Development of the Scalp Hair Assessment PRO™ measure for alopecia areata.

BACKGROUND: Valid patient-reported outcome (PRO) measures are required to evaluate alopecia areata (AA) treatments. OBJECTIVES: To develop a content-valid and clinically meaningful PRO measure to assess AA scalp hair loss with scores comparable with the five-response-level Alopecia Areata Investigator Global Assessment (AA-IGA™). METHODS: A draft PRO measure was developed based on input from 10 clinical experts in AA. The PRO measure was cognitively debriefed, modified and finalized through two rounds of qualitative semistructured interviews with patients with AA who had experienced ≥ 50% scalp hair loss. Data were thematically analysed. RESULTS: Adults (round 1: n = 25; round 2: n = 15) and adolescents aged 15-17 years (round 1: n = 5) in North America participated. All patients named scalp hair loss as a key AA sign or symptom. Patients demonstrated the ability to self-report their current amount of scalp hair using percentages. In round 1 not all patients interpreted the measurement concept consistently; therefore, the PRO was modified to clarify the measurement concept to improve usability. Following modifications, patients in round 2 responded without difficulty to the PRO measure. Patients confirmed that they could use the five-level response scale to rate their scalp hair loss: no missing hair, 0%; limited, 1-20%; moderate, 21-49%; large, 50-94%; nearly all or all, 95-100%. Almost all patients deemed hair regrowth resulting in ≤ 20% scalp hair loss a treatment success. CONCLUSIONS: The Scalp Hair Assessment PRO™ is a content-valid, clinically meaningful assessment of distinct gradations of scalp hair loss for evaluating AA treatment for patients with ≥ 50% hair loss at baseline.

The Alopecia Areata Investigator Global Assessment scale: a measure for evaluating clinically meaningful success in clinical trials.

BACKGROUND: Content-valid and clinically meaningful instruments are required to evaluate outcomes of therapeutic interventions in alopecia areata (AA). OBJECTIVES: To develop an Investigator's Global Assessment (IGA) to interpret treatment response in AA treatment studies. METHODS: Qualitative interviews were conducted in the USA with expert dermatologists and with patients with AA who had experienced ≥ 50% scalp-hair loss. Thematic data analysis identified critical outcomes and evaluated the content validity of the new IGA. RESULTS: Expert clinicians (n = 10) judged AA treatment success by the amount of scalp-hair growth (median 80% scalp hair). Adult (n = 25) and adolescent (n = 5) patients participated. Scalp-hair loss was the most bothersome AA sign/symptom for most patients. Perceived treatment success - short of 100% scalp hair - was the presence of ~ 70-90% scalp hair (median 80%). Using additional clinician and patient insights, the Alopecia Areata Investigator Global Assessment (AA-IGA™) was developed. This clinician-reported outcome assessment is an ordinal, static measure comprising five severity categories of scalp-hair loss. Nearly all clinicians and patients in this study agreed that, for patients with ≥ 50% scalp-hair loss, successful treatment would be hair regrowth resulting in ≤ 20% scalp-hair loss. CONCLUSIONS: We recommend using the Severity of Alopecia Tool to assess the extent (0-100%) of scalp-hair loss. The AA-IGA is a robust ordinal measure providing distinct and clinically meaningful gradations of scalp-hair loss that reflects patients' and expert clinicians' perspectives and treatment expectations. What is already known about this topic? The Severity of Alopecia Tool is widely used to assess the extent of scalp-hair loss in patients with alopecia areata. Guidelines define treatment success as a 50% improvement in scalp hair, and clinical trials have used dynamic thresholds of 50% and 90%. However, there is no clinical consensus on these endpoints, and patient perspectives on treatment success are unknown. What does this study add? Through qualitative interviews with 10 expert dermatologists and 30 patients with alopecia areata who had experienced ≥ 50% scalp-hair loss, we developed the Alopecia Areata Investigator Global Assessment (AA-IGA™) to measure five clinically meaningful gradations of alopecia areata scalp-hair loss that reflects patients' and clinicians' perspectives and expectations of treatment success in alopecia areata treatment studies. What are the clinical implications of this work? The AA-IGA is a robust ordinal measure that can inform clinical evaluation of alopecia areata treatment outcomes. The AA-IGA can be used to determine clinically meaningful treatment success for alopecia areata, with success defined by patients and clinicians as reaching ≤ 20% scalp-hair loss. Linked Comment: Blome. Br J Dermatol 2020; 183:609.

