Axillary lymph-node metabolic activity assessment on 18F-FDG-PET/CT in rheumatoid arthritis patients treated with biologic therapies.

Objective: Recent studies have provided new insights into the role of lymph nodes (LNs) in rheumatoid arthritis (RA). The aim of this study was to evaluate the metabolic activity of the axillary LNs in relation to that of the upper limb joints and the clinical assessment of disease activity in RA patients treated with biologic therapies.Method: 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (18F-FDG-PET/CT) scans were acquired for 64 patients with RA at baseline and after 6 months of biologic therapy, and the patients' clinical status was evaluated. The maximum standardized uptake value (SUVmax), metabolic active volume, and total lesion glycolysis (TLG) were used to assess glucose metabolism in the LNs and 12 joints. Clinical evaluations included serum markers and the Disease Activity Score based on 28-joint count-erythrocyte sedimentation rate (DAS28-ESR).Results: Changes in the SUVmax and TLG for the axillary LNs correlated significantly with those of the ipsilateral wrist joints. There was a positive correlation between the changes in the three metabolic parameters of the axillary LNs and the changes in disease activity after treatment. After 6 months of biologic therapy, all metabolic parameters for the axillary LNs in patients with a DAS28-ESR < 3.2 were significantly lower than those of patients with a DAS28-ESR ≥ 3.2.Conclusion: A relationship between the glucose metabolism of the axillary LNs and the ipsilateral wrist joints was demonstrated by the 18F-FDG-PET/CT parameters. The metabolic activity and active volume of axillary LNs may reflect the therapeutic response to the biologic treatment of RA.

Peptidylarginine deiminase is involved in maintaining the cornified oral mucosa of rats.

BACKGROUND AND OBJECTIVE: Epithelial cells derived from different regions exhibit marked differences in their differentiation capacity, allowing them to provide a suitable protective barrier. We aimed to clarify the role of peptidylarginine deiminase (PAD) in modifying the key epidermal proteins filaggrin (FLG) and keratin 1 (K1) during stratification of the rat palate and buccal mucosa. MATERIAL AND METHODS: We performed immunofluorescence, immunoblotting, PAD activity assays and 2-dimensional electrophoresis, and developed an organotypic culture model. RESULTS: PAD1 expression was highest in the palate, whereas PAD2, PAD3 and PAD4 expression was highest in the skin, suggesting the tissue-specific expression of PAD isozymes that leads to differences in calcium dependency. Immunoblotting showed that the FLG monomer, as well as its degradation products and precursor (proFLG), were most abundantly expressed in the skin but had low expression in the palate, whereas only faint proFLG expression was detected in the buccal mucosa. FLG and K1 were colocalized with PAD1 and were likely to be citrullinated in the cornified layers of the skin; this colocalization was not detected on the palatal surface, and dot-like presence of proFLG that might be citrullinated and that of PAD1 were found in the granules of the palate. Organotypic models derived from the rat palate revealed that PAD inhibition reduced the breakdown of FLG, increased its association with K1 together with epithelial compaction, and decreased permeability in a dye permeability assay. Conversely, PAD stimulation had the opposite effects. CONCLUSION: Citrullination is likely a protein modification that plays an important role in maintaining the structure and function of oral cornified mucosa in a way that is distinctly different from that of the skin.

Protein-altering variants of PTPN2 in childhood-onset Type 1A diabetes.

