Verification of the seizure liability of compounds based on their in vitro functional activity in cultured rat cortical neurons and co-cultured human iPSC-derived neurons with astrocytes and in vivo extrapolation to cerebrospinal fluid concentration.

Methodical screening of safe and efficient drug candidate compounds is crucial for drug development. A high-throughput and accurate compound evaluation method targeting the central nervous system can be developed using in vitro neural networks. In particular, an evaluation system based on a human-derived neural network that can act as an alternative to animal experiments is desirable to avoid interspecific differences. A microelectrode array (MEA) is one such evaluation system, and can measure in vitro neural activity; however, studies on compound evaluation criteria and in vitro to in vivo extrapolation are scarce. In this study, we identified the parameters that can eliminate the effects of solvents from neural activity data obtained using MEA allow for accurate compound evaluation. Additionally, we resolved the issue associated with compound evaluation criteria during MEA using principal component analysis by considering the neuronal activity exceeding standard deviation (SD) of the solvent as indicator of seizurogenic potential. Overall, 10 seizurogenic compounds and three negative controls were assessed using MEA-based co-cultured human-induced pluripotent stem cell-derived neurons and astrocytes, and primary rat cortical neurons. In addition, we determined rat cerebrospinal fluid (CSF) concentrations during tremor and convulsion in response to exposure to test compounds. To characterize the in vitro to in vivo extrapolation and species differences, we compared the concentrations at which neuronal activity exceeding the SD range of the solvent was detectable using the MEA system and rat CSF concentration.

Efficacy of a "provisional incision" for longitudinal flap division after free-flap surgery.

Fix and flap surgery for severe open limb fractures is already a standard treatment. In cases where the fracture is complicated or accompanied by bone defects, secondary surgery is required for fracture sites covered with a myocutaneous flap after the soft tissue condition has stabilized. We applied the delayed procedure concept used for distant flaps and attempted to prevent postoperative myocutaneous flap necrosis by performing a provisional incision prior to the longitudinal incision of the flap. We report the course of five cases of the longitudinal division of the myocutaneous flap using "provisional incision" after free-flap surgery for severe open fracture and verify its usefulness. In this case series, five patients with severe open limb fractures treated from 2020 to 2021 who underwent longitudinal incision of the myocutaneous flap using provisional incision after free-flap surgery were included. The types of flaps used for soft tissue reconstruction in the acute phase, the reasons for the need for secondary surgery, the period from soft tissue reconstruction to additional surgery, and the healing status of soft tissue after secondary surgery were all investigated retrospectively. The types of flaps used for soft tissue reconstruction were latissimus dorsi myocutaneous flap in four cases and anterolateral thigh flap in one case. The breakdown of secondary surgery was osteosynthesis in one case, plate removal in one case, and bone cement removal and autologous bone grafting in three cases. The period from soft tissue reconstruction to secondary surgery ranged from 6 weeks to 4 months. In all cases, the wound healed without necrosis of the myocutaneous flap. For the treatment of severe open limb fractures, longitudinal division of the myocutaneous flap using "provisional incision" is a safer approach to the necessary secondary surgery and reduces the possibility of necrosis of the flap.

Raster plots machine learning to predict the seizure liability of drugs and to identify drugs.

In vitro microelectrode array (MEA) assessment using human induced pluripotent stem cell (iPSC)-derived neurons holds promise as a method of seizure and toxicity evaluation. However, there are still issues surrounding the analysis methods used to predict seizure and toxicity liability as well as drug mechanisms of action. In the present study, we developed an artificial intelligence (AI) capable of predicting the seizure liability of drugs and identifying drugs using deep learning based on raster plots of neural network activity. The seizure liability prediction AI had a prediction accuracy of 98.4% for the drugs used to train it, classifying them correctly based on their responses as either seizure-causing compounds or seizure-free compounds. The AI also made concentration-dependent judgments of the seizure liability of drugs that it was not trained on. In addition, the drug identification AI implemented using the leave-one-sample-out scheme could distinguish among 13 seizure-causing compounds as well as seizure-free compound responses, with a mean accuracy of 99.9 ± 0.1% for all drugs. These AI prediction models are able to identify seizure liability concentration-dependence, rank the level of seizure liability based on the seizure liability probability, and identify the mechanism of the action of compounds. This holds promise for the future of in vitro MEA assessment as a powerful, high-accuracy new seizure liability prediction method.

