Search

A paucity of prenatal data is available concerning prenatal experiences of primigravid women compared with those of multigravid women. Therefore, the objectives of this study were twofold: to compare prenatal personal (detnographic and other descriptive elements, including self-esteem) and psychosocial variables (maternal-fetal attachment, marital satisfaction) and to describe perceived pregnancy experiences for both primigravid and multigravid women. Both quantitative and qualitative data were collected using a descriptive mixed-methods design. The data were part of a larger, longitudinal study focused on adjustment to parenthood in military and civilian couples. Married pregnant women who resided on the east and west coasts ofthe United States were recruited from prenatal care facilities. Participants included 50 pregnant primigravid and 50 multigravid married women recruited during the last trimester of a healthy, uncomplicated pregnancy. The main outcome measures included personal and psychosocial variables (demographics, self-esteem, maternal-fetal attachment, marital satisfaction) and perceived pregnancy experiences. Multigravid women had significantly lower levels of maternal-fetal attachment (p < .00) and marital satisfaction (p < .00) than did primigravid women during their third trimester of pregnancy. The pregnant women's responses clearly reveal that unique and distinct differences exist between the needs of primigravid women and those of multigravid women. Jnnovative prenatal educational interventions tailored to meet the distinct needs of primigravid and multigravid women are suggested. [ABSTRACT FROM AUTHOR]

PurposeTo review the literature on and discuss the role of the primary care provider in assessing and managing overweight children before they become obese.Data SourcesSelected research, national guidelines and recommendations, and the professional experience of the authors.ConclusionsThe focus of primary care involves early detection and family interventions that are designed for lifestyle modifications, specifically for improved nutrition and an increase in regular physical activity, to achieve optimal child health. Early identification and management of children who exceed a healthy weight for height, gender, and age will prevent the increasing incidence of pediatric obesity. Early prevention and management of pediatric overweight and obesity will also decrease the potential for associated medical and psychosocial problems.Implications For PracticePediatric obesity has risen dramatically in the United States during the last two decades; it is a significant child health problem that is preventable and largely under-diagnosed and under-treated. It is essential to discuss prevention of obesity with parents at every well-child visit; treatment should be initiated when patterns of weight gain exceed established percentiles for increasing height for age and gender. [ABSTRACT FROM AUTHOR]

NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice, lacking many components of a mature immune system, are at increased risk of disease. General understanding of potential pathogens of these mice is limited. We describe a high mortality disease outbreak caused by an opportunistic bacterial infection in NSG mice. Affected animals exhibited perianal fecal staining, dehydration, and wasting. Histopathologic lesions included a primary necrotizing enterocolitis, with inflammatory and necrotizing lesions also occurring in the liver, kidneys, heart, and brain of some mice. All affected individuals tested negative for known opportunistic pathogens of immunodeficient mice. We initially identified a member of Enterobacter cloacae complex (ECC) in association with the outbreak by traditional diagnostics. ECC was cultured from extraintestinal organs, both with and without histopathologic lesions, suggesting bacteremia. Infrared spectroscopy and MALDI-TOF mass spectrometry demonstrated that isolates from the outbreak shared molecular features and likely a common origin. We subsequently hypothesized that advanced sequencing methods would identify a single species of ECC associated with clinical disease. Using a novel targeted amplicon-based next-generation sequencing assay, we identified Enterobacter hormaechei in association with this outbreak. Knowledge of this organism as a potential opportunistic pathogen in NSG mice is critical for preclinical studies to prevent loss of animals and confounding of research., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: KN, CW, and KSH are employees of Charles River Laboratories.

Accumulation of amyloid-β peptide (Aβ) aggregates in synapses may contribute to the profound synaptic loss characteristic of Alzheimer's disease (AD). The origin of synaptic Aβ aggregates remains elusive, but loss of endosomal proteostasis may trigger their formation. In this study, we identified the synaptic compartments where Aβ accumulates, and performed a longitudinal analysis of synaptosomes isolated from brains of TgCRND8 APP transgenic mice of either sex. To evaluate the specific contribution of Aβ-degrading protease endothelin-converting enzyme (ECE-1) to synaptic/endosomal Aβ homeostasis, we analyzed the effect of partial Ece1 KO in brain and complete ECE1 KO in SH-SY5Y cells. Global inhibition of ECE family members was used to further assess their role in preventing synaptic Aβ accumulation. Results showed that, before extracellular amyloid deposition, synapses were burdened with detergent-soluble Aβ monomers, oligomers, and fibrils. Levels of all soluble Aβ species declined thereafter, as Aβ42 turned progressively insoluble and accumulated in Aβ-producing synaptic endosomal vesicles with characteristics of multivesicular bodies. Accordingly, fibrillar Aβ was detected in brain exosomes. ECE-1-deficient mice had significantly increased endogenous synaptosomal Aβ42 levels, and protease inhibitor experiments showed that, in TgCRND8 mice, synaptic Aβ42 became nearly resistant to degradation by ECE-related proteases. Our study supports that Aβ accumulating in synapses is produced locally, within endosomes, and does not require the presence of amyloid plaques. ECE-1 is a determinant factor controlling the accumulation and fibrillization of nascent Aβ in endosomes and, in TgCRND8 mice, Aβ overproduction causes rapid loss of Aβ42 solubility that curtails ECE-mediated degradation. SIGNIFICANCE STATEMENT Deposition of aggregated Aβ in extracellular plaques is a defining feature of AD. Aβ aggregates also accumulate in synapses and may contribute to the profound synaptic loss and cognitive dysfunction typical of the disease. However, it is not clear whether synaptotoxic Aβ is mainly derived from plaques or if it is produced and aggregated locally, within affected synaptic compartments. Filling this knowledge gap is important for the development of an effective treatment for AD, as extracellular and intrasynaptic pools of Aβ may not be equally modulated by immunotherapies or other therapeutic approaches. In this manuscript, we provide evidence that Aβ aggregates building up in synapses are formed locally, within synaptic endosomes, because of disruptions in nascent Aβ proteostasis., (Copyright © 2023 the authors.)

Aggregation and accumulation of amyloid-β peptide (Aβ) are a critical trigger for the onset of Alzheimer's disease (AD). While the plaques are the most outstanding Aβ pathological feature, much of the recent research emphasis has been on soluble Aβ species because of their diffusible, proinflammatory, and toxic properties. The focus on soluble aggregated Aβ species has also increased the interest in antibodies that are selective for different Aβ conformations. In the current study, we developed and characterized a new class of monoclonal antibodies (referred to as mAbSL) that are selective for Aβ protofibrils. Cloning and sequencing of the heavy and light chain variable regions for multiple antibodies identified sequence characteristics that may impart the conformational selectivity by the antibodies. Transfection of FreeStyle 293F cells with the plasmids permitted in-house expression and purification of mAbSL antibodies along with non-conformation-selective Aβ monoclonal antibodies (Aβ mAbs). Several of the purified mAbSL antibodies demonstrated significant affinity and selectivity for Aβ42 protofibrils compared with Aβ42 monomers and Aβ42 fibrils. Competition ELISA assays assessing the best overall antibody, mAbSL 113, yielded affinity constants of 7 nM for the antibody-Aβ42 protofibril interaction, while the affinity for either Aβ42 monomers or Aβ42 fibrils was roughly 80 times higher. mAbSL 113 significantly inhibited Aβ42 monomer aggregation by a unique mechanism compared with the inhibition displayed by Aβ mAb 513. Aβ42 protofibril dynamics were also markedly altered in the presence of mAbSL 113, whereby insoluble complex formation and protofibril deposition were stimulated by the antibody at low substoichiometric molar ratios. As the field contemplates the therapeutic effectiveness of Aβ conformation-selective antibodies, the findings presented here demonstrate new information on a monoclonal antibody that selectively targets Aβ protofibrils and impacts Aβ dynamics., (© 2023 International Society for Neurochemistry.)

