Profile of skin barrier proteins (filaggrin, claudins 1 and 4) and Th1/Th2/Th17 cytokines in adults with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) in adults and profile of skin barrier proteins and inflammatory cytokines. OBJECTIVE: Evaluation of the expression of skin barrier proteins such as filaggrin, claudins 1 and 4 and of circulating inflammatory cytokines (Th1/Th2/Th17) in adults with AD. METHODS: Thirty-three adult patients with AD diagnosed according to the Hanifin & Rajkacriteria, and 25 healthy controls were enrolled in the study. AD severity was measured by Eczema Area and Severity Index (EASI). Laboratory assays included immunohistochemistry analysis of skin barrier proteins, such as filaggrin, claudins 1 and 4 and interleukin-17 (IL-17) from skin samples and determination of circulating cytokine levels (IL-2, 4, 5, 6, 10, 17A, TNF and IFN-γ) by flow cytometry (Cytometric Bead Array). RESULTS: We observed a reduced expression of filaggrin and claudin 1 in lesional skin of AD patients, when compared to controls. There was an inverse correlation of filaggrin expression and disease severity. In addition, IL-17 expression was enhanced in AD patients. Similarly, higher levels of inflammatory cytokines (IL-2, 5, 6, 10, 17A and IFN-γ) were found in AD patients. CONCLUSION: Our data reinforce the role of an altered skin barrier in the pathogenesis of AD. Our results show not only reduced expression of filaggrin and claudin 1 in lesional atopic skin but also inverse correlation of filaggrin expression and disease severity. Moreover, elevation of in situ IL-17 and of circulating interleukin levels in AD emphasize the systemic, inflammatory profile of this defective skin barrier dermatosis.

Analysis of T cell receptor Vbeta diversity in peripheral CD4 and CD8 T lymphocytes in patients with autoimmune thyroid diseases.

Autoimmune thyroid diseases are characterized by intrathyroidal infiltration of CD4(+) and CD8(+) T lymphocytes reactive to self-thyroid antigens. Early studies analysing T cell receptor (TCR) Valpha gene usage have shown oligoclonal expansion of intrathyroidal T lymphocytes but not peripheral blood T cells. However, TCR Vbeta diversity of the isolated CD4(+) and CD8(+) T cell compartments in the peripheral blood has not been characterized fully in these patients. We performed complementarity-determining region 3 (CDR3) spectratyping as well as flow cytometric analysis for the TCR Vbeta repertoire in peripheral CD4(+) and CD8(+) T cells from 13 patients with Graves' disease and 17 patients with Hashimoto's thyroiditis. Polyclonal TCR Vbeta repertoire was demonstrated by flow cytometry in both diseases. In contrast, CDR3 spectratyping showed significantly higher skewing of TCR Vbeta in peripheral CD8(+) T cells but not CD4(+) T cells among patients with Hashimoto's thyroiditis compared with healthy adults. We found trends towards a more skewed CDR3 size distribution in those patients having disease longer than 5 years and requiring thyroid hormone replacement. Patients with Graves' disease exhibited no skewing both in CD4(+) and CD8(+) T cells. These findings indicate that clonal expansion of CD8(+) T cells in Hashimoto's thyroiditis can be detected in peripheral blood and may support the role of CD8(+) T cells in cell-mediated autoimmune attacks on the thyroid gland in Hashimoto's thyroiditis.

Score for Neonatal Acute Physiology and Perinatal Extension II as a predictor of retinopathy of prematurity: study in 304 very-low-birth-weight preterm infants.

