Role of T helper 17 cells and T regulatory cells in alopecia areata: comparison of lesion and serum cytokine between controls and patients.

BACKGROUND: Alopecia areata (AA) is an organ-specific autoimmune disease with T-cell-mediated attack of hair follicle autoantigens. As T helper 17 (Th17) cells and T regulatory (Treg) cells are crucially involved in the pathogenesis, the role of Th17 and Treg cytokines has not been studied yet. OBJECTIVE: To determine whether AA is associated with alterations in lesional and serum Th17 and Treg cytokines and studied whether they were associated with clinical type. METHODS: Scalp skin samples from 45 patients and eight normal controls were obtained for PCR specific for IFN-γ, TNF-α, TGF-ß, IL-1, IL-2, IL-4, IL-10, IL-12A, IL-13, IL-17, IL-22 and IL-23. Serum cytokines were measured from 55 patients and 15 normal controls using ELISA. RESULTS: Lesional IL-17 and IL-22 were significantly increased in patient group. Moreover, positive correlations were shown between lesional IL-17, IL-22 and disease severity. Serum IL-1, IL-17, TNF-α and TGF-ß were significantly increased, and positive correlation was shown between serum IL-17 and disease severity. CONCLUSION: These results showed significantly high Th17 cytokines in both lesion and serum in AA patients, which may highlight a functional role of these cytokines in the pathogenesis of AA.

Sixteen-week analysis of unaltered elastomeric chain relating in-vitro force degradation with in-vivo extraction space tooth movement.

INTRODUCTION: The purposes of this study were to evaluate whether unaltered elastomeric chain can continue to move teeth for 16 weeks and to relate it to the amount of force remaining for the same batch of elastomeric chains. METHODS: The in-vivo portion of the study had a sample of 30 paired extraction space sites from 22 subjects who were measured for closure of the space every 28 days. The altered side elastomeric chain served as the control and was replaced at 28-day intervals whereas the experimental side remained unaltered. In the in-vitro portion of the study, 100 each of 2-unit and 3-unit segments of the same batch of elastomeric chains were placed in a water bath, and the force was measured for 20 of each segment length at the 28-day measurement points. RESULTS: Statistically significant amounts of space closure occurred at both the altered and unaltered sites at all measurement time points. The mean space closure at the altered sites was minimally greater than that observed at the paired unaltered sites. The mean differences of space closure between the altered and unaltered sites ranged from a minimum of -0.05 mm at 4 weeks to a maximum of -0.14 mm at 8 weeks. The elastomeric chain force degraded rapidly by 4 weeks but continued a gradual diminution of force to 86 g at 16 weeks. CONCLUSIONS: Unaltered elastomeric chain continued to move teeth into extraction spaces for 16 weeks in this sample from both statistically and clinically significant standpoints. There were minimal and statistically insignificant differences in the mean space closure measurements between the paired altered and unaltered sites. The elastomeric chain force at 16 weeks was less than 100 g, yet at the same time point, teeth continued to move clinically.

Polymorphisms in the promoter regions of the CXCL1 and CXCL2 genes contribute to increased risk of alopecia areata in the Korean population.

Alopecia areata (AA) is a common disease, which causes hair loss in humans. AA has a genetically complex inheritance. This study investigated the possible correlations between single nucleotide polymorphisms (SNPs) in the promoter regions of the chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) (CXCL1) and chemokine (C-X-C motif) ligand 2 (CXCL2) genes and the development of AA in the Korean population. Two hundred and thirty-five AA patients and 240 control subjects were recruited. The specific SNPs occurring in the promoter regions of the CXCL1 and CXCL2 genes (rs3117604, -429C/T and rs3806792, -264T/C, respectively) were genotyped. All data obtained was evaluated using the SNPStats, SPSS 18.0, and the Haploview v.4.2 software platforms. The Odd's ratios (OR), 95% confidence intervals (CI), and P values were calculated using multiple logistic regression models. Analyses of the genetic sequences obtained revealed a significant correlation between the two SNPs and the development of AA (rs3117604, P = 0.0009 in co-dominant model 1, P = 0.01 in co-dominant model 2, P = 0.004 in the dominant model, P = 0.005 in the log-additive model, P = 0.012 in allele distribution; rs3806792, P = 0.036 in co-dominant model 2, P = 0.0046 in the log-additive model). The TT and CC haplotypes were also observed to show a significant association with increased risk of AA (TT haplotype, P = 0.0018; CC haplotype, P = 0.0349). Our data suggests that the CXCL1 and CXCL2 genes may be associated with AA susceptibility.