Three decades of research, both in vitro and in vivo, have demonstrated the conformational heterogeneity that is displayed by the amyloid β peptide (Aβ) in Alzheimer's disease (AD). Understanding the distinct properties between Aβ conformations and how conformation may impact cellular activity remain open questions, yet still continue to provide new insights into protein misfolding and aggregation. In particular, there is interest in the group of soluble oligomeric prefibrillar Aβ species comprising lower molecular weight oligomers up to larger protofibrils. In the current study, a number of strategies were utilized to separate Aβ protofibrils and oligomers and show that the smaller Aβ oligomers have a much different conformation than Aβ protofibrils. The differences were consistent for both Aβ40 and Aβ42. Protofibrils bound thioflavin T to a greater extent than oligomers, and were highly enriched in β-sheet secondary structure. Aβ oligomers possessed a more open structure with significant solvent exposure of hydrophobic domains as determined by tryptophan fluorescence and bis-ANS binding, respectively. The protofibril-selective antibody AbSL readily discerned conformational differences between protofibrils and oligomers. The more developed structure for Aβ protofibrils ultimately proved critical for provoking the release of tumor necrosis factor α from microglial cells. The findings demonstrated a dependency on β-sheet structure for soluble Aβ aggregates to cause a microglial inflammatory response. The Aβ aggregation process yields many conformationally-varied species with different levels of β-structure and exposed hydrophobicity. The conformation elements likely determine biological activity and pathogenicity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Buprenorphine is commonly used to control postoperative pain in rodents. Short-acting formulations of buprenorphine (bup-HCl) require frequent handling and restraint of animals for appropriate dosing, which can be stressful and confound research outcomes. Ethiqa XR (bup-ER) is an FDA-indexed extended-release buprenorphine formulation that is an alternative to bup-HCl in mice and rats. In the current study, we first evaluated the pharmacokinetics of bup-ER in male C57BL/6J mice by sampling blood at 10 time points, ranging from 30 min to 72 h after administration ( n = 3 mice per time point). Average plasma concentrations fell below therapeutic levels at 48 h after administration. We also evaluated the safety of bup-ER when administered prior to surgery in combination with common anesthetics and the efficacy of bup-ER in mouse laparotomy. Anesthetic safety was studied by measuring respiratory rate, rectal temperature, and recovery time in groups of mice ( n = 8) given bup-HCl, bup-ER, or saline in combination with isoflurane or ketamine-xylazine anesthesia. No differences were seen between analgesic treatment groups with either of the general anesthetics. To evaluate efficacy, mice ( n = 10) were randomly allocated to receive either bup-ER (3.25 mg/kg) once presurgically, bup-HCl (0.1 mg/kg) presurgically and then every 8 h, or saline once before surgery. Mice underwent a sham laparotomy and were assessed for pain based on changes in weight, cageside ethogram, nesting consolidation test, rearing frequency, and nociception to von Frey testing at 6, 12, 24, 48, and 72 h after surgery. Cageside ethogram, rearing frequency, and von Frey testing showed significant differences between bup-ER-treated mice and saline controls in the early postoperative period. No significant effects between treatment groups were seen in daily weights or nesting consolidation scores. This study demonstrates that bup-ER can be safely administered before surgery and provides analgesia for up to 48 h after administration based on pharmacokinetic and behavioral data.

Sepsis is a serious medical condition characterized by bacterial infection and a subsequent massive systemic inflammatory response. In an effort to identify compounds that block lipopolysaccharide (LPS)-induced inflammation reported herein is the development of simple Lipid-A analogues that lack a disaccharide core yet still possess potent antagonistic activity against LPS. The structure of the new lead compound was developed based on predictive computational experiments. LPS antagonism by the lead compound was not straightforward, and a biphasic effect was observed suggesting a possibility of more than one binding site. An IC 50 value of 13 nM for the new compound was determined for the possible high affinity site. The combination of computational, synthetic, and biological studies revealed new structural determinants of these simplified analogues. It is expected that the acquired information will aid future design of LPS targeting glycopharmaceuticals.

Objectives: The objective of this analysis was to gauge how the incidence and mortality of uterine cancer in Kentucky have changed from 1995 through 2017. An assessment of the trends in incidence and mortality across different geographic areas and between different races was also performed., Methods: Age-adjusted annual incidence and mortality rates for uterine cancer were obtained from the Kentucky Cancer Registry. A meta-regression framework was used to assess changes in incidence and mortality rates during the time frame and to determine differences in these rates between rural versus urban counties, Appalachian versus non-Appalachian counties, and Black versus White women., Results: The incidence of uterine cancer has significantly increased throughout the state of Kentucky since 1995. Uterine cancer incidence was 10% and 22% higher in rural and Appalachian counties, respectively, compared with urban and non-Appalachian counties ( P < 0.0001) from 1995 through 2017. In contrast, urban and non-Appalachian women had higher or equivalent age-adjusted mortality from uterine cancer, compared with rural and Appalachian women, respectively. The incidence of uterine cancer was significantly higher in White women compared with Black women from 1995 through 2006, but since 2007, there has been no significant difference in uterine cancer incidence based on race. Black women had higher age-adjusted mortality than White women throughout the entire time period examined., Conclusions: The incidence of uterine cancer is higher in rural and Appalachian Kentucky, without a corresponding geographic trend in mortality. Uterine cancer mortality is significantly higher in Black women.

The NLRP3 inflammasome is a key intracellular component of the innate immune response. It is a three-protein complex essential for the production of mature interleukin 1-β. The complex, which is comprised of three proteins, NLRP3, ASC, and pro-caspase-1, has been implicated in the physiological response to pathogenic elements of cardiovascular disease and Alzheimer's disease. Investigations into the properties of the three proteins can be aided by larger-scale recombinant expression to produce adequate amounts. In the current study, a variety of NLRP3 inflammasome proteins were expressed in the ExpiCHO-S mammalian cell system with a particular focus on ASC. ASC fusion proteins with glutathione-S transferase, maltose-binding protein, and SUMO increased solubility and aided in determining the stability and oligomerization propensity of individual ASC domains and full-length ASC. ASC oligomerization was highly sensitive to protein concentration, ionic strength, and mutation. These observations provided strategic ways to enhance protein purification and characterize ASC oligomerization. The ExpiCHO-S expression system consistently produced high-yield recombinant NLRP3 inflammasome proteins which led to a further understanding of ASC oligomerization., (© 2021 American Institute of Chemical Engineers.)