AIM: To evaluate the use of the Score for Neonatal Acute Physiology and Perinatal Extension (SNAPPE-II) at admission to predict the development of retinopathy of prematurity (ROP) among very-low-birth-weight preterm babies. METHODS: A prospective cohort study included 304 infants screened for ROP from July 2004 to October 2007. The main outcomes were the development of any stage ROP and severe ROP. The main variable was the SNAPPE-II obtained at admission. Seventeen risk factors for ROP were studied by univariate analysis (chi(2) and Student's t test). A simple descriptive analysis was used for the SNAPPE-II (mean, median, standard deviation and interquartile range: p25-p75). Logistic regression and receiver-operating characteristic (ROC) curve were calculated for SNAPPE-II. Ophthalmological examinations started at the 6th week of life and were repeated until the 45th week of corrected gestational age (GA). RESULTS: The mean GA and mean birth weight of the whole cohort were 30.3 weeks (+/-2.2) and 1,209.2 g (+/-277.7), respectively. The median SNAPPE-II among non-ROP and ROP patients were 6.0 and 15.0, respectively (p = 0.001). When compared with severe ROP patients (25.0) there was also a significant difference (p = 0.003). After logistic regression, the SNAPPE-II adjusted odds ratio for ROP was 1.024. The area under the ROC curve was 0.62 (95% confidence interval: 0.55-0.70, p < 0.001). The best discriminative cutoff value was 8.5 (sensitivity: 68%; specificity: 54%; positive predictive value: 37.3%; negative predictive value: 80.6%). CONCLUSIONS: The SNAPPE-II values at admission were significantly higher among babies with ROP, suggesting a positive association between higher scores with the development of ROP, but after adjusted logistic regression and ROC curve results, the SNAPPE-II scores at admission did not enhance the assessment of risk for ROP.

Class IIa bacteriocins: biosynthesis, structure and activity.

In the last decade, a variety of ribosomally synthesized antimicrobial peptides or bacteriocins produced by lactic acid bacteria have been identified and characterized. As a result of these studies, insight has been gained into fundamental aspects of biology and biochemistry such as producer self protection, membrane-protein interactions, and protein modification and secretion. Moreover, it has become evident that these peptides may be developed into useful antimicrobial additives. Class IIa bacteriocins can be considered as the major subgroup of bacteriocins from lactic acid bacteria, not only because of their large number, but also because of their activities and potential applications. They have first attracted particular attention as listericidal compounds and are now believed to be the next in line if more bacteriocins are to be approved in the future. The present review attempts to provide an insight into general knowledge available for class IIa bacteriocins and discusses common features and recent findings concerning these substances.

Tumor-suppressive function of mutated gelsolin in ras-transformed cells.

The flat revertant R1, isolated from human activated Ha-ras oncogene-transformed NIH3T3 fibroblasts (EJ-NIH3T3), expresses a variant form of the actin-regulatory protein gelsolin (p92-5.7). We have cloned cDNAs encoding p92-5.7 and identified as the cause of the expression of p92-5.7 a point mutation in codon 321, which results in an amino acid change from proline to histidine. In order to understand the role of p92-5.7 in reversion of ras-transformed cells, cDNAs encoding p92-5.7 or human authentic gelsolin as a control were transfected into EJ-NIH3T3 cells. All the transfectants that produced p92-5.7 and one of three transfectants that produced human authentic gelsolin either lost or reduced tumorigenicity in syngeneic mice. These results demonstrate that mutated gelsolin can suppress a ras tumor and suggest that authentic gelsolin, if expressed at increased levels, may have a similar suppressive potential. Our data propose an important role for gelsolin in cellular signal transduction pathways that involve the mammalian ras proto-oncogene.

Two-year outcome of first-line antiretroviral therapy among HIV-1 vertically-infected children in Hanoi, Vietnam.