Association between TAP1 gene polymorphisms and alopecia areata in a Korean population.

The transporter 1 ATP-binding cassette sub-family B (MDR/TAP) gene (TAP1) is located in the major histocompatibility complex class II region, and forms a heterodimer that plays a key role in endogenous antigen presentation pathways. Investigation of polymorphisms identified in these loci has revealed an association with several autoimmune disorders. Alopecia areata (AA) is a common autoimmune disease resulting from T cell-induced damage to hair follicles. The present study documents for the first time a comparison between the allelic and genotypic frequencies of TAP1 single nucleotide polymorphisms (SNPs) in patients with AA and those of a control group, using a direct sequencing method. Our results suggest an association between a promoter SNP (rs2071480) and susceptibility to this disease.

Unilateral eruptive vellus hair cysts occurring on the face.

Eruptive vellus hair cysts (EVHC) are small, cystic papules that usually occur on the chest and extremities. Their aetiology is unknown. Fewer than 10 cases of a variant form of EVHC that occur exclusively on the face have been reported. We describe a case of EVHC limited to the right side of the face. To the best of our knowledge, no case of unilateral EVHC has been reported.

Concentrações hormonais e desenvolvimento folicular de vacas leiteiras em hipertermia sazonal e aguda / Hormone concentration and follicular development in dairy cows under seasonal and acute hyperthermia

Avaliaram-se as concentrações hormonais e os parâmetros de desenvolvimento folicular de vacas leiteiras expostas ao calor sazonal e agudo. Dividiram-se os animais em quatro grupos: verão (n=5), outono (n=5), inverno com hipertermia aguda (grupo câmara climática, (CC), n=5) e inverno (n=9). Os animais foram abatidos no sétimo dia após a ovulação, e os parâmetros de desenvolvimento folicular avaliados. O líquido folicular do maior folículo foi aspirado e armazenado para posterior análise de hormônios esteróides e inibina. O número de células da granulosa vivas no verão e no outono foi 40 e 45 por cento respectivamente, menor que no inverno (P<0,05). A concentração de estradiol (E2) no inverno foi 62 por cento maior que no outono (P<0,05) e 34 por cento superior ao grupo verão (P<0,06). Houve um aumento na quantidade de androstenediona no verão em relação aos grupos inverno (P<0,08) e outono (P<0,05). A concentração de inibina foi maior no inverno do que no verão e CC (P<0,05). A exposição ao calor sazonal e agudo modificou os parâmetros de desenvolvimento do folículo e as concentrações hormonais no líquido folicular, podendo explicar em parte a queda nas taxas de concepção no verão.

The present study evaluated the seasonal and acute heat stress on follicular development and steroid and inhibin concentrations in follicular fluid, in bovine dominant follicle. Cows were distributed into four treatments: summer (n=5), autumn (n=5), animals heat stressed during the winter (n=5) and winter (n=9). On day 7 of the estrous cycle, animals were slaughtered and parameters related to follicle development were evaluated. The follicular fluid (FF) was aspirated and stored for further hormonal analysis. During the summer, the number of viable granulosa cells was 40 percent lower than during the winter, and there was a 45 percent decrease in this parameter during the autumn (P<0.05). In the winter, estradiol concentration was 62 percent higher than during the autumn (P<0.05) and 42 percent higher than during the summer (P<0.06). There was an increase in androstenedione concentration in summer group, when compared to winter (P<0.08) and autumn (P<0.05) groups. Inhibin concentration was higher in winter groups than summer and winter heat stressed groups (P<0.05). Seasonal and acute heat stress altered developmental parameters in dominant follicle and hormonal concentration in follicular fluid, those effects can partially explain the decrease in conception rates during summer.