Baricitinib in patients with moderate-to-severe atopic dermatitis and inadequate response to topical corticosteroids: results from two randomized monotherapy phase III trials.

BACKGROUND: Baricitinib, an oral selective Janus kinase 1 and 2 inhibitor, effectively reduced atopic dermatitis (AD) severity in a phase II study with concomitant topical corticosteroids. OBJECTIVES: To evaluate the efficacy and safety of baricitinib in patients with moderate-to-severe AD who had an inadequate response to topical therapies. METHODS: In two independent, multicentre, double-blind, phase III monotherapy trials, BREEZE-AD1 and BREEZE-AD2, adults with moderate-to-severe AD were randomized 2 : 1 : 1 : 1 to once-daily placebo, baricitinib 1 mg, 2 mg, or 4 mg for 16 weeks. RESULTS: At week 16, more patients achieved the primary end point of Validated Investigator's Global Assessment of AD (0, 1) on baricitinib 4 mg and 2 mg compared with placebo in BREEZE-AD1 [N = 624; baricitinib 4 mg 16·8% (P < 0·001), 2 mg 11·4% (P < 0·05), 1 mg 11·8% (P < 0·05), placebo 4·8%], and BREEZE-AD2 [N = 615; baricitinib 4 mg 13·8% (P = 0·001), 2 mg 10·6% (P < 0·05), 1 mg 8·8% (P = 0·085), placebo 4·5%]. Improvement in itch was achieved as early as week 1 for 4 mg and week 2 for 2 mg. Improvements in night-time awakenings, skin pain and quality-of-life measures were observed by week 1 for both 4 mg and 2 mg (P ≤ 0·05, all comparisons). The most common adverse events in patients treated with baricitinib were nasopharyngitis and headache. No cardiovascular events, venous thromboembolism, gastrointestinal perforation, significant haematological changes, or death were observed with any baricitinib dosage. CONCLUSIONS: Baricitinib improved clinical signs and symptoms in patients with moderate-to-severe AD within 16 weeks of treatment and induced rapid reduction of itch. The safety profile remained consistent with prior findings from baricitinib clinical development in AD, with no new safety concerns.

Subpolar North Atlantic Overturning and Gyre-Scale Circulation in the Summers of 2014 and 2016.

The Atlantic Meridional Overturning Circulation (AMOC) is a key component of the global climate system through its transport of heat and freshwater. The subpolar North Atlantic (SPNA) is a region where the AMOC is actively developed and shaped though mixing and water mass transformation and where large amounts of heat are released to the atmosphere. Two hydrographic transbasin sections in the summers of 2014 and 2016 provide highly spatially resolved views of the SPNA velocity and property fields on a line from Canada to Greenland to Scotland. Estimates of the AMOC, isopycnal (gyre-scale) transport, and heat and freshwater transport are derived from the observations. The overturning circulation, the maximum in northward transport integrated from the surface to seafloor and computed in density space, has a high range, with 20.6 ± 4.7 Sv in June-July 2014 and 10.6 ± 4.3 Sv in May-August 2016. In contrast, the isopycnal (gyre-scale) circulation was lowest in summer 2014: 41.3 ± 8.2 Sv compared to 58.6 ± 7.4 Sv in 2016. The heat transport (0.39 ± 0.08 PW in summer 2014, positive is northward) was highest for the section with the highest AMOC, and the freshwater transport was largest in summer 2016 when the isopycnal circulation was high (-0.25 ± 0.08 Sv). Up to 65% of the heat and freshwater transport was carried by the isopycnal circulation, with isopycnal property transport highest in the western Labrador Sea and the eastern basins (Iceland Basin to Scotland).