AIM: To examine the contribution of PTPN2 coding variants to the risk of childhood-onset Type 1A diabetes. METHODS: PTPN2 mutation analysis was carried out for 169 unrelated Japanese people with childhood-onset Type 1A diabetes. We searched for coding variants that were absent or extremely rare in the general population and were scored as damaging by multiple in silico programs. We performed mRNA analysis and three-dimensional structural prediction of the detected variants, when possible. We also examined possible physical links between these variants and previously reported risk SNPs as well as clinical information from variant-positive children. RESULTS: One frameshift variant (p.Q286Yfs*24) and two probably damaging missense substitutions (p.C232W and p.R350Q) were identified in one child each. Of these, p.Q286Yfs*24 and p.C232W were hitherto unreported, while p.R350Q accounted for 2/121,122 alleles of the exome datasets. The p.Q286Yfs*24 variant did not encode stable mRNA, and p.C232W appeared to affect the structure of the tyrosine-protein phosphatase domain. The three variants were physically unrelated to known risk SNPs. The variant-positive children manifested Type 1A diabetes without additional clinical features and invariably carried risk human leukocyte antigen alleles. CONCLUSIONS: The results provide the first indication that PTPN2 variants contribute to the risk of Type 1A diabetes, independently of known risk SNPs. PTPN2 coding variants possibly induce non-specific Type 1A diabetes phenotypes in individuals with human leukocyte antigen-mediated disease susceptibility. Our findings warrant further validation.

Spectrophotometer is useful for assessing vitiligo and chemical leukoderma severity by quantifying color difference with surrounding normally pigmented skin.

BACKGROUND: Acquired skin hypopigmentation has many etiologies, including autoimmune melanocyte destruction, skin aging, inflammation, and chemical exposure. Distinguishing lesions from normally pigmented skin is clinically important to precisely assess disease severity. However, no gold standard assessment method has been reported. We aimed to investigate whether spectrophotometers are useful for assessing vitiligo and rhododendrol (4-(4-hydroxyphenol)-2-butanol) (Rhododenol® )-induced leukoderma disease severity by quantifying skin color. METHODS: Mexameter® MX18 and CM-700d spectrophotometer were used for assessing vitiligo/leukoderma by measuring melanin index, L*a*b* color space, and ΔE*ab value, which represents the color difference between two subjects and is calculated by the values of L*a*b*. RESULTS: MX18 and CM-700d can quantitatively distinguish vitiligo/leukoderma from normally pigmented skin based on melanin index. CM-700d consistently quantified the color of vitiligo/leukoderma lesions and surrounding normally pigmented skin in L*a*b* color spaces and ΔE*ab. ΔE*ab is well correlated with melanin index and clinical appearance. CONCLUSION: ΔE*ab has been frequently used in aesthetic dentistry; however, current study is the first to use it in the measurement of skin color. ΔE*ab seems to be a useful parameter to evaluate the color contrast between vitiligo/leukoderma and surrounding normally pigmented skin and can be used to evaluate disease severity and patient's quality of life.

Next-generation sequencing for patients with non-obstructive azoospermia: implications for significant roles of monogenic/oligogenic mutations.

Azoospermia affects up to 1% of adult men. Non-obstructive azoospermia is a multifactorial disorder whose molecular basis remains largely unknown. To date, mutations in several genes and multiple submicroscopic copy-number variations (CNVs) have been identified in patients with non-obstructive azoospermia. The aim of this study was to clarify the contribution of nucleotide substitutions in known causative genes and submicroscopic CNVs in the genome to the development of non-obstructive azoospermia. To this end, we conducted sequence analysis of 25 known disease-associated genes using next-generation sequencing and genome-wide copy-number analysis using array-based comparative genomic hybridization. We studied 40 Japanese patients with idiopathic non-obstructive azoospermia. Functional significance of molecular alterations was assessed by in silico analyses. As a result, we identified four putative pathogenic mutations, four rare polymorphisms possibly associated with disease risk, and four probable neutral variants in 10 patients. These sequence alterations included a heterozygous splice site mutation in SOHLH1 and a hemizygous missense substitution in TEX11, which have been reported as causes of non-obstructive azoospermia. Copy-number analysis detected five X chromosomal or autosomal CNVs of unknown clinical significance, in addition to one known pathogenic Y chromosomal microduplication. Five patients carried multiple molecular alterations. The results indicate that monogenic and oligogenic mutations, including those in SOHLH1 and TEX11, account for more than 10% of cases of idiopathic non-obstructive azoospermia. Furthermore, this study suggests possible contributions of substitutions in various genes as well as submicroscopic CNVs on the X chromosome and autosomes to non-obstructive azoospermia, which require further validation.

