Impact of tocilizumab on N-terminal pro-brain natriuretic peptide levels in patients with active rheumatoid arthritis without cardiac symptoms.

OBJECTIVE: To prospectively investigate the effect of tocilizumab (TCZ) on the levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), as a predictor of congestive heart failure (CHF) in patients with active rheumatoid arthritis (RA). METHOD: Seventy patients with RA (median age 59 years, 86% female) free of cardiovascular disease were treated with TCZ and followed for 24 weeks. The NT-proBNP levels were measured at baseline and week 24. Thirty healthy controls were included for comparison of normal NT-proBNP levels with those of RA patients. RESULTS: The NT-proBNP level was significantly higher in patients with RA than in controls (median 42.5 pg/mL vs 109.0 pg/mL, p < 0.001). NT-proBNP levels decreased by 63% over the 24 weeks of TCZ treatment. Multiple linear regression analysis indicated that the percentage change in the NT-proBNP level was significantly associated with that of the Simplified Disease Activity Index (ß = 0.356, p = 0.014), even after adjusting for the levels of rheumatoid factor, duration of RA, age, and anti-cyclic citrullinated peptide antibody. CONCLUSION: TCZ decreased the NT-proBNP level in patients with RA without preceding cardiovascular disease and CHF. TCZ may have a cardioprotective effect in those with active RA.

Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo.

The respiratory epithelium is a potential site for somatic gene therapy for the common hereditary disorders alpha 1-antitrypsin (alpha 1AT) deficiency and cystic fibrosis. A replication-deficient adenoviral vector (Ad-alpha 1AT) containing an adenovirus major late promoter and a recombinant human alpha 1AT gene was used to infect epithelial cells of the cotton rat respiratory tract in vitro and in vivo. Freshly isolated tracheobronchial epithelial cells infected with Ad-alpha 1AT contained human alpha 1AT messenger RNA transcripts and synthesized and secreted human alpha 1AT. After in vivo intratracheal administration of Ad-alpha 1AT to these rats, human alpha 1AT messenger RNA was observed in the respiratory epithelium, human alpha 1AT was synthesized and secreted by lung tissue, and human alpha 1AT was detected in the epithelial lining fluid for at least 1 week.

Predictors of restenosis after coronary artery stenting.

AIM: The aim of this study was to analyze restenosis after percutaneous coronary intervention, factors related to restenosis after coronary artery stenting and the degree of the risk of restenosis were evaluated. METHODS: The study enrolled 181 patients (249 lesions) who underwent the first coronary artery stenting. Multivariate analysis was performed, and the restenotic index (RI) was calculated by combining the extracted predictors. RESULTS: Among the 181 patients (249 lesions), restenosis occurred in 89 (111 lesions) and did not occur in 92 (138 lesions). Vascular revasculation was performed in 95 restenosed target lesions in 68 patients. The mean period of follow-up angiography after the procedures was 206 days in the restenosis group and 271 days in the non-restenosis group, i.e. significantly shorter in the restenosis group. As a result of multivariate analysis, diabetes mellitus, Cr level, amount of the contrast medium used and stent diameter were selected as significant factors that independently contributed to the restenosis after coronary artery stenting. By combining these factors, the RI was calculated by the following formula for the prediction of restenosis: RI=exp (1.088xCr+0.909xdiabetes mellitus+0.871xcontrast medium+0.591xstent diameter). CONCLUSION: The risk of restenosis after coronary artery stenting can be predicted to an extent according to the RI devised in this study.

Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT.