This review takes a closer look at the structural components of the molecules involved in the processes leading to caspase-1 activation. Interleukins 1β and 18 (IL-1β, IL-18) are well-known proinflammatory cytokines that are produced following cleavage of their respective precursor proteins by the cysteine protease caspase-1. Active caspase-1 is the final step of the NLRP3 inflammasome, a three-protein intracellular complex involved in inflammation and induction of pyroptosis (a proinflammatory cell-death process). NLRP3 activators facilitate assembly of the inflammasome complex and subsequent activation of caspase-1 by autoproteolysis. However, the definitive structural components of active caspase-1 are still unclear and new data add to the complexity of this process. This review outlines the historical and recent findings that provide supporting evidence for the structural aspects of caspase-1 autoproteolysis and activation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Matrix metalloproteinases (MMPs), including MMP-9, are an integral part of the immune response and are upregulated in response to a variety of stimuli. New details continue to emerge concerning the mechanistic and regulatory pathways that mediate MMP-9 secretion. There is significant evidence for regulation of inflammation by dimethyl sulfoxide (DMSO) and 3',5'-cyclic adenosine monophosphate (cAMP), thus investigation of how these two molecules may regulate both MMP-9 and tumor necrosis factor α (TNFα) secretion by human monocytes was of high interest. The hypothesis tested in this study was that DMSO and cAMP regulate MMP-9 and TNFα secretion by distinct mechanisms., Aim: To investigate the regulation of lipopolysaccharide (LPS)-stimulated MMP-9 and tumor necrosis factor α secretion in THP-1 human monocytes by dimethyl sulfoxide and cAMP., Methods: The paper describes a basic research study using THP-1 human monocyte cells. All experiments were conducted at the University of Missouri-St. Louis in the Department of Chemistry and Biochemistry. Human monocyte cells were grown, cultured, and prepared for experiments in the University of Missouri-St. Louis Cell Culture Facility as per accepted guidelines. Cells were treated with LPS for selected exposure times and the conditioned medium was collected for analysis of MMP-9 and TNFα production. Inhibitors including DMSO, cAMP regulators, and anti-TNFα antibody were added to the cells prior to LPS treatment. MMP-9 secretion was analyzed by gel electrophoresis/western blot and quantitated by ImageJ software. TNFα secretion was analyzed by enzyme-linked immuno sorbent assay. All data is presented as the average and standard error for at least 3 trials. Statistical analysis was done using a two-tailed paired Student t -test. P values less than 0.05 were considered significant and designated as such in the Figures. LPS and cAMP regulators were from Sigma-Aldrich, MMP-9 standard and antibody and TNFα antibodies were from R&D Systems, and amyloid-β peptide was from rPeptide., Results: In our investigation of MMP-9 secretion from THP-1 human monocytes, we made the following findings. Inclusion of DMSO in the cell treatment inhibited LPS-induced MMP-9, but not TNFα, secretion. Inclusion of DMSO in the cell treatment at different concentrations inhibited LPS-induced MMP-9 secretion in a dose-dependent fashion. A cell-permeable cAMP analog, dibutyryl cAMP, inhibited both LPS-induced MMP-9 and TNFα secretion. Pretreatment of the cells with the adenylyl cyclase activator forskolin inhibited LPS-induced MMP-9 and TNFα secretion. Pretreatment of the cells with the general cAMP phosphodiesterase inhibitor IBMX reduced LPS-induced MMP-9 and TNFα in a dose-dependent fashion. Pre-treatment of monocytes with an anti-TNFα antibody blocked LPS-induced MMP-9 and TNFα secretion. Amyloid-β peptide induced MMP-9 secretion, which occurred much later than TNFα secretion. The latter two findings strongly suggested an upstream role for TNFα in mediating LPS-stimulate MMP-9 secretion., Conclusion: The cumulative data indicated that MMP-9 secretion was a distinct process from TNFα secretion and occurred downstream. First, DMSO inhibited MMP-9, but not TNFα, suggesting that the MMP-9 secretion process was selectively altered. Second, cAMP inhibited both MMP-9 and TNFα with a similar potency, but at different monocyte cell exposure time points. The pattern of cAMP inhibition for these two molecules suggested that MMP-9 secretion lies downstream of TNFα and that TNFα may a key component of the pathway leading to MMP-9 secretion. This temporal relationship fit a model whereby early TNFα secretion directly led to later MMP-9 secretion. Lastly, antibody-blocking of TNFα diminished MMP-9 secretion, suggesting a direct link between TNFα secretion and MMP-9 secretion., Competing Interests: Conflict-of-interest statement: There are no conflicts of interest to declare by any of the authors., (©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.)

Objectives: The goal of this study was to assess how the incidence and mortality of cervical cancer in Kentucky has changed from 1995 through 2017. Additionally, trends in incidence and mortality across different geographic areas and between different races were evaluated., Methods: Age-adjusted annual incidence and mortality rates for cervical cancer were collected from the Kentucky Cancer Registry (KCR). A quadratic fit model was used to evaluate changes in the incidence and mortality over time and to compare differences in cervical cancer incidence and mortality by: 1) rural versus urban counties, 2) Appalachian versus non-Appalachian counties, and 3) black versus white women., Results: Overall, the incidence of cervical cancer has significantly decreased throughout Kentucky since 1995. When comparing different geographic regions, the incidence was 14% and 23% higher in rural and Appalachian counties, respectively, compared to urban and non-Appalachian counties (p < 0.0001) throughout the study period. The incidence of cervical cancer was significantly higher in black women compared to white women from 1995 through 2007, but since 2008 there has been no significant difference in cervical cancer incidence based on race. Similar to incidence rates, mortality from cervical cancer was 29% higher in Appalachia (p = 0.0004) throughout the studied time period. Black women had higher age-adjusted mortality than white women until 2012, but since that time there has not been a significant difference in cervical cancer mortality based on race., Conclusions: Women residing in rural and Appalachian Kentucky have higher cervical cancer incidence and mortality rates., Competing Interests: Declaration of competing interest The authors wish to report that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Protein aggregation plays a central role in numerous neurodegenerative diseases. The key proteins in these diseases are of significant importance, but their investigation can be challenging due to unique properties of protein misfolding and oligomerization. Alpha-synuclein protein (α-Syn) is the predominant component of Lewy Bodies in Parkinson's disease (PD) and is a member of this class of proteins. Many α-Syn studies are limited by the inability to separate various monomeric, oligomeric, and fibrillar forms of the protein from heterogeneous mixtures. This Editorial Highlight summarizes the impact of a study published in the current issue of Journal of Neurochemistry, in which Lashuel and colleagues developed a simple, rapid centrifugation- and filter-based method for separating, isolating, and quantifying different forms of α-Syn. The researchers used electron microscopy, SDS-PAGE, circular dichroism, and protein assays to carefully validate the method and quantitate α-Syn yields and loss. The publication of this new method will not only aid in future studies of α-Syn, but will likely extend to other proteins that underlie a variety of neurodegenerative diseases., (© 2020 International Society for Neurochemistry.)

Glia have been implicated in Alzheimer's disease (AD) pathogenesis. Variants of the microglia receptor triggering receptor expressed on myeloid cells 2 (TREM2) increase AD risk, and activation of disease-associated microglia (DAM) is dependent on TREM2 in mouse models of AD. We surveyed gene-expression changes associated with AD pathology and TREM2 in 5XFAD mice and in human AD by single-nucleus RNA sequencing. We confirmed the presence of Trem2-dependent DAM and identified a previously undiscovered Serpina3n + C4b + reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral-nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less evident in TREM2-R47H and TREM2-R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences.

This review discusses the profound connection between microglia, neuroinflammation, and Alzheimer's disease (AD). Theories have been postulated, tested, and modified over several decades. The findings have further bolstered the belief that microglia-mediated inflammation is both a product and contributor to AD pathology and progression. Distinct microglia phenotypes and their function, microglial recognition and response to protein aggregates in AD, and the overall role of microglia in AD are areas that have received considerable research attention and yielded significant results. The following article provides a historical perspective of microglia, a detailed discussion of multiple microglia phenotypes including dark microglia, and a review of a number of areas where microglia intersect with AD and other pathological neurological processes. The overall breadth of important discoveries achieved in these areas significantly strengthens the hypothesis that neuroinflammation plays a key role in AD. Future determination of the exact mechanisms by which microglia respond to, and attempt to mitigate, protein aggregation in AD may lead to new therapeutic strategies., (© 2019 International Society for Neurochemistry.)