A clinical and ultrasonographic study of risk factors for elbow injury in young baseball players.

PURPOSE: To determine the risk factors for elbow injury and its association with glenohumeral internal rotation deficit among young baseball players. METHODS: 229 baseball players aged 9 to 14 (mean, 11) years completed a self-administered questionnaire with items related to years of playing baseball, hours of training per weekday, days of training per week, and past and present experience of elbow pain. Two orthopaedic surgeons measured the range of motion of both shoulders and elbows. Another 2 orthopaedic surgeons performed ultrasonography to detect any elbow abnormality such as fragmentation of the medial epicondylar apophysis and osteochondritis dissecans of the capitellum. Using univariate and multivariable analyses, participants with or without elbow abnormality were compared to determine the risk factors for elbow abnormality. RESULTS: Elbow abnormality was detected in 100 of the participants and comprised osteochondritis dissecans of the capitellum (n=18) and fragmentation of the medial epicondylar apophysis (n=82). Elbow abnormality was associated with being a pitcher, past and present experience of elbow pain, loss of elbow extension, and the side-to-side internal rotation difference. The 100 participants with elbow abnormality were stratified into symptomatic (n=57) or asymptomatic (n=43) of elbow pain. Those with elbow abnormality and elbow pain was associated with being a pitcher. CONCLUSION: Being a pitcher was a risk factor for both elbow abnormality and elbow pain. Nonetheless, 43% of baseball players with elbow abnormality were asymptomatic. The use of ultrasonography was effective in detecting elbow abnormality and enabling early treatment.

Association of caspase-14 and filaggrin expression with keratinization of the oral mucosa and reconstruction culture rat models.

BACKGROUND AND OBJECTIVE: Keratinization of the oral mucosa, such as the gingiva, has been shown to be important for periodontal health. Caspase-14 is a protease that plays a role in keratinization of the epidermis. The objective of this study was to investigate whether the expression of caspase-14 is intimately linked with keratinization and to examine the effect of the main component of green tea on the improvement of keratinization in rat oral mucosal preparations. MATERIAL AND METHODS: Histological and immunohistochemical analyses and quantitative mRNA measurements of caspase-14 and its substrate filaggrin were performed using different types of rat epithelial tissue and organotypic reconstruction culture models derived from epithelial cells and fibroblasts taken from the rat oral mucosa. RESULTS: In the skin, palate, buccal mucosa and esophagus, the degree of keratinization appeared to be associated with expression of cytokeratin 10. The relative protein and mRNA expression levels of caspase-14 and filaggrin were consistent with the degree of keratinization in the following order: skin > palate > buccal mucosa > esophagus. The culture models of palatal and buccal mucosa retained a stratified epithelial structure. Expression of caspase-14 appeared to be stronger in the palatal model than in the buccal model. Remarkably, epigallocatechin-3-gallate (EGCG) improved the localization of cytokeratins and increased the expression of caspase-14 and filaggrin. This expression was more intense in the palatal model than in the buccal model, indicating that both models maintain the intrinsic properties of keratinization of the mucosa from where the cultured cells were derived. CONCLUSIONS: These results suggest that keratinization is closely associated with expression of caspase-14 and filaggrin. Our reconstruction models are promising tools for drug evaluation and show that EGCG is beneficial for improving both keratinization and expression of the linked protease in the oral mucosa.

Involvement of IP3-receptor activation in endothelin-1-induced Ca(2+) influx in rat pulmonary small artery.