Recent regulatory changes have placed a major emphasis on in vitro safety testing and alternative models. In regard to skin sensitization tests, dendritic cells (DCs) derived from human peripheral blood have been considered in the development of new in vitro alternatives. Human cell lines have been also reported recently. In our previous study, we suggested that measuring CD86 and/or CD54 expression on THP-1 cells (human monocytic leukemia cell line) could be used as an in vitro skin sensitization method. An inter-laboratory study among two laboratories was undertaken in Japan in order to further develop an in vitro skin sensitization model. In the present study, we used two human cell lines: THP-1 and U-937 (human histiocytic lymphoma cell line). First we optimized our test protocol (refer to the related paper entitled "optimization of the h-CLAT protocol" within this journal) and then we did an inter-laboratory validation with nine chemicals using the optimized protocol. We measured the expression of CD86 and CD54 on the above cells using flow cytometry after a 24h and 48h exposure to six known allergens (e.g., DNCB, pPD, NiSO(4)) and three non-allergens (e.g., SLS, tween 80). For the sample test concentration, four doses (0.1x, 0.5x, 1x, and 2x of the 50% inhibitory concentration (IC(50))) were evaluated. IC(50) was calculated using MTT assay. We found that allergens/non-allergens were better predicted using THP-1 cells compared to U-937 cells following a 24 h and a 48 h exposure. We also found that the 24h treatment time tended to have a better accuracy than the 48 h treatment time for THP-1 cells. Expression of CD86 and CD54 were good predictive markers for THP-1 cells, but for U-937 cells, expression of CD86 was a better predictor than CD54, at the 24h and the 48 h treatment time. The accuracy also improved when both markers (CD86 and CD54) were used as compared with a single marker for THP-1 cells. Both laboratories gave a good prediction of allergen/non-allergen, especially using THP-1 cells. These results suggest that our method, human Cell Line Activation Test (h-CLAT), using human cell lines THP-1 and U-937, but especially THP-1 cells at 24h treatment, may be a useful in vitro skin sensitization model to predict various contact allergens.

Development of an in vitro skin sensitization test using human cell lines: the human Cell Line Activation Test (h-CLAT). I. Optimization of the h-CLAT protocol.

The aim of this study is to optimize the experimental conditions for an in vitro skin sensitization test using the human cell lines THP-1 and U-937. As regards pre-culturing time, the expression of CD86 on DNCB-treated THP-1 cells tended to be higher after 48h and 72h pre-culture compared with other time points evaluated. Next, we investigated the effect of chemical treatment time, and found that induction of CD86 expression on THP-1 cells by DNCB reached a plateau after 24h. Augmentation of CD86 expression is often observed when cells are treated with a subtoxic dose of allergens. To determine the appropriate dose of test samples, the cytotoxicity of test samples to THP-1 and U-937 cells was assessed with MTT assay, and the 50% inhibitory concentration (IC50) of each test sample was calculated. Based on the cytotoxicity assay data, four concentrations in the range between toxic and non-toxic were selected (0.1x, 0.5x, 1x and 2x IC50). Several kinds of antibodies were tested for staining THP-1 and U-937 cells treated with allergens/non-allergens (e.g., DNCB, Ni/SLS), and suitable antibodies for staining CD86 and CD54 were selected. We confirmed that the working dilutions of the selected CD86 and CD54 antibodies were appropriate for use in our method. The effect of an FcR blocking procedure was also evaluated. The mean fluorescence intensity (MFI value) was decreased by the FcR blocking procedure, which indicated that non-specific staining was blocked. Therefore, this procedure should be included in the method. Based on our findings, the protocol for this assay was optimized and the experimental conditions to be used in a future validation study were identified. We propose to call this kind of in vitro skin sensitization test h-CLAT, which is short for human Cell Line Activation Test.

Dynamics of capillary condensation in aerogels.

Dynamics of capillary condensation of liquid 4He in various density silica aerogels was investigated systematically. Interfaces were clearly visible when bulk liquid was rapidly sucked into the aerogel. Time evolution of the interface positions was consistent with the Washburn model and their effective pore radii were obtained. Condensation was a single step in a dense aerogel and two steps in a low density aerogel. Crossover between the two types of condensation was observed in an intermediate density aerogel. Variety of the dynamics may be the manifestation of the fractal nature of aerogels which had a wide range of distribution of pore radii.

Evaluation of paraumbilical vein as a prognostic index of severe liver cirrhotic patients with portal-systemic shunts.