Stereoscopic depth discrimination with contrast windowed stimuli.

Depth discrimination with a shifted contrast window was compared to that with a fixed contrast window. Stereoscopic performance with the fixed window was limited to small disparities and varied with spatial frequency. Performance with the shifted window extended to larger disparities and was more similar for low and high spatial frequencies. The results depended upon window shape, indicating that edge blur is an important factor. Stereoscopic performance with shifted patterns was supported at disparities larger than a phase disparity model might predict, suggesting that a combination of position and phase disparity computations are used for the perception of stereoscopic depth.

UASB reactor for domestic wastewater treatment at low temperatures: a comparison between a classical UASB and hybrid UASB-filter reactor.

The performance of an upflow anaerobic sludge blanket (UASB) reactor and a hybrid UASB-filter reactor was investigated and compared for the treatment of domestic wastewater at different operational temperatures (28, 20, 14 and 10 degrees C) and loading rates. For each temperature studied a constant CODt removal was observed as long as the upflow velocity was lower than 0.35 m/h. At these upflow velocities similar removals were observed for both reactor types at 28 and 20 degrees C, 82 and 72% respectively. However, at 14 and 10 degrees C the UASB reactor showed a better COD removal (70% and 48%, respectively) than the hybrid reactor (60% and 38%). COD removal resulted from biological degradation and solids accumulation in the reactors. At 28 degrees C, a constant 200 g sludge mass was observed in both reactors and COD removal was attributed to biological degradation only. At lower temperatures, solids accumulation was observed in addition to biological degradation with an increase in reactor sludge as the temperature decreased. The decrease in biological degradation at lower temperatures was offset by solids accumulation and explains the similar overall COD removal efficiency observed at 28 degrees C, 20 degrees C and 14 degrees C. The decrease in temperature was also followed by an increase in the effluent TSS concentration in both reactors. At 14 and 10 degrees C a lower effluent TSS concentration and better performance was observed in the UASB reactor.

Temperature effect on UASB reactor operation for domestic wastewater treatment in temperate climate regions.

The performance of an upflow anaerobic sludge blanket (UASB) reactor was investigated for the treatment of domestic wastewater at different operational temperatures (28, 20, 14 and 10 degrees C) and loading rates. For each temperature studied a constant COD(t) removal was observed as long as the upflow velocity was lower than 0.35 m/h: 82% at 28 degrees C, 68% at 14 degrees C and 44% at 10 degrees C. At 20 degrees C the COD removal increased with the HRT, reaching similar values as at 28 degrees C for long HRT. At upflow velocities higher than 0.35 m/h, a reduction in total COD removal was observed due to washout of influent TSS. At 28 degrees C, a constant 200 g sludge mass was observed and COD removal was attributed to biological degradation only. At lower temperatures, COD removal resulted from degradation and solids accumulation in the reactor. The increase in reactor sludge was greater as the temperature decreased and explains the similar overall COD removal efficiency at 28 degrees C, 20 degrees C and 14 degrees C. During the transition from winter to summer conditions (10 degrees C to 28 degrees C), methane production initially increased due to the degradation of accumulated solids. Afterwards, methane production gradually declined and an increase in COD removal was observed, indicating that the TSS accumulated during the winter was exhausted and influent degradation remained.

Inducing blindsight in normal observers.

We attempted to induce blindsight in normal observers, in an effort to replicate and extend the findings of Kolb and Braun (1995). In that demonstration, observers were able to localize a target in the absence of visual awareness, indicated by the lack of a correlation between localization accuracy and confidence ratings. Replication of this work seemed essential, given the failed attempt by Morgan, Mason, and Solomon (1997). A key aspect of the present work was the use of a pointing response, which is believed to have access to the unconscious representations subserving blindsight. In the critical rivalrous condition, the display consisted of Gabor patterns presented dichoptically with orthogonal orientation in each eye. Binocular summation of left and right images combined to give the appearance of a uniform plaid, camouflaging the texture-defined target. Our attempt to demonstrate blindsight in normal observers was unsuccessful, in that the localization accuracy of the texture-defined target and the observers' confidence ratings were positively correlated. Although the replication was unsuccessful, the results are valuable in that they provide closure to this widely publicized but fruitless line of inquiry.