Expressions of PD-L1 and Nectin-4 in urothelial cancer patients treated with pembrolizumab.

OBJECTIVES: Recently, the standard of care for advanced urothelial cancer (UC) has been changed by developing immune-checkpoint inhibitors (ICIs). However, its response rate is limited to 20-30%. The identification of biomarkers to predict the therapeutic effects of ICIs is urgently needed. The present study explored the association between immunohistochemical biomarkers and clinical outcomes in UC patients treated with pembrolizumab. PATIENTS AND METHODS: A total of 85 patients with UC who received pembrolizumab after chemotherapy from January 2018 to May 2020 were retrospectively reviewed. Tumor tissues were obtained for immunohistochemical study from 47 out of 85 patients. The protein expressions of PD-L1, WT1, Nectin-4, CD4, CD8, Foxp3, and CD68 in tumor cells and/or tumor infiltrating lymphocytes were immunohistochemically examined. The associations between protein expressions and overall survival (OS), progression-free survival (PFS), and disease control rate (DCR) were statistically analyzed. RESULTS: Patients with positive PD-L1 in tumor cells showed significantly worse OS (Log-rank test: HR 5.146, p = 0.001, Cox regression analysis: HR 4.331, p = 0.014) and PFS (Log-rank test: HR 3.31. p = 0.022), along with significantly lower DCR (14.3%) compared to the PD-L1 negative patients (67.5%). In addition, patients with strong expression of Nectin-4 in tumor cells showed significantly higher DCR (100%) than the other patients (50%). CONCLUSION: PD-L1 expression in tumor cells was associated with poor prognosis (OS and PFS) and low DCR. Interestingly, the strong expression of Nectin-4 was correlated with high DCR. PD-L1 and Nectin-4 expression in tumor cells could be prognostic biomarkers useful for pembrolizumab in patients with advanced UC.

Principal Component Analysis to Distinguish Seizure Liability of Drugs in Human iPS Cell-Derived Neurons.

Screening for drug discovery targeting the central nervous system requires the establishment of efficient and highly accurate toxicity test methods that can reduce costs and time while maintaining high throughput using the function of an in vitro neural network. In particular, an evaluation system using a human-derived neural network is desirable in terms of species difference. Despite the attention, the microelectrode array (MEA) is attracting among the evaluation systems that can measure in vitro neural activity, an effective analysis method for evaluation of toxicity and mechanism of action has not yet been established. Here we established analytical parameters and multivariate analysis method capable of detecting seizure liability of drugs using MEA measurement of human iPS cell-derived neurons. Using the spike time series data of all drugs, we established periodicity as a new analytical parameter. Periodicity has facilitated the detection of responses to seizurogenic drugs, previously difficult to detect with conventional analytical parameters. By constructing a multivariate analytical method that identifies a parameter set that achieves an arbitrary condition, we found that the parameter set comprising total spikes, maximum frequency (MF), inter- MF interval (IMFI), coefficient of variance of IMFI, and periodicity can uniformly detect the seizure liability of seizurogenic drugs with different mechanisms of action. Seizurogenic drugs were suggested to increase the regularity of the network burst in MEA measurements in human iPS cell-derived neurons.

Trends in the serovar and antimicrobial resistance in clinical isolates of Salmonella enterica from cattle and pigs between 2002 and 2016 in Japan.

