Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.

Development of the Parent-to-Infant Bonding Scale: Validation in Swedish Mothers and Fathers in Community and Clinical Contexts.

Valid measurement instruments are needed to investigate the impact of parental bonding on child health development. The aim was to develop and validate a self-report questionnaire, the Parent-to-Infant Bonding Scale (PIBS) to measure bonding in both mothers and fathers. Internal consistency and construct validity were analysed using data from Swedish parents from both clinical (N = 182), and community (N = 122) population samples. Overall, good or acceptable internal consistency of the PIBS appeared. Convergent validity (against the Postpartum Bonding Questionnaire, analysed in the clinical sample) and discriminant validity (against the mental health constructs of depressive symptoms and anxiety) were demonstrated. The results support the PIBS as a measure of maternal and paternal bonding in community and clinical populations. Assessments of criterion validity in these populations are desirable. The similarities in PIBS measurement properties between the parent groups suggest its usefulness for comparisons between mothers and fathers, and for future investigations of unique and interactive impacts of maternal and paternal bonding on child outcomes using community and clinical cohorts.

Effects of a local auxiliary protein on the two-dimensional affinity of a TCR-peptide MHC interaction.

The affinity of T-cell receptors (TCRs) for major histocompatibility complex molecules (MHCs) presenting cognate antigens likely determines whether T cells initiate immune responses, or not. There exist few measurements of two-dimensional (2D) TCR-MHC interactions, and the effect of auxiliary proteins on binding is unexplored. Here, Jurkat T-cells expressing the MHC molecule HLA-DQ8-glia-α1 and the ligand of an adhesion protein (rat CD2) were allowed to bind supported lipid bilayers (SLBs) presenting fluorescently labelled L3-12 TCR and rat CD2, allowing measurements of binding unconfounded by cell signaling effects or co-receptor binding. The 2D Kd for L3-12 TCR binding to HLA-DQ8-glia-α1, of 14±5 molecules/µm2 (mean±s.d.), was only marginally influenced by including CD2 up to ∼200 bound molecules/µm2 but higher CD2 densities reduced the affinity up to 1.9-fold. Cell-SLB contact size increased steadily with ligand density without affecting binding for contacts at up to ∼20% of total cell area, but beyond this lamellipodia appeared, giving an apparent increase in bound receptors of up to 50%. Our findings show how parameters other than the specific protein-protein interaction can influence binding behavior at cell-cell contacts.

A cell topography-based mechanism for ligand discrimination by the T cell receptor.

The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.

Single-cell measurements of two-dimensional binding affinity across cell contacts.

The two-dimensional (2D) affinity between protein molecules across contacting cells is a key parameter regulating and initiating several cellular processes. However, measuring 2D affinity can be challenging, and experimental data are limited. In addition, the obtained 2D affinities are typically averaged over the cell population. We here present a method to measure 2D affinity on single cells binding to polyhistidine-tagged fluorescent ligands anchored to a supported lipid bilayer (SLB). By decreasing the density of ligands in the SLB using imidazole, a new steady-state accumulation in the contact is obtained, and from this change, both the 2D affinity and the number of receptors on the cell can be determined. The method was validated on an SLB containing rat CD2 binding to the rat CD48 mutant T92A expressed on Jurkat T cells. The addition of imidazole did not influence the average 2D affinity (1/Kd), and the spread in affinities within the cell population was low, Kd = 4.9 ± 0.9 molecules/µm2 (mean ± SD), despite an order of magnitude spread in ligand accumulation because of differences in receptor density. It was also found that cell contact size increased both with ligand density and with the number of receptors per cell but that the contact size stayed approximately constant when lowering the ligand density, above a density of around 10 rat CD2 molecules/µm2, after the contact first had formed, indicative of a heterogeneous process. In summary, this method not only allows for single-cell affinities to be measured, but it can also reduce measurement and analysis time and improve measurement accuracy. Because of the low spread in 2D Kd within the cell population, the analysis can further be restricted to the cells showing the strongest binding, paving the way for using this method to study weak binding events.