A retrospective analysis of 86 HIV-1 vertically-infected Vietnamese children with a follow-up period >24 months after initiating antiretroviral therapy (ART) was performed from 2008 to 2012, to assess the outcome of first-line ART in resource-limited settings. Of the 86 children, 68 (79.1%) were treated successfully (plasma HIV-1 viral load [VL] <1000 copies/ml), and 63 (73.3%) had full viral suppression (VL <400 copies/ml) after 24 months of ART. No significant difference between successfully treated patients and failure groups was observed in VL, CD4(+) T-cell count or clinical stage at baseline; age at ART start; or ART regimen. All 14 children with VL >5000 copies/ml, one of four children with VL 1000-5000 copies/ml and none with VL <1000 copies/ml developed reverse transcriptase inhibitor (RTI)-resistance mutations by 24 months of ART. Y181C and M184V/I were the most dominant non-nucleoside and nucleoside RTI-resistance mutations, respectively (13/15, 86.7%). These findings suggest that VL testing after 24 months of ART can be used to efficiently differentiate ART failures among HIV-1 vertically-infected children in resource-limited settings.

Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis.

Two Pseudomonas aeruginosa genes, termed phaJ1(Pa) and phaJ2(Pa), homologous to the Aeromonas caviae (R)-specific enoyl-CoA hydratase gene (phaJ(Ac)) were cloned using a PCR technique to investigate the monomer-supplying ability for polyhydroxyalkanoate (PHA) synthesis from beta-oxidation cycle. Two expression plasmids for phaJ1(Pa) and phaJ2(Pa) were constructed and introduced into Escherichia coli DH5alpha strain. The recombinants harboring phaJ1(Pa) or phaJ2(Pa) showed high (R)-specific enoyl-CoA hydratase activity with different substrate specificities, that is, specific for short chain-length enoyl-CoA or medium chain-length enoyl-CoA, respectively. In addition, co-expression of these two hydratase genes with PHA synthase gene in E. coli LS5218 resulted in the accumulation of PHA up to 14-29 wt% of cell dry weight from dodecanoate as a sole carbon source. It has been suggested that phaJ1(Pa) and phaJ2(Pa) products have the monomer-supplying ability for PHA synthesis from beta-oxidation cycle.

Control of acetic acid concentration by pH-stat continuous substrate feeding in heterotrophic culture phase of two-stage cultivation of Alcaligenes eutrophus for production of P(3HB) from CO2, H2, and O2 under non-explosive conditions.

A-two stage culture method of hydrogen-oxidizing bacterium, Alcaligenes eutrophus, is used to produce poly-D-3-hydroxybutyrate, P(3HB) from CO2, O2, and H2 without using a very high oxygen transfer rate while maintaining the O2 concentration in gas phase below 6.9 (v/v)% to prevent detonation of the gas mixture. The two-stage method consists of a heterotrophic culture using fructose as carbon source for exponential cell growth and an autotrophic culture for P(3HB) accumulation. We investigated the use of acetic acid as a cheaper carbon source than fructose for the heterotrophic culture in the two-stage method. However, the acetate concentration in the culture system must be maintained at 1.0 g. dm-3 since its inhibitory effect on the cell growth is very strong. Then, high cell density cultivation of A. eutrophus was investigated by pH-stat continuous feeding of acetic acid to control acetate concentration. As a result, acetate concentration was automatically maintained around 1.0 g. dm-3 by using a feed with a composition in CH3COOH/CH3COONH4/KH2PO4 molar ratio of 5:1:0.084. Cell concentration increased to 48.6 g. dm-3 after 21 h of cultivation. The cell mass grown in the fed-batch culture on acetic acid was useful for P(3HB) production from CO2 in the subsequent autotrophic culture stage. Copyright 1999 John Wiley & Sons, Inc.

Papillary adenoma of the distal common bile duct.

A 73-year-old man with a papillary adenoma located in the distal common bile duct is reported. He underwent pylorus-preserving pancreatoduodenectomy. The lesion in the common bile duct featured papillary proliferation of the epithelium and fibrous elements with diffuse infiltration by inflammatory cells. Positive staining for MIB-1 (Ki-67) and p53 was identified in the nuclei of the proliferative epithelium. These findings suggested the malignant potential of this lesion. Further progress in imaging diagnostic techniques should increase the frequency with which such lesions are discovered. Even now, if mural irregularities and defects are found in the extrahepatic biliary system, especially the distal common bile duct, the possibility of such borderline biliary adenoma should be taken into consideration when making a diagnosis.