AIMS: Given the significance of Salmonella enterica in both human and animal health, and a recent global dissemination of Salmonella 4,[5],12:i:-, changes in the prevalent serovars and antimicrobial resistance in clinical isolates of Salmonella from cattle and pigs were investigated in Japan. METHODS AND RESULTS: The serovars and antimicrobial susceptibilities of 1605 Salmonella enterica isolated from cattle (n = 894) and swine (n = 711) between 2002 and 2016 were examined. The most common serovar among all samples was Salmonella Typhimurium. However, its monophasic variant with antigenic structure S. 4,[5],12:i:-, which was first detected in cattle in 2006 and swine in 2010, has been rapidly increasing in incidence and resistance. Resistance rates to cefotaxime and ciprofloxacin were generally low (<10% in the cattle isolates and <5% in the swine isolates); however, isolates resistant to more than five antimicrobials, which often include these antimicrobials, were recently detected in Salmonella Dublin, S. 4,[5],12:i:-, S. Typhimurium, Salmonella Newport, Salmonella Choleraesuis and Salmonella 6,7:c:-. Among them, two S. 4,[5],12:i:- isolates possessed extended-spectrum ß-lactamase-encoding genes; blaSHV-12 or blaCTX-M-55 , respectively, while all the five S. Typhimurium isolates possessed AmpC-type ß-lactamase gene of blaCMY-2 . CONCLUSIONS: S. 4,[5],12:i:- has been rapidly increasing and exhibiting a remarkable change in antimicrobial resistance in Japan. Considering certain serovars are characterized by multidrug resistance including medically important antimicrobials, continuous monitoring and appropriate measures are required to protect public health and veterinary husbandry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a trend in the serovars and antimicrobial resistance in clinical isolates of Salmonella from cattle and pigs in Japan, and showed that there were certain types of Salmonella serovars depending on the animal origin which needs more attention.

Combination of p53-DC vaccine and rAd-p53 gene therapy induced CTLs cytotoxic against p53-deleted human prostate cancer cells in vitro.

Recently, the US FDA approved sipuleucel-T, which is composed of autologous DCs stimulated with a recombinant fusion protein of prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GM-CSF), as the first immunotherapeutic agent for metastatic castration resistant prostate cancer (mCRPC). However, sipuleucel-T demonstrated only modest efficacy in mCPRC patients. Researchers are now investigating the potential of p53 protein as a tumor-associated antigen (TAA) loaded in DC-based cancer vaccine. Approximately half of all tumors overexpress p53, and up to 20% of prostate cancer cells overexpresses p53. In this study, we evaluated the feasibility of combining p53-DC vaccine and rAd-p53 gene therapy, using the p53-overexpressing and non-expressing prostate cancer cells in vitro. We successfully generated the p53-DC vaccine by culturing autologous DCs infected with rAd-p53. This p53-DC vaccine can differentiate CTLs specifically cytotoxic to p53-overexpressing prostate cancer cells. In addition, rAd-p53 infection can induce overexpression of p53 and thus the cytotoxicity of CTLs differentiated by the p53-DC vaccine in p53 non-expressing prostate cancer cells. These findings suggest that this combination therapy using p53-DC vaccine and rAd-p53 gene therapy together may represent a new paradigm for the treatment of mCRPC.

Distinct Excitation to Pulpal Stimuli between Somatosensory and Insular Cortices.