A daytime nap does not increase mnemonic discrimination ability.

It has been proposed that sleep readies the brain for novel learning, and previous work has shown that sleep loss impairs the ability to encode new memories. In the present study, we examined if a daytime nap would increase mnemonic discrimination (MD) performance. MD is the ability to differentiate between memories that are similar but not identical. Participants performed the Mnemonic Similarity Task (MST) twice, once in the morning and once in the afternoon. The goal of this task is to distinguish stimuli that have been seen before from novel stimuli that are similar but not identical. After the morning MST, participants were randomly allocated into either a sleep or a wake group. The sleep group had a 2-hr nap opportunity, whereas the wake group spent a similar amount of time passively resting. All participants then performed a second MST in the afternoon with a novel set of images. Results did not show any support for increased MD ability after a nap. There was, however, a correlation showing that an increase in sleepiness between sessions predicted a decrease in MD performance. Future work must systematically examine how strong sleep manipulations that are needed for sleep to have an effect on encoding ability, as well as which kind of memory tasks that are sensitive to sleep manipulations. More knowledge about the relationship between sleep and the ability to differentiate similar memories from each other is important because impaired MD ability has previously been reported in various groups in which sleep disturbances are also common.

Lipid Bilayer-like Mixed Self-Assembled Monolayers with Strong Mobility and Clustering-Dependent Lectin Affinity.

Glycans at the surface of cellular membranes modulate biological activity via multivalent association with extracellular messengers. The lack of tuneable simplified models mimicking this dynamic environment complicates basic studies of these phenomena. We here present a series of mixed reversible self-assembled monolayers (rSAMs) that addresses this deficiency. Mixed rSAMs were prepared in water by simple immersion of a negatively charged surface in a mixture of sialic acid- and hydroxy-terminated benzamidine amphiphiles. Surface compositions derived from infrared reflection-absorption spectroscopy (IRAS) and film thickness information (atomic force microscopy, ellipsometry) suggest the latter to be statistically incorporated in the monolayer. These surfaces' affinity for the lectin hemagglutinin revealed a strong dependence of the affinity on the presentation, density, and mobility of the sialic acid ligands. Hence, a spacer length of 4 ethylene glycol and a surface density of 15% resulted in a dissociation constant Kd,multi of 1.3 × 10-13 M, on par with the best di- or tri-saccharide-based binders reported to date, whereas a density of 20% demonstrated complete resistance to hemagglutinin binding. These results correlated with ligand mobility measured by fluorescence recovery after photobleaching which showed a dramatic drop in the same interval. The results have a direct bearing on biological cell surface multivalent recognition involving lipid bilayers and may guide the design of model surfaces and sensors for both fundamental and applied studies.

Improvements in Body Composition after 4 Years of Growth Hormone Treatment in Adult-Onset Hypopituitarism Compared to Age-Matched Controls.

BACKGROUND/AIMS: It is unknown whether long-term growth hormone replacement therapy (GHRT) affects body composition in an age- or sex-dependent manner. We aimed to study the effects of 4 years of GHRT on body composition in a large cohort of patients with hypopituitarism compared to a reference population matched by age and sex. METHODS: A total of 964 GH-deficient adults from KIMS (Pfizer International Metabolic Database) with adult-onset hypopituitarism, adequately replaced with all pituitary hormones except for GH at baseline were included. A random sample of the general population (2,301 subjects) from a similar time period was used as reference. Patients and controls were grouped by sex in 5 age cohorts of 10 years. Main outcome measures were changes in BMI and waist circumference after 4 years of GHRT. RESULTS: In younger patients (28-47 years), 4 years of GHRT resulted in a BMI increase similar to that observed in the reference population, but older patients (48-67 years) had significantly less BMI increase than age-matched healthy controls. Significant differences were seen in waist circumference in patients of all age cohorts who showed virtually no change after 4 years of GHRT compared to approximately 4 cm of increase in the reference population. CONCLUSION: Four years of GHRT resulted in improvements in BMI and waist circumference in patients with adult-onset hypopituitarism compared to age-matched controls observed during the same follow-up time. Despite these beneficial effects on body composition, BMI and waist circumference remained higher in patients on GHRT compared to healthy controls.

Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions.

The αß T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D Kd of ∼5,000 molecules/µm(2) This value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination.

A more generalized fear response after a daytime nap.

The aim of this study was to examine how a daytime nap affected the consolidation of fear learning. Participants first underwent fear conditioning during which they were exposed to a large and a small circle. One of these was repeatedly paired with an electric shock (making it the CS+), whereas the other circle was never paired with the shock (the CS-). After a delay interval containing either a nap or wake, participants again viewed the CS+ and the CS- intermixed with eight novel circles that varied in size between the two stimuli seen before, as well as a blue triangle that served as a novel stimulus without prior fear relevance. We examined both fear retention (the difference between the CS+ and the CS-) and fear generalization (responses to the novel stimuli based on their similarity to the original CS+). Contrary to previous studies, results from the participants who acquired a differentiated fear response during the acquisition phase revealed that the wake group showed significantly larger skin conductance responses to the CS+ compared to the CS-, whereas no such difference was present in the sleep group. These results were not driven by differences in explicit memory or by differences in general reactivity. Analyzing responses to the novel stimuli revealed a tendency towards a more generalized response in the sleep group, with no differences between the CS+ and any other stimulus, whereas the wake group showed increased responses to the stimuli depending on their similarity to the original CS+. This effect was however only present when controlling for baseline differences in worry.

Social, educational and vocational outcomes in patients with childhood-onset and young-adult-onset growth hormone deficiency.

OBJECTIVE: Hypopituitarism diagnosed in childhood, adolescence and young adulthood has the potential to affect growth and somatic development. Less is known about the impact of such a diagnosis on other aspects of development. DESIGN: An analysis of the KIMS database (Pfizer International Metabolic Database) was performed to explore social, educational and vocational outcomes of adult patients diagnosed in childhood, adolescence and young adulthood compared with adult-onset controls. PATIENTS: A total of 2952 adult patients diagnosed with hypothalamic pituitary conditions before the age of 25 were divided into two groups: childhood-onset [<16 years (CO)] (n = 1782) and young-adult-onset [16 to <25 years (YAO)] (n = 1170). A total of 1617 adult patients diagnosed with a nonfunctioning pituitary adenoma at the age of 25 or older formed the adult-onset control group (AO). MEASUREMENTS: KIMS Patient Life Situation Form which provided information on social, educational and vocational outcomes. RESULTS: Compared with the AO control group, CO and YAO patients were between 4·5 and 8·0 times more likely to live with their parents in adulthood; CO and YAO patients were also less likely to live in partnership and to have children. The impact on educational and vocational outcomes was less marked than on social outcomes with no significant differences compared with the AO control group. Educational and vocational outcomes showed the lowest level in male and female CO and YAO patients who had been previously diagnosed with a brain tumour. CONCLUSIONS: Social outcomes were more affected than educational and vocational outcomes. Although CO patients are more adversely affected, YAO patients were also failing to achieve social milestones. This has consequences for the delivery of endocrine care in both paediatric and adult services.

Two Swedish screening instruments for exhaustion disorder: cross-sectional associations with burnout, work stress, private life stress, and personality traits.