AIM: The aim of this study was to predict the outcome in severe liver cirrhotic patients with portal-systemic shunts. METHODS: One-hundred and sixteen patients with liver cirrhosis diagnosed as Child-Pugh class B and C with portal-systemic shunts confirmed by abdominal ultrasonography, computed tomography and magnetic resonance imaging were enrolled in this study. Twenty-three factors were evaluated concerning clinical laboratory parameters and extracted prognostic factors using the Cox proportional hazards model, and the prognostic index (PI) was prepared by combining these factors. RESULTS: The cumulative survival rates after admission were 64.6%, 35.6% and 25% after 1, 3 and 5 years, respectively. Using multivariate analysis, age, the presence of hepatocellular carcinoma (HCC), portal vein tumor thrombosis (PVTT) and paraumbilical vein (PUV) shunt were selected as significant prognostic factors that contributed independently to the prognosis of severe liver cirrhotic patients with portal-systemic shunts. The PI was calculated with the following formula using these 4 factors. PI = 0.042 x Age + 0.913 x HCC + 0.989 x PVTT + 1.079 x PUV shunt. The group with a high score for PI was found to die with significantly higher frequency than the group with a low score. CONCLUSIONS: It was found that tumor related factors and PUV shunt were the most important factors for severe liver cirrhotic patients with portal-systemic shunts. The PI is suggested to be an appropriate index to predict the prognosis for these patients.

Prognostic factors of primary biliary cirrhosis detected by health screening.

AIM: The liver cirrhosis is likely to differ in the Japanese and Western populations. Thus, we performed a retrospective cohort analysis by a review of clinical records to clarify prognostic factors after the onset of primary biliary cirrhosis (PBC) detected by health screening. METHODS: The subjects were 52 patients with PBC. Thirty-nine factors were evaluated concerning clinical data and extracted prognostic factors using the Cox proportional hazard model. RESULTS: The mean duration of the follow-up period was 5.1 years, during which 6 (11.5%) of the patients died. The cumulative survival rate after the onset of PBC was 93.4% after 5 year, and 67.8% after 10 years. Multivariate analysis indicated 2 factors, i.e. the body mass index (BMI), and IgG, as independent prognostic factors. Their hazard ratios were 0.399 (per 1 kg/m2 of BMI) and 1.282 (per 100 mg/dL of IgG). The prognostic index (PI) was calculated by the following formula using these 2 factors. PI = 0.919 x BMI+0.249 x IgG. CONCLUSIONS: The prediction of the outcome using PI based on the 2 factors provides additional information for the determination of the therapeutic approach in PBC after health screening.

Effects of storage conditions on forensic examinations of blood samples and bloodstains stored for 20 years.

The effects of various storage conditions on blood identification tests, DNA degradation, and short tandem repeat (STR) typing were evaluated. Bloodstains stored at room temperature, 4 °C, -20 °C, and -80 °C for 20 years; blood samples stored at -20 °C and -80 °C for 20 years; and fresh blood samples were analyzed. Leuco-malachite-green testing, anti-human hemoglobin (Hb) testing (using immunochromatography), and tests for hemoglobin-beta (HBB) mRNA were performed as blood identification tests. DNA degradation was evaluated by quantifying the ratios of 305 and 129 base pair (bp) fragments to 41 bp fragments. STR typing was performed using an AmpFlSTR® Identifiler™ Plus PCR Amplification Kit. All samples were positive in leuco-malachite-green staining and anti-human Hb assays. HBB was not detected in blood samples stored at -20 °C or -80 °C, although this marker was detected in all bloodstains. As indicated by the ratio of 129:41 bp and 305:41 bp DNA fragments, DNA from bloodstains stored at room temperature or 4 °C were significantly degraded compared to DNA from all other samples. STR typing analyses revealed that a portion of the loci was undetected in bloodstains stored at room temperature. Therefore, to prevent DNA degradation during long-term storage, it is recommended that bloodstains and blood be stored at below -20 °C. In addition, because bloodstains are more suitable for detection of blood-specific mRNAs than blood sample, it is desirable that blood is stored as bloodstain for this method.