Somatosensory information from the dental pulp is processed in the primary (S1) and secondary somatosensory cortex (S2) and in the insular oral region (IOR). Stimulation of maxillary incisor and molar initially induces excitation in S2/IOR, rostrodorsal to the mandibular incisor and molar pulp-responding regions. Although S1 and S2/IOR play their own roles in nociceptive information processing, the anatomical and physiological differences in the temporal activation kinetics, dependency on stimulation intensity, and additive or summative effects of simultaneous pulpal stimulation are still unknown. This information contributes not only to understanding topographical organization but also to speculating about the roles of S1 and S2/IOR in clinical aspects of pain regulation. In vivo optical imaging enables investigation of the spatiotemporal profiles of cortical excitation with high resolution. We determined the distinct features of optical responses to nociceptive stimulation of dental pulps between S1 and S2/IOR. In comparison to S1, optical signals in S2/IOR showed a larger amplitude with a shorter rise time and a longer decay time responding to maxillary molar pulp stimulation. The latency of excitation in S2/IOR was shorter than in S1. S2/IOR exhibited a lower threshold to evoke optical responses than S1, and the peak amplitude was larger in S2/IOR than in S1. Unexpectedly, the topography of S1 that responded to maxillary and mandibular incisor and molar pulps overlapped with the most ventral sites in S1 that was densely stained with cytochrome oxidase. An additive effect was observed in both S1 and S2/IOR after simultaneous stimulation of bilateral maxillary molar pulps but not after contralateral maxillary and mandibular molar pulp stimulation. These findings suggest that S2/IOR is more sensitive for detecting dental pulp sensation and codes stimulation intensity more precisely than S1. In addition, contra- and ipsilateral dental pulp nociception converges onto spatially closed sites in S1 and S2/IOR.

Characterization of mesenchymal progenitor cells in crown and root pulp from human mesiodentes.

OBJECTIVE: Mesiodentes are usually found in the central position of the upper or lower jaw as supernumerary teeth. Here, we obtained 10 mesiodentes and three permanent teeth (PT) and separated the dental pulp (DP) from these into crown and root portions. We then characterized and compared the isolated crown portion-derived cells (crown cells) with root portion-derived cells (root cells) using a range of in vitro assays. MATERIALS AND METHODS: Crown cells and root cells were examined for cell surface marker expression, colony-forming unit-fibroblast (CFU-F), cell proliferation, cell cycle characteristics and markers, and osteogenic and adipogenic differentiation. RESULTS: The proportion of CD105-positive cells (CD105(+) cells) in the crown cells vs the root cells varied among the mesiodentes, but not among the PT. When there were more CD105(+) cells in the root cells than in the crown cells, the root cells showed higher CFU-F, proliferation capacity, and osteogenic differentiation capacity. In contrast, when the crown cells contained more CD105(+) cells than the root cells, the crown cells showed the higher CFU-F, proliferation capacity, and osteogenic differentiation capacity. In addition, the sorted CD105(+) cells showed higher CFU-F and proliferation capacity than the sorted CD105(-) cells. CONCLUSION: These results indicated that proportion of CD105(+) cells is an effective means of characterizing DP-derived cells in mesiodentes.

Effectiveness of the analysis of craniofacial morphology and pharyngeal airway morphology in the treatment of children with obstructive sleep apnoea syndrome.

OBJECTIVE: In general, no consensus has been reached regarding the diagnostic criteria for obstructive sleep apnoea syndrome (OSAS) in children and the criteria for selecting treatment are inconsistent. Therefore, the craniofacial and pharyngeal airway morphology of OSAS in children who had undergone drug therapy (non-op group) and OSAS in children who had undergone both drug therapy and surgical therapy (adenotonsillectomy) (op group) were compared. The purpose of this study was to examine the effectiveness of craniofacial morphology and pharyngeal airway morphology analysis in the treatment of children with OSAS. METHODS: The craniofacial and pharyngeal airway morphology of the control group, the non-op group and the op group were compared to examine the differences of each group. The comparison used Mann-Whitney's U test. RESULTS: A comparison between the non-op and the op groups showed significant differences in the facial axis, mandibular plane angle, ramus plane to the SN (porion and orbit) point, point Pog (pogonion) to the McNamara line, anteroposterior dysplasia indicator (APDI), D-AD1 [the distance between the posterior nasal spine (PNS) point and the nearest adenoid tissue, measured along the PNS-Ba (basion) point plane], D-AD2 (the distance between the PNS point and the nearest adenoid tissue, measured along a line from the PNS point perpendicular to the S (sella turcica)-Ba point plane), upper pharynx and soft palatal length. The op group showed significantly lower values of APDI than the non-op group, indicating that the op group showed a significant occlusion of class II, and that the mandibular bone was positioned posteriorly relative to the maxillary bone. CONCLUSIONS: The op group showed a significant posterior position and backward rotation of the mandibular bone, stenosis of the nasopharyngeal airway and an elongated soft palate compared with the non-op group, and it was speculated that there was a high probability of the necessity of surgical therapy (adenotonsillectomy) when a morphological factor played a major role as a cause of obstructive sleep apnoea. We recommend craniofacial morphology analysis and pharyngeal airway morphology analysis in the diagnosis and treatment planning of OSAS children.