AIMS: To examine the relationships of two screening instruments recently developed for assessment of exhaustion disorder (ED) with some other well-known inventories intended to assess ED-related concepts and self-reports of job demands, job control, job support, private life stressors, and personality factors. METHODS: A cross-sectional population sample ( n = 1355) completed: the Karolinska Exhaustion Disorder Scale (KEDS), Self-reported Exhaustion Disorder Scale (s-ED), Shirom-Melamed Burnout Questionnaire (SMBQ), Utrecht Work Engagement Scale (UWES-9), Job Content Questionnaire (JCQ), Big Five Inventory (BFI), and items concerning family-to-work interference and stress in private life. RESULTS: Compared to participants without any indication of ED, participants classified as having ED on KEDS or s-ED had higher scores on all four SMBQ subscales, lower scores on the UWES-9 subscales vigor and dedication, higher JCQ job demands scores, lower JCQ job support scores, higher degrees of family-to-work interference and stress in private life, and higher BFI neuroticism and openness scores. In addition, participants classified as having ED on KEDS had lower scores on the UWES-9 absorption subscale, the JCQ job control scale, and lower BFI extraversion, agreeableness and conscientiousness scores, compared to the subgroup not classified as having ED. CONCLUSIONS: As expected, we observed an overall pattern of associations between the ED screening inventories KEDS and s-ED and measures of burnout, work engagement, job demands-control-support, stress in private life, family-to-work interference, and personality factors. The results suggest that instruments designed to assess burnout, work engagement, and ED share common ground, despite their conceptual differences.

Nanoscale, Voltage-Driven Application of Bioactive Substances onto Cells with Organized Topography.

With scanning ion conductance microscopy (SICM), a noncontact scanning probe technique, it is possible both to obtain information about the surface topography of live cells and to apply molecules onto specific nanoscale structures. The technique is therefore widely used to apply chemical compounds and to study the properties of molecules on the surfaces of various cell types. The heart muscle cells, i.e., the cardiomyocytes, possess a highly elaborate, unique surface topography including transverse-tubule (T-tubule) openings leading into a cell internal system that exclusively harbors many proteins necessary for the cell's physiological function. Here, we applied isoproterenol into these surface openings by changing the applied voltage over the SICM nanopipette. To determine the grade of precision of our application we used finite-element simulations to investigate how the concentration profile varies over the cell surface. We first obtained topography scans of the cardiomyocytes using SICM and then determined the electrophoretic mobility of isoproterenol in a high ion solution to be -7 × 10(-9) m(2)/V s. The simulations showed that the delivery to the T-tubule opening is highly confined to the underlying Z-groove, and especially to the first T-tubule opening, where the concentration is ∼6.5 times higher compared to on a flat surface under the same delivery settings. Delivery to the crest, instead of the T-tubule opening, resulted in a much lower concentration, emphasizing the importance of topography in agonist delivery. In conclusion, SICM, unlike other techniques, can reliably deliver precise quantities of compounds to the T-tubules of cardiomyocytes.

Sleep and the generalization of fear learning.

Fear conditioning is an important survival mechanism, as is the ability to generalize learned fear responses to stimuli that are similar to the original conditioned stimulus. Overgeneralization of fear learning, prominent in many anxiety disorders, is however highly maladaptive. Because sleep is involved in the consolidation of fear learning, and in active processing of information, the present study explored the effect of sleep on generalization of fear learning. Participants watched a random sequence of pictures of a small and a big circle, one of them coupled with an aversive sound. Then, after a delay period containing either a nap or wake, generalization was examined as participants watched the two circles again, together with eight novel circles that gradually varied in size between the former two. Results showed that the fear response increased as a function of similarity to the conditioned response. However, there was no difference in the degree of generalization between the sleep and the wake group.

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy.

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.

The Lund University Checklist for Incipient Exhaustion-a cross-sectional comparison of a new instrument with similar contemporary tools.