We examined the endothelin-1 (ET-1)-induced increase in the intracellular free Ca(2+) concentration ([Ca(2+)]i) in fura-2-loaded rat pulmonary small arteries. ET-1 (30 nM) elicited a long-lasting increase in [Ca(2+)]i in physiological salt solution (PSS). In subsequent experiments, arteries were pretreated with BQ-788, an ETB-specific blocker, to allow us to focus on responses mediated via the ETA receptor, the existence of which was confirmed by immunohistochemistry. In Ca(2+)-free PSS, ET-1 evoked a small transient increase in [Ca(2+)]i, indicating Ca(2+) release from the SR (sarcoplasmic reticulum). After a switch to PSS (containing 2mM CaCl2), ET-1 elicited a long-lasting increase in [Ca(2+)]i that was not inhibited by 1 µM nicardipine, an L-type Ca(2+)-channel inhibitor, suggesting involvement of a Ca(2+)-influx pathway independent of that channel. In arteries preincubated with 30 µM cyclopiazonic acid (CPA) or 2 µM thapsigargin (TG), the ET-1-induced Ca(2+)-release was greatly reduced, and the induced Ca(2+)-influx was attenuated. U-73122, a phospholipase C (PLC) inhibitor, had inhibitory effects similar to those of CPA and TG on the ET-1-induced Ca(2+)-release and Ca(2+)-influx, whereas U-73343, an inactive analogue of U-73122, had no such effects. Two putative membrane-permeable IP3-receptor blockers, 2-aminoethoxydiphenyl borate (2APB, 50 µM) and Xestospongin C (20 µM), (a) almost completely inhibited the ET-1-induced Ca(2+)-release and Ca(2+)-influx, and (b) reduced the ET-1-induced contraction. These results indicate that in rat pulmonary small arteries, ET-1 induces receptor-operated Ca(2+) influx via the ETA receptor, and that this influx interacts with InsP3-receptor activation.

Identification and characterization of novel multiple bacteriocins produced by Lactobacillus sakei D98.

AIM: To characterize novel multiple bacteriocins produced by Lactobacillus sakei D98. METHODS AND RESULTS: Lactobacillus sakei D98 isolated from Shubo (rice malt) produced at least three bacteriocins. Using three purification steps, three novel antimicrobial peptides termed sakacin D98a, sakacin D98b and sakacin D98c were purified from the culture supernatant. Amino acid and DNA sequencing analysis revealed that the sakacins D98a, D98b and D98c are novel class IIa-like or class IId bacteriocins. In particular, sakacin D98b has a variant pediocin-box sequence, YANGVXC (with Ala instead of Gly), and a different location for the disulfide bridge (Cys(11) and Cys(18)) from that found in other class IIa bacteriocins. CONCLUSIONS: Three novel bacteriocins were identified from Lactobacillus sakei D98. Their antimicrobial spectra and intensities indicate that these sakacins would have different modes of action. In addition, sakacin D98b showed low inhibitory activity against Listeria, probably due to the differences in amino acids and position of the disulfide bridge compared with the other class IIa bacteriocins. SIGNIFICANCE AND IMPACT OF STUDY: Sakacins D98a and D98c are novel bacteriocins belonging to class IId bacteriocins. On the other hand, sakacin D98b, a class IIa-like bacteriocin, has a unique internal structure and activity spectrum.

Imaging findings of neurogenic tumours in the head and neck region.

OBJECTIVE: The aim of this study was to describe the CT, MRI and ultrasonography findings of five cases of neurogenic tumours in the head and neck region. METHODS: Five neurogenic tumours were analysed with respect to their CT value, the presence of cystic change, target sign, lobulation, connection to the nerve and vascularity. RESULTS: The contrast-enhanced CT (ECT) of the schwannomas demonstrated either a mass with low enhancement (two out of three cases), which reflected the predominant Antoni B components, or a mass with cystic changes, which was an Antoni A-based schwannoma displaying cystic changes (one out of three cases). On MRI, all tumours showed homogeneous and isointense signals for muscle on T1 weighted images (T1 WIs). T2 weighted images (T2 WIs) and gadolinium (Gd)-enhanced T1 WIs demonstrated target sign in both schwannomas. Ultrasound examination showed a well-defined, ovoid or round hypoechoic mass. The direct connection to the nerve was demonstrated in two of the five cases. Lobulation was observed in only one of the five cases and cystic changes were observed in one of the five cases. In all of the cases, no vascularity was seen in power Doppler images (PDIs) obtained percutaneously. CONCLUSIONS: Low-enhanced areas on ECTs can be specific for schwannomas, which suggests the predominance of Antoni B components. The target sign on T2 WIs and Gd-enhanced T1 WIs can be specific, which can be used to differentiate the two different components (Antoni A and Antoni B). The direct connection to the nerve can be a specific finding for neurogenic tumours; however, at present the sensitivity is 40%.