Effect of in vitro maturation of mouse oocytes on the health and lifespan of adult offspring.

BACKGROUND: In vitro maturation of oocytes can, in some circumstances, provide an alternative approach to gonadotrophin-induced maturation in clinical settings. However, the consequences of these protocols on the long-term health of offspring are unknown. Here, the long-term health status and lifespans of offspring produced by in vitro maturation of mouse oocytes was compared with that of oocytes induced to mature in vivo using gonadotrophin treatment. METHODS: Mouse oocytes were matured in vitro using both an established optimized system and in the absence of amino acids to produce a suboptimal condition for maturation. Oocytes induced to mature in vivo with gonadotrophins constituted the control group. All metaphase II oocytes were fertilized in vitro and transferred at the 2-cell stage to the oviducts of pseudo-pregnant foster mothers for development to term. Offspring were subjected to a wide variety of physiological and behavioral tests for the first year of life and natural lifespan determined. RESULTS: There was no difference among the groups in lifespan or in most of the physiological and behavioral analyses. However, the pulse rate and cardiac output were slightly, but significantly, reduced in the optimized in vitro matured group compared with the in vivo matured group (P = 0.0119 and P = 0.0197, respectively). Surprisingly, these decreases were largely abrogated in the in vitro group matured without amino acids. CONCLUSIONS: Evidence presented here using a mouse model suggests that the in vitro maturation of oocytes has minimal effects on the long-term health of offspring. However, a finding of slight reductions in pulse rate and cardiac output may focus future clinical attention.

Inhibition of antimicrobial peptides by group A streptococci: SIC and DRS.

SIC (streptococcal inhibitor of complement) is a 31 kDa protein secreted by a few highly virulent strains of GAS (group A streptococci), predominantly by the M1 strain. Initially described as an inhibitor of the membrane attack complex of complement, it has turned out to be a polyfunctional inhibitor of the innate mucosal immune response. The SIC protein sequence contains three domains: an N-terminal SRR (short repeat region), followed by three longer tandem repeats [LRR (long repeat region)] and a C-terminal PRR (proline-rich region). SIC inhibits the antibacterial activity of a wide range of antimicrobial peptides and proteins: i.e. lysozyme, SLPI (secretory leucocyte proteinase inhibitor), LL-37, hNP-1 (human neutrophil peptide-1) and the human beta-defensins 1, 2 and 3. Analysis of the functional properties of recombinant domains of SIC shows that binding and inhibition of lysozyme and human beta-defensin-3 require the SRR+LRR, as does binding to SLPI. Complement inhibition is confined to the SRR. M12 GAS secrete a protein 'distantly related to SIC' (DRS). DRS contains a C-terminal PRR which is significantly similar to that of SIC, but it has no central LRR and the N-terminal SRR is very different. DRS inhibits human beta-defensin-3, but has no effect on lysozyme, SLPI or complement.

Pneumococcal meningitis: clinical course and resource use in Western Australian children.

OBJECTIVES: Paediatric pneumococcal meningitis causes high rates of neurological sequelae and is presenting challenges due to the emergence of antibiotic resistance. Diagnosis can be difficult and management is frequently complicated and resource intensive. This population-based study outlines the diagnosis, clinical course and initial resource use of Western Australian (WA) children diagnosed between 1990 and 2000. METHODS: The charts of all rural and metropolitan children discharged from all WA hospitals with International Classification of Disease 9 and 10 codes of pneumococcal or streptococcal meningitis from the Health Department's Hospital Morbidity Data System were reviewed retrospectively. RESULTS: Ninety-four episodes of pneumococcal meningitis were confirmed (median age 12.4 months). Initial diagnosis was often difficult with a suggestive prodrome in less than one quarter of cases and a median of two reviews until diagnosis (range 1-7). The median duration of hospitalization was 11.6 days, 41.5% were admitted to the Intensive Care Unit (ICU), and 47.9% had seizures. There were eight related deaths and neurological sequelae occurred in 24. Initial hospitalization cost on average $A 19,900. Penicillin resistance was first noted in 1993 and occurred in 4.6% of isolates. Most cases (84.5%) were caused by serotypes contained in the currently available seven-valent conjugate vaccine. CONCLUSIONS: Pneumococcal meningitis is often difficult to diagnose, necessitates long and frequently complicated in-patient stays and has high rates of neurological sequelae. It is possible to prevent most cases with new conjugate vaccines.