High expression of post-treatment Ki-67 status is a risk factor for locoregional recurrence following breast-conserving surgery after neoadjuvant chemotherapy.

INTRODUCTION: During the past decade, neoadjuvant chemotherapy (NAC) has been increasingly used in patients to reduce large tumors to a size eligible for breast-conserving therapy (BCT). However, the association between NAC and Ki-67 has not yet been fully elucidated. The aim of this study was to evaluate the prognostic significance of Ki-67, specifically after NAC followed by BCT, particularly in terms of locoregional recurrence (LRR). METHODS: A total 217 patients who received BCT after NAC were retrospectively analyzed. In these patients, immunohistochemistry analyses defined four tumor subtypes, Luminal A, Luminal B, Triple negative, and HER2 type. Ki-67 was examined by immunohistochemistry in both pretreatment core needle samples and post-treatment surgical excision specimens. High Ki-67 expression was defined as >20%. The prognostic factors LRR, locoregional relapse-free survival (LRRFS), and overall survival (OS) were analyzed. RESULTS: In total, LRR developed in 14 patients, and the 5 year-LRRFS was 94.2%. Post-treatment high Ki-67 expression, triple negative, the presence of lymphovascular invasion, and histological grade 3 were significantly high in LRR for prognostic factors (P < 0.05). There were significant differences in Kaplan-Meier method for LRRFS curves according to these three factors for patients receiving BCT following NAC (P < 0.05). There was a significant difference in the 5 year-OS for patients with and without LRR (41.7% vs. 93.9%, P < 0.001). CONCLUSION: Post-treatment high Ki-67 expression could be one of the important prognostic factors of LRR, and require careful follow-up on LRR at the time of surveillance.

Relationship between DNA degradation ratios and the number of loci detectable by STR kits in extremely old seminal stain samples.

The relationships between DNA degradation ratios and the number of detected loci were explored in extremely old seminal stains evaluated using three short tandem repeat (STR) kits: the AmpFlSTR® Identifiler™ PCR Amplification Kit (Identifiler), the AmpFlSTR® Yfiler™ PCR Amplification Kit (Yfiler), and the AmpFlSTR® MiniFiler™ PCR Amplification Kit (MiniFiler). DNA degradation ratios based on 41, 129, and 305bp DNA fragments were calculated (129:41 and 305:41), and the relationships between the ratios and storage duration were also explored. Using the Identifiler kit, the number of loci detected was strongly correlated with the 129:41 ratio (r=0.887), whereas the correlation with the 305:41 ratio was moderate (r=0.656). Using the Yfiler kit, the DYS385 amplicon was detected in all samples, suggesting that DYS385 may be resistant to degradation. The number of detected loci was strongly correlated with the 129:41 ratio (r=0.768), and moderately so with the 305:41 ratio (r=0.515). MiniFiler detected at least seven loci in all samples. In samples that did not yield full profiles, the undetected loci were D7S820 and D21S11, or D21S11 only, suggesting that these loci might be easily degraded. The number of loci detected using STR kits correlated with the DNA degradation ratios. In particular, the 129:41 ratio was particularly useful for estimating the number of loci detectable by STR kits. On the other hand, we suggest that storage duration cannot be accurately estimated using DNA degradation ratios; these ratios were not strongly correlated with storage duration (129:41; r=-0.698, 305:41; r=-0.550). However, the ratios may allow the identification of samples that have been stored for more than 40years.

Gene transfer to freshly isolated human respiratory epithelial cells in vitro using a replication-deficient adenovirus containing the human cystic fibrosis transmembrane conductance regulator cDNA.