CDK4 and cyclin D1 allow human myogenic cells to recapture growth property without compromising differentiation potential.

In vitro culture systems of human myogenic cells contribute greatly to elucidation of the molecular mechanisms underlying terminal myogenic differentiation and symptoms of neuromuscular diseases. However, human myogenic cells have limited ability to proliferate in culture. We have established an improved immortalization protocol for human myogenic cells derived from healthy and diseased muscles; constitutive expression of mutated cyclin-dependent kinase 4, cyclin D1 and telomerase immortalized human myogenic cells. Normal diploid chromosomes were preserved after immortalization. The immortalized human myogenic cells divided as rapidly as primary human myogenic cells during the early passages, and underwent myogenic, osteogenic and adipogenic differentiation under appropriate culture conditions. The immortalized cells contributed to muscle differentiation upon xenotransplantation to immunodeficient mice under conditions of regeneration following muscle injury. We also succeeded in immortalizing cryopreserved human myogenic cells derived from Leigh disease patients following primary culture. Forced expression of the three genes shortened their cell cycle to < 30 h, which is similar to the doubling time of primary cultured human myogenic cells during early passages. The immortalization protocol described here allowed human myogenic cells to recapture high proliferation activity without compromising their differentiation potential and normal diploidy.

Cardiac hamartoma in a young squirrel monkey who died suddenly.

A case of cardiac hamartoma in a 2-month-old squirrel monkey is reported. The monkey showed a loss of appetite and died suddenly. Microscopically, an encapsulated nodular lesion was found at the right atrial wall. The lesion consisted of irregularly shaped, slender myocytes intermingled with a few fibroblasts and collagen fibers. Neither nuclear atypia nor inflammatory cell infiltrate was seen. The constituting cells had stratified striations in the cytoplasm and reacted immunohistochemically for desmin, indicating the nature of myocytes. Based on the above findings, a diagnosis of cardiac hamartoma was made. This is the first case of cardiac hamartoma in this species.

Identification and characterization of novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15.

AIM: To characterize novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15. METHODS AND RESULTS: Leuconostoc pseudomesenteroides QU 15 isolated from Nukadoko (rice bran bed) produced novel bacteriocins. By using three purification steps, four antimicrobial peptides termed leucocin A (ΔC7), leucocin A-QU 15, leucocin Q and leucocin N were purified from the culture supernatant. The amino acid sequences of leucocin A (ΔC7) and leucocin A-QU 15 were identical to that of leucocin A-UAL 187 belonging to class IIa bacteriocins, but leucocin A (ΔC7) was deficient in seven C-terminal residues. Leucocin Q and leucocin N are novel class IId bacteriocins. Moreover, the DNA sequences encoding three bacteriocins, leucocin A-QU 15, leucocin Q and leucocin N were obtained. CONCLUSIONS: These bacteriocins including two novel bacteriocins were identified from Leuc. pseudomesenteroides QU 15. They showed similar antimicrobial spectra, but their intensities differed. The C-terminal region of leucocin A-QU 15 was important for its antimicrobial activity. Leucocins Q and N were encoded by adjacent open reading frames (ORFs) in the same operon, but leucocin A-QU 15 was not. SIGNIFICANCE AND IMPACT OF STUDY: These leucocins were produced concomitantly by the same strain. Although the two novel bacteriocins were encoded by adjacent ORFs, a characteristic of class IIb bacteriocins, they did not show synergistic activity.

Effects of rehydration on dentin strengthened by heating or UV irradiation.