Pneumococcal meningitis in Western Australian children: epidemiology, microbiology and outcome.

OBJECTIVES: Pneumococcal meningitis is now vaccine-preventable but continues to cause high rates of neurological sequelae internationally. Population-based epidemiology, outcome and microbiology data are necessary to target vaccination strategies. This study outlines these key areas for Western Australian children diagnosed 1990-2000. METHODS: The charts of all rural and metropolitan children with International Classification of Disease 9 and 10 discharge codes of pneumococcal or streptococcal meningitis from the Health Department's Hospital Morbidity Data System were reviewed. RESULTS: Over 10.5 years, 94 episodes were confirmed. The average annual incidence for children under 2 years was 13.45 per 100 000 and 0.66 per 100 000 for children 2 years or older. Indigenous children had an almost seven-fold increased risk compared to non-Indigenous (with 78.55 per 100 000 in the under two-year Indigenous group). Eight children died and 24 of the survivors had neurological sequelae. Penicillin resistance occurred in four of 87 isolates. One quarter of the cohort qualify under the current Australian policy of vaccination of high-risk children with seven-valent conjugate (7vPCV) vaccine. Most isolates (49/58) were 7vPCV serotypes, however, Indigenous populations were less well-covered (58.3% covered vs 91.3% of isolates from non-Indigenous children). Indigenous coverage would be improved to 75% with 11-valent conjugate vaccine. CONCLUSIONS: Indigenous children and those under 2 years are most affected by pneumococcal meningitis and remain primary vaccination targets. Three quarters of these children would not be protected by a policy of vaccination of only high-risk children with 7vPCV--improved protection requires higher valencies for Indigenous populations and universal infant vaccination.

Antimicrobial peptides: mediators of innate immunity as templates for the development of novel anti-infective and immune therapeutics.

Antimicrobial molecules are ancient and essential small cationic molecules of the host defence system which are found in a wide variety of species. They display antimicrobial activity against a wide range of bacteria, fungi and viruses, an activity that has been mostly attributed to the disruption of microbial membranes. In this article, we will review the "classical" functions of 3 classes of antimicrobial molecules, namely defensins, cathelicidins, and the four-disulfide core proteins secretory leukocyte proteinase inhibitor (SLPI) and elafin. In addition to the study of their expression in a variety of cell types and the regulation of their production, we will also describe novel properties of these molecules that have been highlighted by recent studies. These include their ability to chemoattract a variety of inflammatory, immune and other cell types (neutrophils, macrophages, monocytes, lymphocytes, mast cells, epithelial cells) in vitro and in vivo. In addition, we will discuss the potential use of these newly discovered properties for therapeutic or vaccination purposes, using protein- or gene-transfer based methodologies. Finally, we will examine in an extensive fashion the strategies used by microorganisms to circumvent and subvert host defence mechanisms, such as the modifications of cell membranes and walls, the secretion of inactivating proteins and proteases and the down-regulation of expression of antimicrobial molecules. Increased understanding of the mechanisms used by both the host and the microbes to 'win the battle' may ultimately lead to new therapeutic strategies aimed to treat infectious diseases.

Adverse skin and joint reactions associated with oral antibiotics in children: the role of cefaclor in serum sickness-like reactions.