Microvesicles (MVs) and exosomes comprise a class of cell-secreted particles termed extracellular vesicles (EVs). These cargo-holding vesicles mediate cell-to-cell communication and have recently been implicated in neurodegenerative diseases such as Alzheimer's disease (AD). The two types of EVs are distinguished by the mechanism of cell release and their size, with the smaller exosomes and the larger MVs ranging from 30 to 100 nm and 100 nm to 1 μm in diameter, respectively. MV numbers are increased in AD and appear to interact with amyloid-β peptide (Aβ), the primary protein component of the neuritic plaques in the AD brain. Because microglial cells play such an important role in AD-linked neuroinflammation, we sought to characterize MVs shed from microglial cells, better understand MV interactions with Aβ, and determine whether internalized Aβ may be incorporated into secreted MVs. Multiple strategies were used to characterize MVs shed from BV-2 microglia after ATP stimulation. Confocal images of isolated MVs bound to fluorescently labeled annexin-V via externalized phosphatidylserine revealed a polydisperse population of small spherical structures. Dynamic light scattering measurements yielded MV diameters ranging from 150 to 600 nm. Electron microscopy of resin-embedded MVs cut into thin slices showed well-defined uranyl acetate-stained ring-like structures in a similar diameter range. The use of a fluorescently labeled membrane insertion probe, NBD C 6 -HPC, effectively tracked MVs in binding experiments, and an Aβ ELISA confirmed a strong interaction between MVs and Aβ protofibrils but not Aβ monomers. Despite the lesser monomer interaction, MVs had an inhibitory effect on monomer aggregation. Primary microglia rapidly internalized Aβ protofibrils, and subsequent stimulation of the microglia with ATP resulted in the release of MVs containing the internalized Aβ protofibrils. The role of MVs in neurodegeneration and inflammation is an emerging area, and further knowledge of MV interaction with Aβ may shed light on extracellular spread and influence on neurotoxicity and neuroinflammation.

Aggregation and accumulation of amyloid-β peptide (Aβ) is a key component of Alzheimer's disease (AD). While monomeric Aβ appears to be benign, oligomers adopt a biologically detrimental structure. These soluble structures can be detected in AD brain tissue by antibodies that demonstrate selectivity for aggregated Aβ. Protofibrils are a subset of soluble oligomeric Aβ species and are described as small (< 100 nm) curvilinear assemblies enriched in β-sheet structure. Our own in vitro studies demonstrate that microglial cells are much more sensitive to soluble Aβ42 protofibrils compared to Aβ42 monomer or insoluble Aβ42 fibrils. Protofibrils interact with microglia, trigger Toll-like receptor signaling, elicit cytokine transcription and expression, and are rapidly taken up by the cells. Because of the importance of this Aβ species, we sought to develop an antibody that selectively recognizes protofibrils over other Aβ species. Immunization of rabbits with isolated Aβ42 protofibrils generated a high-titer anti serum with a strong affinity for Aβ42 protofibrils. The antiserum, termed AbSL, was selective for Aβ42 protofibrils over Aβ42 monomers and Aβ42 fibrils. AbSL did not react with amyloid precursor protein and recognized distinct pathological features in AD transgenic mouse brain slices. Competition studies with an Aβ antibody that targets residues 1-16 indicated that the conformational epitope for AbSL involved the N-terminal region of protofibrils in some manner. The newly developed antibody may have potential diagnostic and therapeutic uses in AD tissue and patients, and targeting of protofibrils in AD may have beneficial effects. Read the Editorial Highlight for this article on page 621. Cover Image for this issue: doi. 10.1111/jnc.13827., (© 2017 International Society for Neurochemistry.)

One pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid-β peptide (Aβ) in the affected brain. While there are numerous deleterious effects of Aβ accumulation, there is general agreement that a sustained inflammatory response to aggregated Aβ contributes to progressive neurodegeneration in AD and microglial cells play a significant role in this process. Our laboratory and others have shown that small soluble aggregates of Aβ activate a microglia-mediated inflammatory response. One component of the response involves internalization of extracellular Aβ, and this process is likely very sensitive to Aβ structure. In this study we analyzed the proclivity of microglia for internalization of Aβ42 monomers and protofibrils using fluorescently-labeled Aβ. Both Aβ42 species were labeled directly via amino linkage with an Alexa Fluor 488 tetrafluorophenyl ester (AF488-TFP) and then isolated individually by chromatography. Aβ42 protofibrils retained their size and morphological properties after labeling but monomers had a much higher stoichiometry of labeling compared to protofibrils. Primary murine microglia internalized AF488-Aβ42 protofibrils rapidly and in significant amounts compared to AF488-Aβ42 monomers. Microglial internalization of protofibrils was dependent on time and concentration, and corresponded with tumor necrosis factor α secretion. In competition studies, unlabeled Aβ42 protofibril internalization, detected by immunostaining, did not diminish AF488-protofibril uptake. Internalized AF488-Aβ42 protofibrils were found widely dispersed in the cytosol with some lysosomal accumulation but little degradation. These studies highlight the sensitivity that microglia exhibit to Aβ structure in the internalization process and emphasize their affinity for soluble Aβ protofibrils., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid β (Aβ)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP(-/-)) microglial cultures, oligomeric Aβ was unable to stimulate increased secretion from mAPP(-/-) cells. This was consistent with an ability of oligomeric Aβ to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aβ produced less microgliosis in mAPP(-/-) mice compared with wild-type mice. The mAPP(-/-) mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aβ plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease., Significance Statement: A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid β (Aβ) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aβ stimulation of microglial activation is one source of brain inflammatory changes during disease. Aβ is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aβ are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aβ production to drive the microgliosis associated with AD brains., (Copyright © 2016 the authors 0270-6474/16/368471-16$15.00/0.)