E10A, an adenovirus-carrying endostatin gene, dramatically increased the tumor drug concentration of metronomic chemotherapy with low-dose cisplatin in a xenograft mouse model for head and neck squamous-cell carcinoma.

Most cancer chemotherapeutic agents are administered at the maximum-tolerated dose (MTD) in short cycles with treatment breaks. However, MTD-based chemotherapies are often associated with significant toxicity and treatment breaks allow the opportunity for tumor regrowth and acquisition of chemoresistance. To minimize these drawbacks, a metronomic strategy, in which chemotherapeutics are administered at doses significantly below the MTD without treatment breaks, has been suggested by many investigators. The antitumor effect of metronomic chemotherapy may be partially due to inhibition of tumor angiogenesis, and it could be enhanced by a combination therapy, including antiangiogenic agents. In this study, we evaluated the synergistic effect of E10A, an adenovirus carrying the endostatin gene, the most potent inhibitors of tumor angiogenesis, in combination with weekly low-dose cisplatin in a xenograft mouse model for head and neck squamous-cell carcinoma. The E10A induced mRNA and protein expressions of endostatin in H891 cells in vitro. E10A significantly enhanced the in vivo tumor growth inhibitory effect of cisplatin. Immunohistochemical analysis with a TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling) assay and anti-CD31 antibodies revealed that the combination of E10A and cisplatin induced high levels of cell apoptosis and inhibited tumor angiogenesis. Importantly, E10A increased the platinum concentrations in tumors to fivefold higher than that induced by cisplatin alone.

Efficient antitumor effects of carrier cells loaded with a fiber-substituted conditionally replicating adenovirus on CAR-negative tumor cells.

Carrier cells delivering a conditionally replicating adenovirus (CRAd), which selectively replicates in tumor cells and induces tumor cell lysis, have promising potential for treatment of cancer because CRAd-loaded carrier cells evade inhibition by neutralizing anti-adenovirus (Ad) antibodies and because the carrier cells are locally retained at the injection point after local injection. A previous study by Hamada et al. demonstrated that carrier cells (CRAd-containing cell fragments derived from the carrier cells) are engulfed into the target cells, probably through a pathway independent of the primary receptor for Ad, the coxsackievirus and Ad receptor (CAR) (Mol Ther, 15: 1121-1128; 2007); however, it remains to be elucidated whether carrier cells infected with a conventional CRAd, which is composed of subgroup-C Ad serotype-5 (Ad5), mediate antitumor effects on CAR-negative cells. In order to examine whether carrier cells delivering a conventional CRAd (Carrier-F5) induce lysis of CAR-negative tumor cells, CAR-positive and CAR-negative tumor cells were incubated with Carrier-F5. Carrier-F5 mediated efficient killing of CAR-positive tumor cells; however, CAR-negative tumor cells were almost refractory to Carrier-F5. On the other hand, carrier cells loaded with a fiber-substituted CRAd containing fiber proteins of Ad serotype-35 (Ad35) (CRAd-F35), which binds to human CD46 for infection, showed efficient killing of both CAR-positive and CAR-negative tumor cells. Intra-tumoral injection of carrier cells loaded with CRAd-F35 (Carrier-F35) also resulted in efficient regression of both CAR-positive and CAR-negative tumors. These results demonstrated that the expression levels of receptors for Ad are an important factor for CRAd-loaded carrier cell-mediated cancer therapy, and that Carrier-F35 would have potential as a cancer treatment for not only CAR-positive tumors but also CAR-negative tumors.