Cystic fibrosis (CF) results from mutations of the CF transmembrane conductance regulator (CFTR) gene and subsequent defective regulation of cAMP-stimulated chloride (Cl-) permeability across the apical membrane of epithelial cells. In vitro transfer of normal CFTR cDNA corrects this defect, and studies in experimental animals have shown successful gene transfer to airway epithelium in vivo using a recombinant adenoviral vector containing the human CFTR cDNA (AdCFTR), supporting the feasibility of in vivo AdCFTR-mediated gene therapy for the respiratory manifestations of CF. One step in applying this therapy to CF patients is to evaluate the safety and efficacy of AdCFTR-mediated gene transfer in the actual target for human gene therapy, human airway epithelium. The present study demonstrates that AdCFTR restores cAMP-stimulated Cl- permeability in human CF bronchial epithelial cells. In addition, the study utilizes freshly isolated human airway epithelial cells from the nose and/or bronchi of normal individuals and/or individuals with CF to demonstrate that after in vitro AdCFTR-mediated gene transfer: (i) AdCFTR DNA does not replicate as a function of dose and time; (ii) CF epithelial cells express AdCFTR-mediated normal human CFTR mRNA; and (iii) CF epithelial cells, including terminally differentiated ciliated cells (the most common airway epithelial cell type), express the normal human CFTR protein. Together, these data support the use of AdCFTR in human gene therapy trials and suggest that biologic efficacy should be achievable in vivo.

The mystery of the trichothecene 3-O-acetyltransferase gene. Analysis of the region around Tri101 and characterization of its homologue from Fusarium sporotrichioides.

The trichothecene 3-O-acetyltransferase gene, Tri101, plays a pivotal role for the well-being of the type B trichothecene producer Fusarium graminearum. We have analyzed the cosmids containing Tri101 and found that this resistance gene is not in the biosynthetic gene cluster reported so far. It was located between the UTP-ammonia ligase gene and the phosphate permease gene which are not related to trichothecene biosynthesis. These two 'house-keeping' genes were also linked in Fusarium species that do not produce trichothecenes. The result suggests that the isolated occurrence of Tri101 is attributed to horizontal gene transfer and not to the reciprocal translocation of the chromosome containing the gene cluster. Interestingly, 3-O-acetylation was not always a primary self-defensive strategy for all the t-type trichothecene producers; i.e. the type A trichothecene producer Fusarium sporotrichioides did not acetylate T-2 toxin in vivo although the fungus possessed a functional 3-O-acetyltransferase gene. Thus Tri101 appears to be a defense option which the producers have independently acquired in addition to their original resistance mechanisms.

Inhibitory effects of 3-nitrophloroglucinecarboxylic acid derivatives on Epstein-Barr virus early antigen induction.

The inhibitory effects of two series of 3-nitrophloroglucinecarboxylic acid derivatives, 3-nitro-2,4,6-trihydroxythiobenzamides (II) and 3-nitro-phloroglucinecarboxylates (III), on Epstein-Barr virus early antigen (EBV-EA) induction were examined using Raji cells. Some of them strongly inhibited the induction, N-nonyl and O-decyl derivatives being the most potent inhibitors among the thioamides and esters, respectively. These results suggest the possibility that these two 3-nitrophloroglucinecarboxylic acid derivatives may be listed as novel inhibitors of tumor promotion.

Inhibitory effects of 3-nitro-2,4,6-trihydroxybenzamides on Epstein-Barr virus early antigen induction.

Inhibitory effects of a series of 3-nitro-2,4,6-trihydroxybenzamides on Epstein-Barr virus early antigen (EBV-EA) induction were examined using Raji cells. Some of the tested compounds showed highly inhibitory activity, the N-octyl amide derivative being the most active among them. These results suggest the possibility that 3-nitro-2,4,6-trihydroxybenzamides might be listed as novel inhibitors of tumor promotion.

The birth prevalence of malformation syndromes in Tokyo infants: a survey of 14,430 newborn infants.

