expression in patients with renal cell carcinoma is associated with several clinicopathological factors, and appeared to play an important role in tumor cell proliferation and MMP-2 expression, but is not a significant prognostic factor.