Type I collagen, the major organic component of human dentin, plays an important role in regulating the mechanical strength of dentin. Collagen in dentin can be strengthened by heating. We hypothesized that UV irradiation could produce similar strengthening effects and might maintain the strength of dentin after rehydration. Beam-shaped dentin specimens from the crowns of human third molars were subjected to flexural testing. Flexural strengths were two and three times greater than those in the control group after 5 minutes' UV irradiation and heating to 140 degrees C, respectively. After 30 days of rehydration, the heated specimens reverted to their original strength, whereas the UV specimens were 69% stronger than the original. Raman spectra of dental collagen were unchanged after heating, whereas several peaks, including a C-C bond in a proline ring, were amplified by UV irradiation. It is concluded that dentin strengthened by UV irradiation retains strength after rehydration because of chemical changes in collagen.

Uracil-tegafur and tamoxifen vs cyclophosphamide, methotrexate, fluorouracil, and tamoxifen in post-operative adjuvant therapy for stage I, II, or IIIA lymph node-positive breast cancer: a comparative study.

BACKGROUND: It has been reported that treatment with uracil-tegafur (UFT) has shown significantly better survival and relapse-free survival (RFS) than surgery alone. Therefore, we compared UFT with a combination therapy of cyclophosphamide, methotrexate, and fluorouracil (CMF) in patients who had undergone curative surgery for axillary lymph node-positive breast cancer. METHODS: A total of 377 node-positive patients with stage I, II, or IIIA disease were registered from September 1996 through July 2000 and were randomly assigned to either 6 cycles of CMF or 2 years of UFT. In both arms, tamoxifen (TAM) was concurrently administered for 2 years. The primary end point in this study was the non-inferiority of UFT to CMF. RESULTS: No statistically significant difference between the two groups was observed with regard to the 5-year RFS rate (72.2% in the UFT and 76.3% in the CMF). Adverse event profiles differed between the two groups, with a significantly lower incidence of leukopenia and anaemia in the UFT group, as well as anorexia, nausea/vomiting, stomatitis, and alopecia, which have implications for quality of life. CONCLUSION: UFT administered in combination with TAM holds promise in the treatment of lymph node-positive early breast cancer. On stratified analysis, the recurrence rate in the UFT group was found to be better in oestrogen receptor (ER)-positive patients. Tegafur-based treatment should be evaluated by a prospective randomised trial conducted in ER-positive patients.

Standard steroid treatment for autoimmune pancreatitis.

OBJECTIVE: To establish an appropriate steroid treatment regimen for autoimmune pancreatitis (AIP). METHODS: A retrospective survey of AIP treatment was conducted in 17 centres in Japan. The main outcome measures were rate of remission and relapse. RESULTS: Of 563 patients with AIP, 459 (82%) received steroid treatment. The remission rate of steroid-treated AIP was 98%, which was significantly higher than that of patients without steroid treatment (74%, 77/104; p<0.001). Steroid treatment was given for obstructive jaundice (60%), abdominal pain (11%), associated extrapancreatic lesions except the biliary duct (11%), and diffuse enlargement of the pancreas (10%). There was no relationship between the period necessary to achieve remission and the initial dose (30 mg/day vs 40 mg/day) of prednisolone. Maintenance steroid treatment was given in 377 (82%) of 459 steroid-treated patients, and steroid treatment was stopped in 104 patients. The relapse rate of patients with AIP on maintenance treatment was 23% (63/273), which was significantly lower than that of patients who stopped maintenance treatment (34%, 35/104; p = 0.048). From the start of steroid treatment, 56% (55/99) relapsed within 1 year and 92% (91/99) relapsed within 3 years. Of the 89 relapsed patients, 83 (93%) received steroid re-treatment, and steroid re-treatment was effective in 97% of them. CONCLUSIONS: The major indication for steroid treatment in AIP is the presence of symptoms. An initial prednisolone dose of 0.6 mg/kg/day, is recommend, which is then reduced to a maintenance dose over a period of 3-6 months. Maintenance treatment with low-dose steroid reduces but dose not eliminate relapses.