OBJECTIVE: To review presentations to Princess Margaret Hospital Emergency Department (PMH ED) with adverse joint and skin reactions associated with the use of oral antibiotics, to describe the clinical course of children with cefaclor-related serum sickness-like reactions (cefaclor SSLR) and compare these with cases reported to the Adverse Drug Reactions Advisory Committee (ADRAC). METHODS: Twelve-month retrospective review of presentations to a tertiary paediatric ED (42,000 visits annually) via an ED computer database search and review of medical charts of children presenting with joint or skin reactions. Telephone interviews were conducted with the caregivers of children with cefaclor SSLR. RESULTS: Adverse skin or joint reactions occurred in 150 children; 70 after cefaclor alone, 10 after cefaclor in combination with other antibiotics and 70 after other antibiotic courses. SSLR occurred in 44 children; 32 after cefaclor alone, five after cefaclor in combination with other antibiotics and seven after other single antibiotics. In children with cefaclor SSLR, otitis media was the most common indication (59.4%), another 18.8% had viral illnesses. Prolonged sequelae occurred in four children, a situation not previously reported. Sixty reports of paediatric cefaclor SSLR were made to ADRAC during the study period, none originated from PMH ED. CONCLUSIONS: Cefaclor was associated with 53.3% of oral antibiotic related skin and joint adverse reactions and 84.1% of SSLR. The indications for its use in paediatric illness require careful reconsideration. ADRAC data under-represents the incidence of cefaclor SSLR.

Spatially localized generation of nucleotide sequence-specific DNA damage.

Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have been used in conjunction with laser-induced two-photon excitation (TPE) to damage a specific DNA target sequence. To demonstrate that TPE can initiate photochemistry resulting in psoralen-DNA photoadducts, target DNA sequences were incubated with psoTFOs to form triple-helical complexes and then irradiated in liquid solution with pulsed 765-nm laser light, which is half the quantum energy required for conventional one-photon excitation, as used in psoralen + UV A radiation (320-400 nm) therapy. Target DNA acquired strand-specific psoralen monoadducts in a light dose-dependent fashion. To localize DNA damage in a model tissue-like medium, a DNA-psoTFO mixture was prepared in a polyacrylamide gel and then irradiated with a converging laser beam targeting the rear of the gel. The highest number of photoadducts formed at the rear while relatively sparing DNA at the front of the gel, demonstrating spatial localization of sequence-specific DNA damage by TPE. To assess whether TPE treatment could be extended to cells without significant toxicity, cultured monolayers of normal human dermal fibroblasts were incubated with tritium-labeled psoralen without TFO to maximize detectable damage and irradiated by TPE. DNA from irradiated cells treated with psoralen exhibited a 4- to 7-fold increase in tritium activity relative to untreated controls. Functional survival assays indicated that the psoralen-TPE treatment was not toxic to cells. These results demonstrate that DNA damage can be simultaneously manipulated at the nucleotide level and in three dimensions. This approach for targeting photochemical DNA damage may have photochemotherapeutic applications in skin and other optically accessible tissues.

Streptococcal inhibitor of complement (SIC) inhibits the membrane attack complex by preventing uptake of C567 onto cell membranes.

Streptococcal inhibitor of complement (SIC) was first described in 1996 as a putative inhibitor of the membrane attack complex of complement (MAC). SIC is a 31 000 MW protein secreted in large quantities by the virulent Streptococcus pyogenes strains M1 and M57, and is encoded by a gene which is extremely variable. In order to study further the interactions of SIC with the MAC, we have made a recombinant form of SIC (rSIC) in Escherichia coli and purified native M1 SIC which was used to raise a polyclonal antibody. SIC prevented reactive lysis of guinea pig erythrocytes by the MAC at a stage prior to C5b67 complexes binding to cell membranes, presumably by blocking the transiently expressed membrane insertion site on C7. The ability of SIC and clusterin (another putative fluid phase complement inhibitor) to inhibit complement lysis was compared, and found to be equally efficient. In parallel, by enzyme-linked immunosorbent assay both SIC and rSIC bound strongly to C5b67 and C5b678 complexes and to a lesser extent C5b-9, but only weakly to individual complement components. The implications of these data for virulence of SIC-positive streptococci are discussed, in light of the fact that Gram-positive organisms are already protected against complement lysis by the presence of their peptidoglycan cell walls. We speculate that MAC inhibition may not be the sole function of SIC.