A survey of the birth prevalence of congenital anomalies among newborn infants in Japan is under way at a large maternity hospital in Tokyo. Of 14,430 consecutive newborn babies (7,455 M; 6,975 F), 33 had a multiple congenital anomalies (MCA) syndrome. These included 2 with trisomy 13 (including a mosaic), 3 with trisomy 18 (including 1 mosaic), 16 with trisomy 21 (including 1 mosaic), 1 with cri-du-chat syndrome, 1 with 5p partial trisomy, 1 with Hallermann-Streiff syndrome, 1 with Treacher-Collins syndrome, 1 with achodroplasia, 2 with arthrogryposis multiplex congenita, 1 with hemihypertrophy, 1 with Wiedemann-Beckwith syndrome, 1 with asplenia syndrome, 1 with Klippel-Trenaunay-Weber syndrome, and 1 with probable Marfan's syndrome. Except for one infant with Ullrich-Turner syndrome, cases with sex-chromosome aberrations could not be diagnosed neonatally on a clinical basis.

Fiberoptic bronchoscopic laser photoradiation for tumor localization in lung cancer.

Hematoporphyrin derivative (HpD) administration and laser photoradiation were combined to clarify the system's clinical applicability in localizing lung cancer tumors. It was used in 16 cases of lung cancer and in one case of severely atypical squamous metaplasia. Tumors were irradiated 48 hours after intravenous injection of 2.0 to 4.0 mg/kg HpD and fluorescence observed. Fluorescence was seen in 13 lung cancer cases in the case of squamous metaplasia, but results were negative in three of the cases of lung cancer. Among the positive cases was one of occult cancer and two invading the trachea, difficult to recognize by conventional fiberoptic bronchoscopy, as was the severely atypical squamous metaplasia. In the three lung cancer cases not fluorescing, the tumors were obscured by blood, necrotic tissue, or normal mucosa. The method holds promise in the diagnosis of malignant tumors. Nevertheless, it needs further refinement and more studies to elucidate definitive differentiation between malignant and severely atypical, nonmalignant tissue.

Quantitative cytochemical differences between young and old patients with lung cancer.

In order to study differences in the tumor growth and frequency of metastases between younger and older patients with lung cancer, we investigated the nuclear DNA and nuclear protein according to age by means of cytophotometry after combined staining with Feulgen and Naphthol Yellow S. On the basis of Feulgen-Naphthol Yellow S staining method, 13 patients less than 40 years old and 16 patients older than 70 years were investigated. Our results showed no significant difference in nuclear DNA contents between young and old patients, but there were significantly higher nuclear protein contents (p less than 0.05) and nuclear protein to nuclear DNA (NP/DNA) ratios (p less than 0.001) in young patients than in old patients. This suggests that young patients may have higher tumor proliferation (high nuclear protein contents and NP/DNA ratios). The lower nuclear protein content and NP/DNA ratio of older cases is in keeping with the general phenomenon of slower tumor growth and less frequent metastases in such cases.

Cerebral angiography and ultrasonic quantitative flow measurement of common carotid artery in patients with cerebral infarction.

Blood flow rate and velocity in the common carotid artery were measured with an ultrasonic quantitative flow measurement (QFM) system and cerebral angiography was performed in 98 patients about four weeks after the onset of cerebral infarction. Angiography revealed complete occlusion in 13 patients (CO group), visible stenosis in 45 patients (S group), and no apparent stenosis in 40 patients (N group). In the CO group, damaged-side blood flow (DF; 4.80 ml/sec) and velocity (DV; 10.5 cm/sec) were significantly lower (P less than 0.01) than intact-side blood flow (7.19 ml/sec) and velocity (14.86 cm/sec). Both DF and DV were significantly lower in group CO than group S (7.55 ml/sec and 15.04 cm/sec) and in group S than group N (9.32 ml/sec and 18.07 cm/sec). Degree of stenosis in the internal carotid and middle cerebral arteries was significantly associated with reduced DF (P less than 0.05). Of the 24 patients with a mean DF of under 6.5 ml/sec, 15 had stenosis of 75% or more; of the 74 patients with a mean DF of 6.5 ml/sec or more, 66 had stenosis of less than 25%. The results indicate that blood flow determined by QFM reflects the degree of occlusion or stenosis in the intracranial trunk arteries and may thus provide a practical, noninvasive method of assessing the severity of cerebrovascular lesions.