Antimicrobial resveratrol tetramers from the stem bark of Vatica oblongifoliassp. oblongifolia.

Two new resveratrol tetramers, hopeaphenol A (1) and isohopeaphenol A (2), along with the known vaticaphenol A (3), were isolated from the stem bark of Vatica oblongifolia ssp. oblongifolia through bioassay-guided fractionation. The structures and their relative stereochemistry were determined by spectroscopic techniques. Compounds 1 and 3 demonstrated moderate activity against methicillin-resistant Staphylococcus aureus and Mycobacterium smegmatis.

Characteristics of protein splicing in trans mediated by a semisynthetic split intein.

Protein splicing in trans results in the ligation of two protein or peptide segments linked to appropriate intein fragments. We have characterized the trans-splicing reaction mediated by a naturally expressed, approximately 100-residue N-terminal fragment of the Mycobacterium tuberculosis intein and a synthetic peptide containing the 38 C-terminal intein residues, and found that the splicing reaction was very versatile and robust. The efficiency of splicing was nearly independent of temperature between 4 and 37 degrees C and pH between 6.0 and 7.5, with only a slight decline at pH values as high as 8.5. In addition, there was considerable flexibility in the choice of the C-terminal intein fragment, no significant difference in protein ligation efficiency being observed between reactions utilizing the N-terminal fragment and either the naturally expressed 107-residue C-terminal portion of the intein, much smaller synthetic peptides, or the 107-residue C-terminal intein fragment modified by fusion of a maltose binding protein domain to its N-terminus. The ability to use different types of the C-terminal intein fragments and a broad range of reaction conditions make protein splicing in trans a versatile tool for protein ligation.

Protein splicing in vitro with a semisynthetic two-component minimal intein.

Protein splicing elements, or inteins, catalyze their own excision from flanking polypeptide sequences, or exteins, thereby leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. A trans-splicing system, using separately purified and expressed N- and C-terminal intein fragments of about 100 amino acids each, fused to appropriate exteins, was recently derived from the Mycobacterium tuberculosis RecA intein (Mills, K. V., Lew, B. M., Jiang, S.-Q., and Paulus, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3543-3548). We have replaced the C-terminal intein fragment of this system with synthetic peptides comprising 35-50 of the C-terminal residues of the RecA intein. The N-terminal intein fragment and the synthetic peptide were reconstituted by renaturation from guanidinium chloride. In the absence of added reductants, a disulfide-linked dimer of the N-terminal fragment and the peptide accumulated and could be induced to splice by reduction of its disulfide bond. The intermediate and spliced products were identified by polyacrylamide gel electrophoresis, mass spectrometry, and derivatization with thiol-reactive biotin followed by Western blotting with a streptavidin-enzyme conjugate. This is the first example of protein splicing involving a synthetic intein fragment and opens the way for studying the active site structure and function of the intein by the use of different synthetic peptides, including ones with non-natural amino acids.

Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein.

Protein splicing involves the self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. To study the enzymology of this interesting process we have expressed and purified N- and C-terminal segments of the Mycobacterium tuberculosis RecA intein, each approximately 100 amino acids long, fused to appropriate exteins. These fragments were reconstituted into a functional protein splicing element by renaturation from 6 M urea. When renaturation was carried out in the absence of thiols, the reconstituted splicing element accumulated as an inactive disulfide-linked complex of the two intein fragments, which could be induced to undergo protein splicing by reduction of the disulfide bond. This provided a useful tool for separately investigating the requirements for the reconstitution of the intein fragments to yield a functional protein splicing element and for the protein splicing process per se. For example, the pH dependence of these processes was quite different, with reconstitution being most efficient at pH 8.5 and splicing most rapid at pH 7.0. The availability of such an in vitro protein splicing system opens the way for the exploration of intein structure and the unusual enzymology of protein splicing. In addition, this trans-splicing system is a potential protein ligase that can link any two polypeptides fused to the N- and C-terminal intein segments.