The magnitude and duration of ambulatory blood pressure reduction following acute exercise.

The purpose of this study was to observe the magnitude and duration of the ambulatory blood pressure (BP) reduction following exercise and to identify the peak intervals of BP reduction throughout the 24-h diurnal period. Subjects were 25 normo- (N = 116.7/ 78.2+/-10.0/7.2 mm Hg) and 21 hypertensive (H = 140.8/96.9+/-13.9/9.6 mm Hg) adults. Twenty-four hour ambulatory blood pressures (SBP = systolic and DBP = diastolic) were recorded following exercise (E = 50 min @ 50% VO2 max) and during a non-exercise control day (C). The 24-h pressures were compared between the E and C days for (1) duration and magnitude of the BP reduction following exercise, and for (2) the time of day for the diurnal patterns to exhibit reductions in BP. No BP differences were found for N between E and C days. Significant reductions in BP were found for 24-h average SBP (decrease 6.8 mm Hg) and DBP (decrease 4.1 mm Hg), daytime (06.00-22.00 hrs) SBP (decrease 6.9 mm Hg) and DBP (decrease 3.3 mm Hg), and sleep (22.00-06.00) SBP (decrease 5.1 mm Hg) and DBP (decrease 4.4 mm Hg) for H subjects only. H also demonstrated an 11 h reduction in SBP (chi = decrease 8.3+/-2.2 mm Hg) and 4h reduction in DBP (chi = decrease 6.0+/-1.7 mm Hg) following exercise. For the diurnal variation, the peak interval of reduction in SBP (chi = 17.0+/-2.6 mm Hg) was for 11 h; from 11.00-21.00 hrs. For DBP, a significant reduction (chi = decrease 5.7+/-0.7 mm Hg) was found for 5 h; from 11.00-15.00 h. Thus, exercise reduces both systolic and diastolic BP for a significant length of time postexercise as well as reduces pressures during the time of day that typically exhibits higher diurnal pressures.

Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein.

The crystal structure of a blue emission variant (Y66H/Y145F) of the Aequorea victoria green fluorescent protein has been determined by molecular replacement and the model refined. The crystallographic R-factor is 18.1% for all data from 20 to 2.1 A, and the model geometry is excellent. The chromophore is non-native and is autocatalytically generated from the internal tripeptide Ser65-His66-Gly67. The final electron density maps indicate that the formation of the chromophore is complete, including 1,2 dehydration of His66 as indicated by the planarity of the chromophore. The chromophore is in the cis conformation, with no evidence for any substantial fraction of the trans configuration or uncyclized apoprotein, and is well-shielded from bulk solvent by the folded protein. These characteristics indicate that the machinery for production of the chromophore from a buried tripeptide unit is not only intact but also highly efficient in spite of a major change in chromophore chemical structure. Nevertheless, there are significant rearrangements in the hydrogen bond configuration around the chromophore as compared to wild-type, indicating flexibility of the active site. pH titration of the intact protein and the chromopeptide (pKa1 = 4.9 +/- 0.1, pKa2 = 12.0 +/- 0.1) suggests that the predominant form of the chromophore in the intact protein is electrically neutral. In contrast to the wild-type protein [Chattoraj, M., King, B. A., Bublitz, G. U., & Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A., 8362-8367], femtosecond fluorescence up-conversion spectroscopy of the intact protein and a partially deuterated form strongly suggests that excited-state proton transfer is not coupled to fluorescence emission.

A comparison of 24-h average blood pressures and blood pressure load following exercise.