Biochemical and genetic evidence for production of enterocins A and B by Enterococcus faecium WHE 81.

Enterococcus faecium WHE 81, isolated from cheese, has been reported to produce a bacteriocin called "enterocin 81" [J. Appl. Microbiol. 85 (1998) 521.]. Purification of "enterocin 81" was carried out using ammonium sulfate precipitation, desalting on ODP-90 reverse-phase column, and purification through SP Sepharose HP cation exchange and C2/C18 reverse-phase chromatographies. The antimicrobial was eluted from the C2/C18 column as four individually active fractions, designated A81, B81, C81 and D81. The purification procedure used proved particularly efficient for the bacteriocin in fraction D81, with a yield of 46%, while only 3.8% the bacteriocin in fraction B81 could be collected. MALDI-TOF mass spectrometry of the bacteriocins in fractions B81 and D81 showed respective masses of 4,833.0 and 5,462.2 Da. Amino acid sequencing of the two peptides revealed two class-II bacteriocins whose sequences were similar to those of enterocin A and enterocin B, respectively. Using proper primers, chromosomal fragments of 212 and 216 bp enclosing bacteriocin structural genes were PCR-amplified. Cloning of the amplicons and their sequencing revealed two genes with sequences identical to the structural genes of enterocins A and B, respectively. It was therefore clearly established that E. faecium WHE 81 produces bacteriocins respectively identical to enterocins A and B. Our results, combined with data from previous reports, suggest that the two bacteriocins may be widespread among enterococcal strains and may play an important role in controlling the growth of pathogens and other undesirable bacteria in certain fermented food products.

Electroencephalographical study of the Rett syndrome with special reference to the monorhythmic theta activities in adult patients.

The long-term course of the EEG in the Rett syndrome is documented with reference to past studies. The monorhythmic theta activities in the waking state which were characteristically seen in adults with the Rett syndrome were investigated in patients aged between 27 and 28, using auto power spectra. They were often suppressed when the patients were excited or strained. Then the suppression was thought to elevate their conscious levels. The theta activities were often suppressed by calling, but not easily suppressed by other stimuli, especially the somatosensory stimuli of pain and touch. There were also abnormal findings in the ABR and blink reflexes. In the Rett syndrome we, therefore, suspect there are disturbances in the brain stem functions especially in the ascending reticular activating system which is related to elevation of the conscious level.

Multimodality evoked potentials in severe athetoid cerebral palsy: correlation with clinical features and all-night polygraphical data.

We examined the correlation between the clinical and electrophysiological features, short latency somatosensory evoked potentials (SSEPs), auditory brainstem responses (ABRs), electrically elicited blink reflexes (BRs) and all-night polysomnographical examination (PSG) data in eight patients with severe athetoid cerebral palsy (ACP). Absence ABRs were observed in cases who had suffered from severe neonatal hyperbilirubinemia (posticteric ACP), and in most of them gaze abnormalities and a significant reduction in rapid eye movements during REM sleep, as observed on PSG, coexisted. Prolongation of the interpeak latency, N13-N20, of SSEPs existed concurrently with disturbed late components of BRs in two cases of posticteric ACP. The phasic contractions of the submental muscle during sleep were impaired in most of the patients. Multimodality evoked potentials together with PSGs seem to be useful for assessing brainstem dysfunctions in ACP and might also be of use for elucidating the pathogenesis of the episodic sudden death in ACP.

Longitudinal observation of electroencephalograms in the Rett syndrome.