Seasonal and acute heat stress effects on steroid production by dominant follicles in cows.

The present study concerned the seasonal and acute effects of heat stress on steroid concentrations in follicular fluid and on steroid production by granulosa and theca interna cells, in bovine dominant follicles. Three groups of cows were studied: summer (n = 5), autumn (n = 5) and winter (n = 9) cows. During the winter season, another group of cows was acutely heat-stressed from days 3 through 5 of the estrous cycle (n = 5). On day 7 of the estrous cycle, follicular fluid from first-wave dominant follicles was aspirated, and dispersed granulosa and theca cells from each seasonal group were incubated for 18 h at normothermic (37.5 degrees C) or high (40.5 degrees C) temperatures. Cells were incubated in media only or in media containing testosterone (300 ng ml-1, for granulosa cells) or forskolin (4 micrograms ml-1, for theca cells). In follicular fluid the 17 beta-estradiol concentration was high (P < 0.05) in winter and low in autumn, and summer, the androstenedione concentration was high in summer (P < 0.05), low in autumn, and intermediate in winter. During the winter season, acute in vivo heat stress increased follicular fluid androstenedione and decreased estradiol to levels comparable with those prevailing in summer. Basal and forskolin-stimulated androstenedione production by theca cells was higher (P < 0.05) in the winter group than in the summer and autumn groups, and also higher than in the cows that were heat-stressed during winter, which suggests that theca cell function is susceptible to chronic (summer), short-term (winter) and delayed (autumn) heat stresses. In vitro incubation at high temperature (40.5 degrees C) reduced the high, forskolin-stimulated androstenedione production in winter (P < 0.05). Estradiol production by granulosa cells was high in winter and autumn, and low in summer (P < 0.05). Acute heat stress in winter did not alter estradiol production relative to winter controls, whereas a high incubation temperature (40.5 degrees C) reduced (P < 0.05) estradiol production only in the autumn, when the highest production rate was recorded. The results indicate a differential effect of heat stress on the functions of granulosa and theca cells. Both concurrent and delayed effects of heat stress on the steroidogenic capacity of ovarian follicles in cattle are presented.

Effect of heat stress on follicular development during the estrous cycle in lactating dairy cattle.

In this study we examined, in two experiments, patterns of follicular development and dominance under conditions of heat stress. Estrous cycles were programmed to include two follicular waves (wave 1 and 2). On Day 1 of the estrous cycle (Day 0 = estrus), cows were assigned randomly to cooled (C; n = 6) or heat-stressed (H; n = 6) groups. In experiment 1, on Day 12 prostaglandin (PG) F2 alpha was injected and a controlled intravaginal drug release device (1.9 g progesterone) was inserted (this was removed on Day 17). In experiment 2, PGF 2 alpha was injected on Day 14. Ovarian structures were examined daily by ultrasonography, and blood samples were collected at each scanning. Cycle lengths were 20 and 17 days in experiments 1 and 2, respectively. Mean maximal body temperatures were higher (p < 0.01) in H (40.3 degrees C) than in C (38.8 degrees C) cows. In experiment 1, the rate of increase in number of large follicles (> or = 10 mm) was greater in H than in C cows (p < 0.01), resulting in 53% more large follicles in H cows during wave 1; this was associated with a lower (p < 0.05) number of medium-sized (6-9 mm) follicles between Days 7 and 10 of the cycle. Heat stress hastened (p < 0.02) the decrease in size of the first-wave dominant follicle and hastened (p < 0.01) the emergence of the second dominant (preovulatory) follicle by 2 days.(ABSTRACT TRUNCATED AT 250 WORDS)