BACKGROUND: Stress-related health problems (e.g., work-related exhaustion) are a societal concern in many postindustrial countries. Experience suggests that early detection and intervention are crucial in preventing long-term negative consequences. In the present study, we benchmark a new tool for early identification of work-related exhaustion-the Lund University Checklist for Incipient Exhaustion (LUCIE)-against other contextually relevant inventories and two contemporary Swedish screening scales. METHODS: A cross-sectional population sample (n = 1355) completed: LUCIE, Karolinska Exhaustion Disorder Scale (KEDS), Self-reported Exhaustion Disorder Scale (s-ED), Shirom-Melamed Burnout Questionnaire (SMBQ), Utrecht Work Engagement Scale (UWES-9), Job Content Questionnaire (JCQ), Big Five Inventory (BFI), and items concerning work-family interference and stress in private life. RESULTS: Increasing signs of exhaustion on LUCIE were positively associated with signs of exhaustion on KEDS and s-ED. The prevalence rates were 13.4, 13.8 and 7.8 %, respectively (3.8 % were identified by all three instruments). Increasing signs of exhaustion on LUCIE were also positively associated with reports of burnout, job demands, stress in private life, family-to-work interference and neuroticism as well as negatively associated with reports of job control, job support and work engagement. CONCLUSIONS: LUCIE, which is intended to detect pre-stages of ED, exhibits logical and coherent positive relations with KEDS and s-ED as well as other conceptually similar inventories. The results suggest that LUCIE has the potential to detect mild states of exhaustion (possibly representing pre-stages to ED) that if not brought to the attention of the healthcare system and treated, may develop in to ED. The prospective validity remains to be evaluated.

The Lund University Checklist for Incipient Exhaustion: a prospective validation of the onset of sustained stress and exhaustion warnings.

BACKGROUND: The need for instruments that can assist in detecting the prodromal stages of stress-related exhaustion has been acknowledged. The aim of the present study was to evaluate whether the Lund University Checklist for Incipient Exhaustion (LUCIE) could accurately and prospectively detect the onset of incipient exhaustion and to what extent work stressor exposure and private burdens were associated with increasing LUCIE scores. METHODS: Using surveys, 1355 employees were followed for 11 quarters. Participants with prospectively elevated LUCIE scores were targeted by three algorithms entailing 4 quarters: (1) abrupt onset to a sustained Stress Warning (n = 18), (2) gradual onset to a sustained Stress Warning (n = 42), and (3) sustained Exhaustion Warning (n = 36). The targeted participants' survey reports on changes in work situation and private life during the fulfillment of any algorithm criteria were analyzed, together with the interview data. Participants untargeted by the algorithms constituted a control group (n = 745). RESULTS: Eighty-seven percent of participants fulfilling any LUCIE algorithm criteria (LUCIE indication cases) rated a negative change in their work situation during the 4 quarters, compared to 48 % of controls. Ratings of negative changes in private life were also more common in the LUCIE indication groups than among controls (58 % vs. 29 %), but free-text commentaries revealed that almost half of the ratings in the LUCIE indication groups were due to work-to-family conflicts and health problems caused by excessive workload, assigned more properly to work-related negative changes. When excluding the themes related to work-stress-related private life compromises, negative private life changes in the LUCIE indication groups dropped from 58 to 32 %, while only a negligible drop from 29 to 26 % was observed among controls. In retrospective interviews, 79 % of the LUCIE indication participants confirmed exclusively/predominantly work stressors, while 6 % described a predominance of private life stressors. CONCLUSIONS: Negative changes in the work situation were the most prominent change related to a sustained increase in LUCIE scores. The findings seem to confirm that LUCIE is a potentially useful tool for clinical screening of incipient work-related exhaustion.

Hydrodynamic Forces on Macromolecules Protruding from Lipid Bilayers Due to External Liquid Flows.