In vitro and in vivo inhibitory effect of three Cox-2 inhibitors and epithelial-to-mesenchymal transition in human bladder cancer cell lines.

BACKGROUND: Although the anti-tumour effect of cyclooxygenase-2 (Cox-2) inhibitors in invasive bladder cancer has been confirmed, its mechanisms of action are unclear. Recently, the concept of an epithelial-to-mesenchymal transition (EMT) promoting carcinoma progression has been suggested, and a key feature of the EMT is the downregulation of E-cadherin. In this study, we investigated the effect of Cox-2 inhibitors on reversal EMT and tumour growth inhibition in bladder cancer cells. METHODS: We used three Cox-2 inhibitors, etodolac, celecoxib and NS-398 and three human bladder cancer cell lines, T24, 5637 and KK47, in this study. T24 xenograft tumour mouse model was used in the in vivo study. RESULTS: Within the clinical drug concentrations, only etodolac showed the in vitro growth inhibition in T24 not in the other cell lines. Etodolac reduced SNAIL mRNA and vimentin cell surface expression, and induced E-cadherin mRNA and E-cadherin cell surface expression, in T24. Etodolac also most strongly inhibited the cell migration of T24 in vitro and showed the highest tumour growth inhibition in T24 tumour in vivo. CONCLUSION: Etodolac at clinical doses exhibited induced in vitro and in vivo anti-tumour effects and reversal effect of EMT in T24. These results suggest that etodolac is a good candidate for an anti-tumour or chemopreventive reagent for high-grade bladder cancer.

In vivo morphometric analysis of inflammatory condylar changes in rat temporomandibular joint.

OBJECTIVE: Injection of complete Freund's adjuvant (CFA) into the temporomandibular joint (TMJ) causes acute swelling around the joint and subsequent morphological alterations in the condyle. We aimed to evaluate changes in the three-dimensional architecture of the condyle induced with CFA. MATERIALS AND METHODS: The CFA was injected into the unilateral TMJ of rats and morphological changes in the condyle were assessed repeatedly for 14 days by in vivo micro-CT. RESULTS: Osseous abnormalities of condyle were first observed at 3-5 days after CFA injection on the tomographic images, and the condylar deformation became more obvious thereafter. Among 12 condyles examined at 14 days postinjection, osteophytosis was observed in all of the specimens and bone erosion coexisted in five condyles. None of the saline-treated condyles showed architectural changes. Significant changes were detected in the mesiolateral and rostrocaudal widths of the CFA-treated condyles at 10-14 days postinjection (P < 0.01). The extent of both condylar bone formation and resorption was greater in the CFA-injected TMJs than in saline-injected TMJs (P < 0.05). CONCLUSION: These results indicate that CFA causes dynamic morphological changes in the condyle and that our experimental approach will provide new insights into the subacute inflammatory processes in the TMJ.

Insulin facilitates repetitive spike firing in rat insular cortex via phosphoinositide 3-kinase but not mitogen activated protein kinase cascade.