The long-term electroencephalographical observations were documented during the course of the Rett syndrome in 8 patients. The abnormal EEG findings changed with age and the clinical stage. In the premonitory stage (birth-1.5 years of age), EEG seemed to be normal. In the acute exacerbation stage (1.5-5 years old), occipital dominant alpha wave-like activity was characteristic and persisted for a few years, but it disappeared afterwards along with the seizure activity. At around 4 years of age, when the whole spectrum of symptoms manifested, the EEG background activity in the waking stage showed higher amplitude, lower frequency and more irregular than normal. Sleep spindles were hardly observed, while frequent seizure activity was found on sleep EEG. Before the onset of epileptic attacks, paroxysmal discharges, such as diffuse spike-and-wave complexes, were noted during sleep recordings. In the chronic stage (after age 6), a monotonous theta rhythm (MTR), which was not influenced by either opening or closing of the eyes but attenuated only by a big noise or strong pain stimuli, characteristically dominated the waking tracing. The MTR was generalized over both hemispheres. After age 20, the MTR tended to be more localized to the centro-parietal area. This suggests that a reduction in responsiveness to visual and other stimuli occurs in this disease, which may be attributable to a dysfunction of the reticular activating system in the brainstem.

The disposition of benzoylecgonine in maternal and fetal rats.

We tested our hypothesis that pregnancy alters the pharmacokinetic profile of benzoylecgonine, and that this metabolite accumulates in the fetus longer than in the mother. Chronically catheterized near-term pregnant and nonpregnant female Sprague-Dawley rats received an intravenous infusion of benzoylecgonine over a period of 30 min. Adult or fetal blood and tissue samples were obtained either at the end of the infusion or 6 h postinfusion for analysis of benzoylecgonine and other cocaine metabolite concentrations via gas chromatography/mass spectrometry (GC/MS). Pregnancy altered benzoylecgonine pharmacokinetics. At the end of the infusion, benzoylecgonine concentration in the fetal plasma was markedly lower than in the maternal plasma with a fetal/maternal ratio of 0.14+/-0.01. A significantly lower concentration of benzoylecgonine was found in both maternal and fetal brain at 0 h postinfusion, with tissue/plasma concentration ratios of 0.04 and 0.24, respectively, suggesting that benzoylecgonine does not readily penetrate into the brain. At 6 h, the fetal concentration of benzoylecgonine was significantly higher than in the corresponding maternal blood and tissues. Ecgonine methyl ester, a metabolite of benzoylecgonine was found in the maternal liver, but not in the fetus. In addition, the amniotic fluid concentration of benzoylecgonine became significantly higher in the 6-h postinfusion samples as compared to the end of infusion value, suggesting that repeated intrauterine exposure to cocaine may cause an accumulation of benzoylecgonine in the fetus.

Development of a novel method for feeding a mixture of L-lactic acid and acetic acid in fed-batch culture of Ralstonia eutropha for poly-D-3-hydroxybutyrate production.

Fermentative production of poly-D-3-hydroxybutyrate [P(3HB)] from a mixture of L-lactic acid and acetic acid by Ralstonia eutropha was investigated. For fed-batch culture with cell density, it is necessary to control the concentration of these organic acids in the culture medium below the inhibitory level for cell growth. Therefore, a novel feeding method, termed the computer-controlled pH-stat substrate feeding method, was developed using the rate of increase of the pH (pH-increasing rate) of the culture medium as an indicator for feed control. The pH-increasing rate, which was calculated every minute by a pH meter-linked computer, represented secondary information regarding substrate consumption by cells. When the pH-increasing rate decreased to 5% of the maximum increasing rate, acidic substrate solution was fed into the fermentor until the pH was reduced to 7.00. Using this feeding strategy, the cell concentration and PHA content obtained in 42 h were 75.0 g/l and 73.1% (w/w), respectively, resulting in a high P(3HB) productivity of 1.30 g/l.h.

Class IIa bacteriocins from lactic acid bacteria: antibacterial activity and food preservation.

In the last decade, a variety of ribosomally synthesized antimicrobial peptides, or bacteriocins, produced by lactic acid bacteria have been identified and characterized. As a result of these studies, insight has been gained into various fundamental aspects of biology and biochemistry such as bacteriocin processing and secretion, mechanisms of cell immunity, and structure-function relationships. In parallel, there has been a growing awareness that bacteriocins may be developed into useful antimicrobial food additives. Class IIa bacteriocins can be considered as the major subgroup of bacteriocins from lactic acid bacteria, not only because of their large number, but also because of their significant biological activities and potential applications. The present review provides an overview of the knowledge available for class IIa bacteriocins and discusses common features and recent findings concerning these substances. The activity and potential food applications of class IIa bacteriocins are a major focus of this review.