Recent findings suggest that the senile plaques in Alzheimer's disease may contain soluble amyloid-β peptide (Aβ) fibril precursors along with insoluble fibrils. These soluble Aβ species, including oligomers and protofibrils, have been well-studied in vitro and are formed via non-covalent self-assembly of Aβ monomers. While both 40- and 42-residue forms of Aβ are observed in the human body, the majority of the Aβ aggregation work has been conducted on Aβ42 or Aβ40 separately, with relatively few investigations of mixtures. In order to study the effect of different combinations of Aβ40 and Aβ42 on protofibril formation, mixtures of either dry solid peptide, or purified Aβ40 and Aβ42 monomer solutions were mixed together and protofibril/monomer distributions were quantified. Increases in the Aβ42/Aβ40 ratio increased protofibril formation but the presence of Aβ40 in the mixed Aβ solutions had a significant negative impact on protofibril formation compared to equivalent solutions of pure Aβ42. Protofibril size was less affected, but β-sheet structure increased with protofibrils formed from higher Aβ42/Aβ40 ratio solutions. Direct measurement of Aβ42/Aβ40 ratios by C-terminal-selective ELISA found very little Aβ40 incorporated into protofibrils. The cumulative data emphasizes the critical importance of Aβ42, yet establishes Aβ40 as a regulator of Aβ42 aggregation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Some of the pathological hallmarks of the Alzheimer's disease brain are senile plaques composed of insoluble amyloid-β protein (Aβ) fibrils. However, much of the recent emphasis in research has been on soluble Aβ aggregates in response to a growing body of evidence that shows that these species may be more neurotoxic than fibrils. Within this subset of soluble aggregated Aβ are protofibrils and oligomers. Although each species has been widely investigated separately, few studies have directly compared and contrasted their physical properties. In this work, we examined well-recognized preparations of Aβ(1-42) oligomers and protofibrils with multiangle (MALS) and dynamic (DLS) light scattering in line with, or following, size-exclusion chromatography (SEC). Multiple SEC-MALS analyses of protofibrils revealed molecular weight (Mw) gradients ranging from 200 to 2600 kDa. Oligomeric Aβ species are generally considered to be a smaller and more nascent than protofibrils. However, oligomer Mw values ranged from 225 to 3000 kDa, larger than that for protofibrils. Root-mean-square radius (Rg) values correlated with the Mw trends with protofibril Rg values ranging from 16 to 35 nm, while oligomers produced one population at 40-43 nm with a more disperse population from 22 to 39 nm. Hydrodynamic radius (RH) measurements by DLS and thioflavin T fluorescence measurements indicated that protofibrils and oligomers had commonalities, yet electron microscopy revealed morphological differences between the two. SEC-purified Aβ(1-42) monomer at lower concentrations was slower to nucleate but formed protofibrils (1500 kDa) or soluble protofilaments (3000 kDa) depending on the buffer type. The findings from these studies shed new light on the similarities and differences between distinct soluble aggregated Aβ species.

Neuroinflammation triggered by accumulation of amyloid-β protein (Aβ) is a significant component of the Alzheimer's disease (AD) brain. Senile plaques composed of Aβ attract and activate microglia cells resulting in cytokine secretion and a proinflammatory environment. The mechanism by which Aβ activates microglia is complex and involves numerous cellular components. One receptor potentially involved in Aβ recognition and the ensuing microglia proinflammatory response is CD47. Since there is significant interest in soluble aggregated Aβ species, we sought to determine if CD47 plays a key role in microglia cytokine release stimulated by soluble Aβ(1-42) protofibrils. Pretreatment of primary murine microglia with the CD47 antagonist peptide 4N1K significantly and potently inhibited both tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) secretion stimulated by Aβ(1-42) protofibrils. 4N1K displayed toxicity to the microglia but only at concentrations much higher than the observed inhibition. Surprisingly, 4N1K also potently inhibited TNFα secretion triggered by lipopolysaccharide which is not known to signal through CD47. Treatment of the microglia with a neutralizing anti-CD47 antibody failed to block the Aβ protofibril response even though comparable samples were completely inhibited by 4N1K. Finally, Aβ(1-42) protofibrils stimulated similar levels of secreted TNFα production in both wild-type and CD47(-/-) microglia and 4N1K still potently inhibited the Aβ protofibril response even in the CD47(-/-) microglia. The overall findings demonstrated that the microglial proinflammatory response to Aβ(1-42) protofibril is not dependent on CD47 and that 4N1K exhibits CD47-independent inhibitory activity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Neuroinflammation is a characteristic feature of the Alzheimer's disease (AD) brain. Significant inflammatory markers such as activated microglia and cytokines can be found surrounding the extracellular senile plaques predominantly composed of amyloid-β protein (Aβ). Several innate immune pathways, including Toll-like receptors (TLRs) and the NLRP3 inflammasome, have been implicated in AD inflammation. Aβ plays a primary role in activating these pathways which likely contributes to the progressive neurodegeneration in AD. In order to better understand the complexities of this interaction we investigated the inflammatory response of primary microglia to Aβ(1-42) protofibrils. Aβ(1-42) protofibrils triggered a time- and MyD88-dependent process that produced tumor necrosis factor alpha (TNFα) and interleukin-1β (IL-1β) mRNA, and intracellular pro and mature forms of IL-1β protein. The accumulation of both IL-1β forms indicated that Aβ(1-42) protofibrils were able to prime and activate the NLRP3 inflammasome. Surprisingly, Aβ-induced accumulation of intracellular mature IL-1β did not translate into greater IL-1β secretion. Instead, we found that Aβ elicited a quantized burst of secreted IL-1β and this process occurred even prior to Aβ priming of the microglia suggesting a basal level of either pro or mature IL-1β in the cultured primary microglia. The IL-1β secretion burst was rapid but not sustained, yet could be re-evoked with additional Aβ stimulation. The findings from this study demonstrated multiple sites of IL-1β regulation by Aβ(1-42) protofibrils including TLR/MyD88-mediated priming, NLRP3 inflammasome activation, and modulation of the IL-1β secretory process. These results underscore the wide-ranging effects of Aβ on the innate immune response., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Microglial cells play a critical role in the propagation of neuroinflammation in the central nervous system. Microglia sense and respond to environmental signals including chemical, physical and biological cues from the surrounding cell/tissue components. In this project, our goal was to examine the effects of surface texture on BV-2 microglia morphology and function by comparing flat and nanoporous gold (np-Au) surfaces to the more conventional glass. The biocompatibility of np-Au with microglia was evaluated using functional cell assays and high resolution imaging with scanning electron microscopy (SEM). Microglia seeded on glass, ultra-flat gold (UF-Au), ultra-thin (UT) np-Au and np-Au monolith were adherent to all surfaces and their viability was not compromised as assessed by multiple toxicity assays. SEM revealed detailed morphological characteristics of adherent microglia and indicated few dramatic changes as a result of the different surfaces. Microglia proliferation was hampered by np-Au monolith but less by UT np-Au and not at all on UF-Au or glass. Microglial activation, measured by tumor necrosis factor α (TNFα) production, was fully functional (and equivalent) on all gold surfaces compared to glass. The present findings should help further the understanding of basic microglia biology on textured surfaces and more fully evaluate np-Au as a multi-functional biocompatible material. The knowledge obtained and technology developed will have a significant impact in the fabrication of nanoelectronic devices, chemical sensor development, porous nanostructured materials for BioMEMs/NEMs integration, and functional biomaterial coatings for drug delivery.

The purpose of this project was to evaluate the efficacy of group diabetes care for an underserved population using a patient-centered approach with the inclusion of interactive diabetes self-management education. In place of the traditional office visit, patients attended three group visits. Improvements in diabetes knowledge and patient-perceived self-efficacy resulted.

Soluble aggregated forms of amyloid-β protein (Aβ) have garnered significant attention recently for their role in Alzheimer's disease (AD). Protofibrils are a subset of these soluble species and are considered intermediates in the aggregation pathway to mature Aβ fibrils. Biological studies have demonstrated that protofibrils exhibit both toxic and inflammatory activities. It is important in these in vitro studies to prepare protofibrils using solution conditions that are appropriate for cellular studies as well as conducive to biophysical characterization of protofibrils. Here we describe the preparation and characterization of Aβ(1-42) protofibrils in modified artificial cerebrospinal fluid (aCSF) and demonstrate their prominent binding and activation of microglial cells. A simple phosphate/bicarbonate buffer system was prepared that maintained the ionic strength and cell compatibility of F-12 medium but did not contain numerous supplements that interfere with spectroscopic analyses of Aβ protofibrils. Reconstitution of Aβ(1-42) in aCSF and isolation with size exclusion chromatography (SEC) revealed curvilinear β-sheet protofibrils <100 nm in length and hydrodynamic radii of 21 nm. Protofibril concentration determination by BCA assay, which was not possible in F-12 medium, was more accurately measured in aCSF. Protofibrils formed and isolated in aCSF, but not monomers, markedly stimulated TNFα production in BV-2 and primary microglia and bound in significant amounts to microglial membranes. This report demonstrates the suitability of a modified aCSF system for preparing SEC-isolated Aβ(1-42) protofibrils and underscores the unique ability of protofibrils to functionally interact with microglia.