The insular cortex (IC) processes gustatory and visceral information, which functionally correlate to feeding behavior. Insulin, a well-known hormone controlling glucose metabolism, is released by elevation of blood glucose concentration following feeding behavior. The IC expresses dense insulin receptors and receives projection from the hypothalamus, which monitors changes in glucose concentration. Therefore, it is likely that insulin modulates neural properties in the IC. However, little is known about the effects of insulin on electrophysiological properties of the neocortex including the IC. To explore the effects of insulin on subthreshold responses and action potential properties in the IC, intracellular recording with sharp glass electrodes was performed from IC pyramidal cells using slice preparations. Although application of insulin (100 nM) had little effect on the resting membrane potential, input resistance and rheobase, insulin significantly increased the frequency of repetitive spike firing in response to a long depolarizing current pulse injection: the slope of the frequency-current curve was increased from 23.7±2.3 Hz/nA to 29.5±3.4 Hz/nA. Insulin slightly decreased the action potential threshold without affecting the amplitude of medium-duration and slow afterhyperpolarization (sAHP) s. The insulin-induced facilitation of repetitive spike firing was dose-dependent and blocked by pre-application of 200 nM lavendustin A, a tyrosine kinase inhibitor. Moreover, when combined with 200 nM wortmannin, a phosphoinositide 3-kinase (PI3-K) inhibitor, or 500 nM deguelin, an inhibitor of protein kinase B (PKB/Akt) downstream of PI3-K, insulin failed to increase the frequency of repetitive spike firing. In contrast, co-application of insulin and (10 µM) PD 98059, an inhibitor of mitogen activated protein kinase (MAPK), exerted facilitation of repetitive spike firing. These results suggest that acute insulin-induced facilitation of firing frequency is at least partially induced by hyperpolarizing effects on the action potential threshold, and that this facilitation is induced by activation of PI3-K but not MAPK cascade.

Two new variants of G6PD deficiencies in Singapore.

Identification of mutations in G6PD gene is performed as an epidemiologic investigation of G6PD deficiency in many countries. In order to understand the hereditary background of G6PD deficiency in a population, screening of mutations is required not only in exonic regions but also for intron and promoter regions. One hundred male neonatal samples diagnosed as with G6PD deficiency by newborn screening in Singapore were used in this study. The multiplex PCR using the multiple tandem forward primers and common reverse primer (MPTP) method was carried out to detect the common 11 mutations in south-east Asia such as Gaohe 95A>G, Orissa 131C>G, Vanua-Lava 383T>C, Mahidol 487G>A, Mediterranean 563C>T, Coimbra 592C>T, Viangchan 871G>A, Chatham 1003G>A, Union 1360C>T, Canton 1376G>T and Kaiping 1388G>A. Samples whose mutations were unidentified by MPTP method were scanned at cording region, intron and promoter region by direct sequencing.Out of 100 samples, 90 samples (90.0%) were identified with one of the above mentioned common mutations. Eight out of 10 samples whose mutations were unidentified by MPTP method carried exonic mutations which had been previously reported such as Murcia 209A>G, Quing Yuan 392G>T, Nankang 517T>C, Chinese5 1024C>T. Two novel mutations were identified in these samples: one had a novel mutation (25C>T); the remaining sample carried a 49 bp deletion in intron 12.

Diagnostic accuracy of microcomputed tomography for osseous abnormalities in the rat temporomandibular joint condyle.

OBJECTIVES: Our aim was to investigate the diagnostic accuracy of in vivo micro-CT for osseous abnormalities of the rat temporomandibular joint (TMJ) condyle, using macroscopic observations as the "gold standard". METHODS: A 30 TMJ arthritis model was prepared by injecting inflammatory complete Freund's adjuvant (CFA) into one side of the TMJ cavities of rats. The TMJ condyles were then imaged using micro-CT. The samples were macroscopically evaluated for osseous abnormalities, including erosions, osteophytes, flattening and concavity. The micro-CT images were independently assessed for abnormalities using the same criteria. Images in three planes were produced using the micro-XYZ technique with the micro-CT equipment. RESULTS: According to the macroscopic observations, 26 of the 60 rat condyles showed osseous abnormalities. The micro-XYZ images detected abnormalities in 25 of the condyles. The condyle diagnostic accuracy of micro-CT was 0.98, the sensitivity was 0.96 and the specificity was 1.0. CONCLUSIONS: Good diagnostic results were obtained using micro-CT. It is therefore an effective technique for the evaluation of osseous abnormalities in the rat TMJ condyle.

Promoter polymorphisms in the IRF3 gene confer protection against systemic lupus erythematosus.