Investigation of the utility of pineapple juice and pineapple waste material as low-cost substrate for ethanol fermentation by Zymomonas mobilis.

The utility of the juice of rotten or discarded pineapples and the waste material of the production of pineapple juice as low-cost substrates for ethanol production by Zymomonas mobilis was investigated. Z. mobilis ATCC 10988 produced 59.0 g.l(-1) ethanol in undiluted pineapple juice without nutritional supplementation and without the regulation of the pH while 42.5 g.l(-1) ethanol was obtained using a 125 g.l(-1) sucrose medium supplemented with 10 g.l(-1) yeast extract and mineral salts. Ethanol fermentation using unhydrolyzed and enzymatically hydrolyzed pineapple waste material was also investigated under various culture conditions. When a 15% (v/v) dilution of unhydrolyzed waste material without nutritional supplementation was used, more than 3.5 g.l(-1) ethanol was produced. When the media containing 15, 30, and 40% (v/v) of the hydrolyzate consisting of a 60% (v/v) suspension of pineapple waste material were used, final concentrations of ethanol were 5.0 g.l(-1), 7.6 g.l(-1), and 9.3 g.l(-1), respectively. These results suggest that pineapple juice and the waste material can be useful low-cost substrates for ethanol production by Z. mobilis without supplementation with expensive organic nitrogen complexes such as yeast extract and without the regulation of the pH during cultivation, leading to the reduction in the production costs.

Optimization of L-lactic acid feeding for the production of poly-D-3-hydroxybutyric acid by Alcaligenes eutrophus in fed-batch culture.

We investigated optimization of the feeding of L-lactic acid for the production of poly-D-3-hydroxybutyric acid [P(3HB)] by Alcaligenes eutrophus in a fed-batch culture system. An acidic substrate solution was fed automatically so as to maintain the pH of the culture liquid at 7.0. Feeding of a substrate solution containing 45% (w/v) L-lactic acid, 6.2% (w/v) sodium L-lactate, 5.8% (w/v) ammonia water and 1.8% (w/v) potassium phosphate [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10], allowed the L-lactate concentration in the culture liquid to be maintained at approximately 2 g/l and the cell concentration reached 27.4 g/l after 15 h of cultivation. To promote P(3HB) production, a two-stage fed-batch culture consisting of a culture for cell growth and one for P(3HB) accumulation was carried out. When the substrate solution, whose C N molar ratio was 23, was fed during the P(3HB) accumulation phase, the cell concentration and the P(3HB) content in the cells reached 103 g/l and 57.6% (w/w), respectively, in 51.5 h.

Extractive acetone-butanol-ethanol fermentation using methylated crude palm oil as extractant in batch culture of Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564).

The possibility of employing methylated crude palm oil (CPOE) as an extraction solvent to reduce end-product inhibition and to enhance solvent productivity in acetone-butanol-ethanol (ABE) fermentation was evaluated using oleyl alcohol as the standard butanol extractant. Fermentation was carried out at an initial glucose concentration of 90 g/l. CPOE did not inhibit the growth of the fermentative organism. Without solvent extraction, butanol production ceased after 30 h at a concentration of 15.4 g/l limiting cell growth to 3.98 g/l and glucose consumption to 62%. Applying CPOE as the extraction solvent, about 47% of the total butanol produced was extracted, glucose consumption was increased to 83% and relatively high glucose consumption rates and solvent productivities were obtained. Butanol production increased to 20.9 g/l; total ABE solvents and yield also increased from 21.2 g/l and 38% (in conventional fermentation) to 29.8 g/l and 40.4%, respectively.