Accumulation of aggregated amyloid-β protein (Aβ) is an important feature of Alzheimer's disease. There is significant interest in understanding the initial steps of Aβ aggregation due to the recent focus on soluble Aβ oligomers. In vitro studies of Aβ aggregation have been aided by the use of conformation-specific antibodies which recognize shape rather than sequence. One of these, OC antiserum, recognizes certain elements of fibrillar Aβ across a broad range of sizes. We have observed the presence of these fibrillar elements at very early stages of Aβ incubation. Using a dot blot assay, OC-reactivity was found in size exclusion chromatography (SEC)-purified Aβ(1-42) monomer fractions immediately after isolation (early-stage). The OC-reactivity was not initially observed in the same fractions for Aβ(1-40) or the aggregation-restricted Aβ(1-42) L34P but was detected within 1-2weeks of incubation. Stability studies demonstrated that early-stage OC-positive Aβ(1-42) aggregates were resistant to 4M urea or guanidine hydrochloride but sensitive to 1% sodium dodecyl sulfate (SDS). Interestingly, the sensitivity to SDS diminished over time upon incubation of the SEC-purified Aβ(1-42) solution at 4°C. Within 6-8days the OC-positive Aβ42 aggregates were resistant to SDS denaturation. The progression to, and development of, SDS resistance for Aβ(1-42) occurred prior to thioflavin T fluorescence. In contrast, Aβ(1-40) aggregates formed after 6days of incubation were sensitive to both urea and SDS. These findings reveal information on some of the earliest events in Aβ aggregation and suggest that it may be possible to target early-stage aggregates before they develop significant stability., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Senile plaques composed of amyloid-β protein (Aβ) are an unshakable feature of the Alzheimer's disease (AD) brain. Although there is significant debate on the role of the plaques in AD progression, there is little disagreement on their role in stimulating a robust inflammatory response within the context of the disease. Significant inflammatory markers such as activated microglia and cytokines are observed almost exclusively surrounding the plaques. However, recent evidence suggests that the plaque exterior may contain a measurable level of soluble Aβ aggregates. The observations that microglia activation in vivo is selectively stimulated by distinct Aβ deposits led us to examine what specific form of Aβ is the most effective proinflammatory mediator in vitro. We report here that soluble prefibrillar species of Aβ(1-42) were better than fibrils at inducing microglial tumor necrosis factor α (TNFα) production in either BV-2 and primary murine microglia. Reconstitution of Aβ(1-42) in NaOH followed by dilution into F-12 media and isolation with size exclusion chromatography (SEC) revealed classic curvilinear β-sheet protofibrils 100 nm in length. The protofibrils, but not monomers, markedly activated BV-2 microglia. Comparisons were also made between freshly isolated protofibrils and Aβ(1-42) fibrils prepared from SEC-purified monomer. Surprisingly, while isolated fibrils had a much higher level of thioflavin T fluorescence per mole, they were not effective at stimulating either primary or BV-2 murine microglia compared to protofibrils. Furthermore, SEC-isolated Aβ(1-40) protofibrils exhibited significantly less activity than concentration-matched Aβ(1-42). This report is the first to demonstrate microglial activation by SEC-purified protofibrils, and the overall findings indicate that small, soluble Aβ(1-42) protofibrils induce much greater microglial activation than mature insoluble fibrils.

Amyloid-β protein (Aβ) is the principal component of the neuritic plaques found in Alzheimer's disease. The predominant Aβ morphology in the plaques is fibrillar which has prompted substantial in vitro work to better understand the molecular organization of Aβ fibrils. In the current study, tryptophan substitutions were made at Aβ(1-40) position 19 (F19W) or 20 (F20W) to ascertain environmental differences between the two residues in the fibril structure. Kinetic studies revealed similar rates of fibril formation between Aβ(1-40) F19W and F20W and both peptides formed typical amyloid fibril structures. Aβ(1-40) F19W fibrils displayed a significant tryptophan fluorescence blue-shift in λ(max) (33nm) compared to monomer while Aβ(1-40) F20W fibrils had a much smaller shift (9nm). Fluorescence quenching experiments with water-soluble acrylamide and KI demonstrated that both W19 and W20 were much less accessible to quenching in fibrils compared to monomer. Lipid-soluble TEMPO quenched the fluorescence of Aβ(1-40) F19W fibrils more effectively than F20W fibrils in agreement with the fluorescence blue-shift results. These findings demonstrate distinct environments between Aβ(1-40) residues 19 and 20 fibrils and indicate that while W20 accessibility is compromised in Aβ fibrils it resides in a much less hydrophobic environment than W19., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A signature feature of Alzheimer's disease is the accumulation of plaques, composed of fibrillar amyloid-beta protein (Abeta), in the brain parenchyma. Structural models of Abeta fibrils reveal an extensive beta-sheet network with a hydrophobic core extending throughout the fibril axis. In this study, phenylalanines in the Abeta(1-40) sequence were substituted with tryptophan residues at either position 4 (F4W) or 19 (F19W) to probe the fibril environment. The F4W substitution did not alter self-assembly kinetics, while the F19W change slightly lengthened the lag phase without hindering fibril formation. The tryptophan fluorescence of Abeta(1-40) F19W, but not Abeta(1-40) F4W, underwent a marked blue shift during fibril formation and this shift was temporally correlated with thioflavin T binding. Isolated Abeta(1-40) F19W fibrils exhibited the largest fluorescence blue shifts consistent with W19 insertion into the Abeta(1-40) fibril inner core and direct probing of the substantially hydrophobic environment therein., (2009 Elsevier Inc. All rights reserved.)

Pathological studies have determined that fibrillar forms of amyloid-beta protein (Abeta) comprise the characteristic neuritic plaques in Alzheimer's disease (AD). These studies have also revealed significant inflammatory markers such as activated microglia and cytokines surrounding the plaques. Although the plaques are a hallmark of AD, they are only part of an array of Abeta aggregate morphologies observed in vivo. Interestingly, not all of these Abeta deposits provoke an inflammatory response. Since structural polymorphism is a prominent feature of Abeta aggregation both in vitro and in vivo, we sought to clarify which Abeta morphology or aggregation species induces the strongest proinflammatory response using human THP-1 monocytes as a model system. An aliquot of freshly reconstituted Abeta(1-42) in sterile water (100 microM, pH 3.6) did not effectively stimulate the cells at a final Abeta concentration of 15 microM. However, quiescent incubation of the peptide at 4 degrees C for 48-96 h greatly enhanced its ability to induce tumor necrosis factor-alpha (TNFalpha) production, the level of which surprisingly declined upon further aggregation. Imaging of the Abeta(1-42) aggregation solutions with atomic force microscopy indicated that the best cellular response coincided with the appearance of fibrillar structures, yet conditions that accelerated or increased the level of Abeta(1-42) fibril formation such as peptide concentration, temperature, or reconstitution in NaOH/PBS at pH 7.4 diminished its ability to stimulate the cells. Finally, depletion of the Abeta(1-42) solution with an antibody that recognizes fibrillar oligomers dramatically weakened the ability to induce TNFalpha production, and size-exclusion separation of the Abeta(1-42) solution provided further characterization of an aggregated species with proinflammatory activity. The findings suggested that an intermediate stage Abeta(1-42) fibrillar precursor is optimal for inducing a proinflammatory response in THP-1 monocytes.