In order to identify a novel candidate gene in systemic lupus erythematosus (SLE), we analysed a panel of six genes encoding molecules involved in the type I interferon (IFN) system. We first identified variants in the five genes related to type I IFN pathway by sequencing. Genotyping of a panel of eight selected single-nucleotide polymorphisms (SNPs) in six candidate genes (TLR9, MYD88, IRF3, IRF7, IFNB1, IFNA17) was performed in 137 patients with SLE and matched with 152 healthy controls using polymerase chain reaction-restriction fragment length polymorphism analysis. In functional assay, quantitative real-time polymerase chain reaction was performed to assess constitutive IRF3 mRNA expression in peripheral blood mononuclear cells from healthy subjects with different IRF3 promoter haplotypes. Among eight SNPs genotyped, an IRF3 SNP at -925 was found to be associated with SLE after correction for multiple tests (corrected P=0.016). Of total five IRF3 SNPs genotyped, the promoter IRF3 SNPs -925A/G and -776C/T showed the most significant association with SLE. With regard to -925A/G, the frequency of GG genotype was significantly decreased among SLE patients compared with the control group (1.5% vs. 9.9%; chi(2)=10.0, P=0.0015, odds ratio 0.12, 95% confidence interval 0.027-0.554). Our experimental data indicated that constitutive IRF3 mRNA expression was significantly lower in cells carrying the minor G-T/G-T haplotype pair compared with those carrying the major A-C haplotype. In conclusion, we showed that the promoter SNPs of the IRF3 gene were significantly associated with resistance against SLE.

FK506-protective effects against trimethyltin neurotoxicity in rats: hippocampal expression analyses reveal the involvement of periarterial osteopontin.

There is little information on the molecular mechanisms in FK506-mediated neuroprotection. In the present study, we investigated the protective effect of FK506, an immunosuppressant and neuroprotectant, on trimethyltin (TMT)-induced neurotoxicity in the rat hippocampus. Histologically, TMT-induced neuronal damage was partially prevented by FK506 in the hippocampal CA1 region, but not in CA3. FK506 treatment significantly reduced the number of apoptotic cells in CA1, but not in CA3, and also prevented induction of cognitive deficits by TMT. Microarray analysis of the rat hippocampus detected 14 genes with TMT-induced alteration of mRNA expression that was rescued by FK506 treatment. Subsequent quantitative RT-PCR analysis confirmed elevated mRNA levels for four inflammatory genes, glutathione S-transferase, lysozyme, matrix Gla protein, and osteopontin after TMT treatment. Upregulation of these genes was reversed by FK506 treatment at 5 days postgavage. Immunohistochemistry revealed that FK506 reduced osteopontin (OPN) induction by TMT in the periarterial area at 5 days postgavage. Our data suggest that inflammatory gene expression is involved in TMT-induced damage to the hippocampal CA1 region, resulting in apoptosis, and that this process is initiated by periarterial OPN activation, and can be alleviated by FK506.

Mutation screening of the CARD15 gene in sarcoidosis.

CARD15 was first identified as a susceptibility gene for Crohn's disease. More recently, CARD15 mutations were shown to be associated with the pediatric granulomatous inflammatory diseases, Blau syndrome and early-onset sarcoidosis (EOS). The aim of the present study was to evaluate whether CARD15 variants also play a role in patients with ordinary sarcoidosis other than EOS. We enrolled 135 Japanese sarcoidosis patients with uveitis as well as 95 healthy individuals and performed mutation analysis by direct sequencing of CARD15 exon 4. Direct DNA sequencing in the sarcoidosis patients showed eight CARD15 variants, including five novel mutations (13402C>T, 13543C>T, 13775C>A, 13937G>A, and 14079C>T). Compared with healthy individuals, CARD15 mutations are not common in the Japanese patients with sarcoidosis. Based on the results, we examined the clinical manifestations in patients with sarcoidosis according to their CARD15 mutations. Sarcoidosis patients with these mutations have no specific clinical features with regard to course of the disease or disease severity. Our results indicate that in general, CARD15 mutations may not contribute to the risk of sarcoidosis.