It has previously been observed that an externally applied hydrodynamic shear flow above a fluid lipid bilayer can change the local concentration of macromolecules that are associated with the lipid bilayer. The external liquid flow results in a hydrodynamic force on molecules protruding from the lipid bilayer, causing them to move in the direction of the flow. However, there has been no quantitative study about the magnitude of these forces. We here use finite element simulations to investigate how the magnitude of the external hydrodynamic forces varies with the size and shape of the studied macromolecule. The simulations show that the hydrodynamic force is proportional to the effective hydrodynamic area of the studied molecule, Ahydro, multiplied by the mean hydrodynamic shear stress acting on the membrane surface, σhydro. The parameter Ahydro depends on the size and shape of the studied macromolecule above the lipid bilayer and scales with the cross-sectional area of the molecule. We also investigate how hydrodynamic shielding from other surrounding macromolecules decreases Ahydro when the surface coverage of the shielding macromolecules increases. Experiments where the protein streptavidin is anchored to a supported lipid bilayer on the floor of a microfluidic channel were finally performed at three different surface concentrations, Φ = 1%, 6%, and 10%, where the protein is being moved relative to the lipid bilayer by a liquid flow through the channel. From photobleaching measurements of fluorescently labeled streptavidin we found the experimental drift data to be within good accuracy of the simulated results, less than 12% difference, indicating the validity of the results obtained from the simulations. In addition to giving a deeper insight into how a liquid flow can affect membrane-associated molecules in a lipid bilayer, we also see an interesting potential of using hydrodynamic flow experiments together with the obtained results to study the size and the intermolecular forces between macromolecules in membranes and lipid bilayers.

Long-term treatment with pegvisomant as monotherapy in patients with acromegaly: experience from ACROSTUDY.

OBJECTIVE: To evaluate use of pegvisomant, a growth hormone (GH) receptor antagonist, as monotherapy in ACROSTUDY, a global safety surveillance study set in 14 countries (373 sites). METHODS: A descriptive analysis of safety, magnetic resonance imaging (MRI) reading, and treatment outcomes in 710 subjects who received at least 1 pegvisomant dose as monotherapy during and up to 5 years follow-up in ACROSTUDY. RESULTS: Subjects received a mean of 5.4 years of pegvisomant and were followed in ACROSTUDY for a mean of 3.8 years. A total of 1,255 adverse events (AEs) were reported in 345 subjects (48.6%). Serious AEs (SAEs) were reported in 133 (18.7%) subjects, including 22 deaths, none of which were attributed to pegvisomant use. Of 670 (94%) subjects with at least 1 liver function test (LFT) reported in ACROSTUDY, 8 (1.2%) had reported increases in transaminases >3 times the upper limit of normal (ULN). No liver failure was reported. Based on central MRI reading, 12 of 542 subjects (2.2%) had a confirmed increase or increase/decrease in tumor size. Injection-site reactions were reported in 2.3%. At 5 years of therapy, insulin-like growth factor 1 (IGF-1) level was reported normal in 67.5% (mean dose 17.2 mg/day) and elevated in 29.9% (mean dose 19.8 mg/day). Subjects on 20 mg per day or more rose from 36% at 3 years to 41% at 5 years of therapy. CONCLUSIONS: ACROSTUDY data indicate that pegvisomant used as sole medical therapy is safe and effective for patients with acromegaly. The reported low incidence of pituitary tumor size increase and liver enzyme elevations are reassuring and support the positive benefit-risk of pegvisomant therapy.

Hydrodynamic trapping of molecules in lipid bilayers.

In this work we show how hydrodynamic forces can be used to locally trap molecules in a supported lipid bilayer (SLB). The method uses the hydrodynamic drag forces arising from a flow through a conical pipette with a tip radius of 1-1.5 µm, placed approximately 1 µm above the investigated SLB. This results in a localized forcefield that acts on molecules protruding from the SLB, yielding a hydrodynamic trap with a size approximately given by the size of the pipette tip. We demonstrate this concept by trapping the protein streptavidin, bound to biotin receptors in the SLB. It is also shown how static and kinetic information about the intermolecular interactions in the lipid bilayer can be obtained by relating how the magnitude of the hydrodynamic forces affects the accumulation of protein molecules in the trap.