Amyloid-beta (Abeta) is a naturally occurring 40- or 42-residue peptide fragment with a primary role in Alzheimer's disease (AD). Aggregated Abeta accumulates as both dense core plaques and diffuse deposits in the brains of AD patients. Abeta plaques are surrounded by activated microglia, some of which are believed to be derived from peripheral blood monocytes that have infiltrated the central nervous system and differentiated into phagocytes in response to Abeta. We have modeled this process using THP-1 human monocytes and found Abeta(1-42) to be as effective as phorbol myristate acetate at differentiating THP-1 monocytes based on cell adhesion, fibronectin binding, CD11b cell-surface expression, and morphological changes. Cell adhesion studies and atomic force microscopy imaging revealed an inverse correlation between Abeta(1-42)-induced monocyte maturation and aggregation progression. Freshly reconstituted Abeta(1-42) solutions were the most effective, yet continued aggregation reduced, and eventually abolished, the ability to induce monocyte adhesion. Abeta(1-40), lower aggregation concentrations of Abeta(1-42), and an aggregation-restricted Abeta(1-42) L34P mutant had little effect on monocyte adhesion under the same conditions as Abeta(1-42). These findings implicated an oligomeric, but not monomeric or fibrillar, Abeta(1-42) aggregation species in the monocyte maturation process. The rapidly-formed Abeta(1-42) oligomers were distinct from Abeta-derived diffusible ligands which did not elicit significant THP-1 monocyte adhesion. These data demonstrate that a specific oligomeric Abeta(1-42) aggregation species can potently initiate the THP-1 monocyte maturation process.

The primary molecules for mediating the innate immune response are the Toll-like family of receptors (TLRs). Recent work has established that amyloid-beta (Abeta) fibrils, the primary components of senile plaques in Alzheimer's disease (AD), can interact with the TLR2/4 accessory protein CD14. Using antibody neutralization assays and tumor necrosis factor alpha release in the human monocytic THP-1 cell line, we determined that both TLR2 and TLR4 mediated an inflammatory response to aggregated Abeta(1-42). This was in contrast to exclusive TLR ligands lipopolysaccharide (LPS) (TLR4) and tripalmitoyl cysteinyl seryl tetralysine (Pam(3)CSK(4)) (TLR2). Atomic force microscopy imaging showed a fibrillar morphology for the proinflammatory Abeta(1-42) species. Pre-treatment of the cells with 10 microg/mL of a TLR2-specific antibody blocked approximately 50% of the cell response to fibrillar Abeta(1-42), completely blocked the Pam(3)CSK(4) response, and had no effect on the LPS-induced response. A TLR4-specific antibody (10 microg/mL) blocked approximately 35% of the cell response to fibrillar Abeta(1-42), completely blocked the LPS response, and had no effect on the Pam(3)CSK(4) response. Polymyxin B abolished the LPS response with no effect on Abeta(1-42) ruling out bacterial contamination of the Abeta samples. Combination antibody pre-treatments indicated that neutralization of TLR2, TLR4, and CD14 together was much more effective at blocking the Abeta(1-42) response than the antibodies used alone. These data demonstrate that fibrillar Abeta(1-42) can trigger the innate immune response and that both TLR2 and TLR4 mediate Abeta-induced tumor necrosis factor alpha production in a human monocytic cell line.

The deposition of aggregated amyloid-beta (Abeta) peptides in the brain as senile plaques is a pathological hallmark of Alzheimer's disease (AD). Several lines of evidence indicate that fibrillar and, in particular, soluble aggregates of these 40- and 42-residue peptides are important in the etiology of AD. Recent studies also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we review our recent reports that Abeta(1-40) in vitro can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta(1-40) in low ionic strength buffers. These aggregates were quite stable and disaggregated to only a limited extent on dilution. A second class of soluble Abeta aggregates was generated at polar-nonpolar interfaces. Aggregation in a two-phase system of buffer over chloroform occurred more rapidly than in buffer alone. In buffered 2% hexafluoroisopropanol (HFIP), microdroplets of HFIP were formed and the half-time for aggregation was less than 10 minutes. Like Abeta protofibrils, these interfacial aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. However, electron microscopy and atomic force microscopy revealed very different morphologies. The HFIP aggregates formed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP these aggregates initially were very unstable and disaggregated completely within 2 minutes. However, their stability increased as they progressed to fibers. It is important to determine whether similar interfacial Abeta aggregates are produced in vivo.

The brains of Alzheimer's disease (AD) patients contain large numbers of amyloid plaques that are rich in fibrils composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the etiology of AD. Recent reports also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we demonstrate that Abeta-(1-40) can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta-(1-40) at low ionic strength. Dilution of these aggregation reactions induced disaggregation to monomers as measured by size exclusion chromatography. Protofibril concentrations monitored by thioflavin T fluorescence decreased in at least two kinetic phases, with initial disaggregation (rate constant approximately 1 h(-1)) followed by a much slower secondary phase. Incubation of the reactions without agitation resulted in less disaggregation at slower rates, indicating that the protofibrils became progressively more stable over time. In fact, protofibrils isolated by size exclusion chromatography were completely stable and gave no disaggregation. A second class of soluble Abeta aggregates was generated rapidly (<10 min) in buffered 2% hexafluoroisopropanol (HFIP). These aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. Electron microscopy and atomic force microscopy revealed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP, these aggregates initially were very unstable and disaggregated completely within 2 min. However, their stability increased as they progressed to fibers. Relative to Abeta protofibrils, the HFIP-induced aggregates seeded elongation by Abeta monomer deposition very poorly. The techniques used to distinguish these two classes of soluble Abeta aggregates may be useful in characterizing Abeta aggregates formed in vivo.

Accumulation of aggregated amyloid-beta peptide (Abeta) in the brain is a pathological hallmark of Alzheimer's disease (AD). In vitro studies indicate that the 40- to 42-residue Abeta peptide in solution will undergo self-assembly leading to the transient appearance of soluble protofibrils and ultimately to insoluble fibrils. The Abeta peptide is amphiphilic and accumulates preferentially at a hydrophilic/hydrophobic interface. Solid surfaces and air-water interfaces have been shown previously to promote Abeta aggregation, but detailed characterization of these aggregates has not been presented. In this study Abeta(1-40) introduced to aqueous buffer in a two-phase system with chloroform aggregated 1-2 orders of magnitude more rapidly than Abeta in the buffer alone. The interface-induced aggregates were released into the aqueous phase and persisted for 24-72 h before settling as a visible precipitate at the interface. Thioflavin T fluorescence and circular dichroism analyses confirmed that the Abeta aggregates had a beta-sheet secondary structure. However, these aggregates were far less stable than Abeta(1-40) protofibrils prepared in buffer alone and disaggregated completely within 3 min on dilution. Atomic force microscopy revealed that the aggregates consisted of small globules 4-5 nm in height and long flexible fibers composed of these globules aligned roughly along a longitudinal axis, a morphology distinct from that of Abeta protofibrils prepared in buffer alone. The relative instability of the fibers was supported by fiber interruptions apparently introduced by brief washing of the AFM grids. To our knowledge, unstable aggregates of Abeta with beta-sheet structure and fibrous morphology have not been reported previously. Our results provide the clearest evidence yet that the intrinsic beta-sheet structure of an in vitro Abeta aggregate depends on the aggregation conditions and is reflected in the stability of the aggregate and the morphology observed by atomic force microscopy. Resolution of these structural differences at the molecular level may provide important clues to the further understanding of amyloid formation in vivo.