Although the use of 24-h ambulatory blood pressure monitoring has been recommended in the study of blood pressure and exercise, consistent results have not been found for average 24-h systolic or diastolic blood pressures. Systolic load and diastolic load (the percentage of pressures >140/90 mm Hg during daytime hours and >120/80 mm Hg during sleep) have recently been identified as an important variable, but has had limited use with exercise. The purpose of this study was to compare the average systolic and diastolic pressures to systolic and diastolic loads from 24-h data recorded after a 50-min treadmill walk at 50% VO2max to data from a nonexercise control day. Subjects were 36 normotensive (116.9 +/- 10.7/77.0 +/- 8.9 mm Hg) and 25 hypertensive (141.0 +/- 13.7/96.6 +/- 9.0 mm Hg) adults. No significant differences were found for systolic and diastolic pressures or loads between the control and exercise days for normotensives. Even though no significant changes were found for any of the average systolic and diastolic pressures between the control and exercise days for the hypertensives, significant reductions were found in systolic load for 24-h (-25.7%), day (6 AM to 10 PM, -23.1%), work (6 AM to 5 PM, -22.9%), and leisure (5 PM to 10 PM, -26.7%) periods; and in diastolic load for the work (-22.5%) period. Thus, the measurement of systolic and diastolic load may be more sensitive than average systolic and diastolic blood pressures for the detection of 24-h ambulatory blood pressure changes with exercise in borderline hypertension.

Effect of exercise on milk immunoglobulin A.

The major immunoglobulin (Ig) in human secretions is IgA. The immune properties of breast milk are well documented; however, the immunological influence of maximal exercise has not been established. The objective of this study was to investigate the role that exercise has on breast milk IgA and IgA subclasses. Breast milk was collected from 17 lactating women (4.6 +/- 2.3 months postpartum) before and after randomized exercise and control periods. The exercise treatment was a maximal graded treadmill test (VO2max = 30.3 +/- 5.7 mL x min-1 x kg-1). Milk was collected at rest, the breasts were emptied, and samples obtained 10, 30, and 60 min following either exercise or 30-min control rest periods. IgA concentrations were established by enzyme-linked immunosorbent assay. The results indicated that samples taken 10 and 30 min after the exercise period had significantly lower (P < or = 0.05) milk IgA concentrations (21.0 +/- 1.8 and 21.8 +/- 1.4 microg x mL-1, respectively) than the corresponding control resting samples (52.8 +/- 3.5 and 79.3 +/- 7.7 microg x mL-1). The exercise samples were similar to the control samples at 60 min (134.0 +/- 24.6 and 116.0 +/- 15.4 microg x mL-1, respectively), indicating that by 1 h, milk IgA production had recovered. The IgA1 data showed a similar significant decrease (P < or = 0.05) at 10 min postexercise, which also returned to control concentrations by the 30- and 60-min collection intervals. There was no significant change in the milk IgA2 concentrations at any of the time points studied. Milk IgA concentrations increased significantly in both exercise and resting control groups after the breasts were emptied, suggesting that breast emptying stimulated milk IgA synthesis. The results provide evidence that exercise alters milk IgA and IgA1 concentrations for 10-30 min after exhaustive exercise, but recovers by 1 h, and provide additional support for exercise effects on the mucosal immune system.

Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer.

The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromophore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be readily monitored by sensitive fluorescence methods without the need to add exogenous fluorescent dyes. The excited state dynamics of GFP were studied following photo-excitation of each of its two strong absorption bands in the visible using fluorescence upconversion spectroscopy (about 100 fs time resolution). It is shown that excitation of the higher energy feature leads very rapidly to a form of the lower energy species, and that the excited state interconversion rate can be markedly slowed by replacing exchangeable protons with deuterons. This observation and others lead to a model in which the two visible absorption bands correspond to GFP in two ground-state conformations. These conformations can be slowly interconverted in the ground state, but the process is much faster in the excited state. The observed isotope effect suggests that the initial excited state process involves a proton transfer reaction that is followed by additional structural changes. These observations may help to rationalize and motivate mutations that alter the absorption properties and improve the photo stability of GFP.