Nordihydroguaiaretic acid (NDGA) was observed by Ono et al. (J Neurochem 87:172-181, 2002) to decrease the fluorescence of thioflavin T associated with freshly extended amyloid beta-protein (Abeta) fibrils. They concluded that NDGA could disaggregate Abeta fibrils into aggregates that were larger than monomers or oligomers and did not bind thioflavin T. Such an effect could be of therapeutic importance in the treatment of Alzheimer's disease. In the current study, we confirmed that NDGA induces a decrease in the fluorescence of thioflavin T associated with Abeta(1-40) fibrils and extended this observation to Abeta(1-40) protofibrils. However, attempts to identify protofibril disaggregation products using dynamic light scattering, electron microscopy, and size exclusion chromatography failed to demonstrate any decrease in aggregate size or concentration or a parallel increase in Abeta monomers or small oligomers when protofibrils were incubated with excess NDGA. We propose instead that the decreases in thioflavin T fluorescence resulted from either displacement or conformational alteration of thioflavin T upon the binding of NDGA to these aggregates. In fact, the same equilibrium fluorescence values were observed regardless of the order in which NDGA, thioflavin T, and Abeta protofibrils were added to the incubation. Although NDGA failed to disaggregate Abeta protofibrils, it did inhibit direct protofibril-protofibril association but did not alter protofibril elongation by monomer addition. These results suggest that NDGA might bind along the lateral surface of Abeta protofibrils. In addition, the binding of NDGA to Abeta protofibrils increased their nonspecific adherence to Superdex 75 resin and diminished their effects on cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Objective: To describe maternal perceptions about postpartum return to the workplace., Design and Setting: The research framework was McCubbin and McCubbin's Resiliency Model of Family Stress, Adjustment, and Adaptation. Employed, postpartum women, who resided in six different states, were recruited from day care and primary care settings to participate in this descriptive study., Participants: Seventy-four employed, married women, who returned to the workplace within one year after childbirth., Main Outcome Measure: As part of a larger study, perceptions about combining multiple roles after return to the workplace are the focus for this report., Results: Participants responded to open-ended questions about their return to the workplace during the first months after giving birth. Content analysis of those responses resulted in two major categories, Resiliency Challenges: Negative Aspects (role conflict/overload, family stress, family/child issues, finances, psychosocial issues) and Resiliency Building: Positive Aspects (social support, maternal role satisfaction, positive adaptation, career role satisfaction)., Conclusions: The data support the premise that employed women view returning to the workforce as having more challenges than they expected because the experience was viewed as being mostly negative. Preparing for return to the workplace is an important, but neglected, topic that needs to be addressed and defined more clearly in the literature. Therefore, future research is needed to identify resources and interventions that will help women experience fewer challenges associated with postpartum employment.

The peptide KLVFF-K6 was observed by Lowe et al. to simultaneously enhance amyloid beta-protein (Abeta) fibrillogenesis and decrease cellular toxicity, as measured in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. It was postulated that accelerated Abeta aggregation and precipitation induced by KLVFF-K6 may lead to an increase in less toxic insoluble fibrils at the expense of more toxic soluble protofibrils. In a previous study, we distinguished between two modes of protofibril growth: elongation by monomer deposition and direct protofibril-protofibril association. These growth mechanisms could be resolved by varying Abeta monomer and NaCl concentrations. Using assays designed to isolate these distinct modes of protofibril growth, we report here that larger Abeta aggregates formed in the presence of KLVFF-K6 resulted from enhanced protofibril association. 3H-Radiomethylated KLVFF-K6 bound to associated protofibrils with an apparent Kd of 180 nM, and concentrations of free [3H]KLVFF-K6 in this range were sufficient to convert soluble protofibrils to sedimentable fibrils. However, promotion of Abeta protofibril association by KLVFF-K6 had no effect on Abeta-induced decreases in cellular MTT reduction. Therefore, our data do not support the proposal that insoluble fibrils formed with KLVFF-K6 are less toxic than soluble protofibrils. KLVFF-K6 did not alter rates of protofibril elongation by monomer deposition. In contrast, when added to Abeta monomers isolated with the use of size-exclusion chromatography, KLVFF-K6 inhibited fibrillogenesis, as measured by thioflavin T fluorescence, and this inhibition was paralleled by a failure to alter cellular MTT reduction.

Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary components of these plaques are 40- and 42-residue peptides, denoted A beta(1-40) and A beta(1-42), that are derived by proteolysis of cellular amyloid precursor protein. Synthetic A beta(1-40) and A beta(1-42) form amyloid fibrils in vitro that share many features with the amyloid in plaques. Soluble intermediates in A beta fibrillogenesis, termed protofibrils, have been identified previously, and here we describe the in vitro formation and isolation of A beta(1-40) protofibrils by size exclusion chromatography. In some experiments, the A beta(1-40) was radiomethylated to better quantify various A beta species. Mechanistic studies clarified two separate modes of protofibril growth, elongation by monomer deposition and protofibril-protofibril association, that could be resolved by varying the NaCl concentration. Small isolated protofibrils in dilute Tris-HCl buffers were directed along the elongation pathway by addition of A beta(1-40) monomer or along the association pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrils with initial molecular masses M(w) of (7-30) x 10(3) kDa grew to M(w) values of up to 250 x 10(3) kDa by these two growth processes. However, the mass per unit length of the associated protofibrils was about 2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer deposition with the elongated, associated, and initial protofibril pools were identical when equal number concentrations of original protofibrils were compared, indicating that the original number of protofibril ends had not been altered by the elongation or association processes. Atomic force microscopy revealed heterogeneous initial protofibrils that became more rodlike following the elongation reaction. Our data indicate that protofibril elongation in the absence of NaCl results from monomer deposition only at the ends of protofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril association occurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactions that result in a relatively disordered fibril structure.

Purpose: To review the literature on and discuss the role of the primary care provider in assessing and managing overweight children before they become obese., Data Sources: Selected research, national guidelines and recommendations, and the professional experience of the authors., Conclusions: The focus of primary care involves early detection and family interventions that are designed for lifestyle modifications, specifically for improved nutrition and an increase in regular physical activity, to achieve optimal child health. Early identification and management of children who exceed a healthy weight for height, gender, and age will prevent the increasing incidence of pediatric obesity. Early prevention and management of pediatric overweight and obesity will also decrease the potential for associated medical and psychosocial problems., Implications for Practice: Pediatric obesity has risen dramatically in the United States during the last two decades; it is a significant child health problem that is preventable and largely under-diagnosed and under-treated. It is essential to discuss prevention of obesity with parents at every well-child visit; treatment should be initiated when patterns of weight gain exceed established percentiles for increasing height for age and gender.

The beta-site amyloid precursor protein-cleaving enzyme (BACE) cleaves the amyloid precursor protein to produce the N terminus of the amyloid beta peptide, a major component of the plaques found in the brains of Alzheimer's disease patients. Sequence analysis of BACE indicates that the protein contains the consensus sequences found in most known aspartyl proteases, but otherwise has only modest homology with aspartyl proteases of known three-dimensional structure (i.e., pepsin, renin, or cathepsin D). Because BACE has been shown to be one of the two proteolytic activities responsible for the production of the Abeta peptide, this enzyme is a prime target for the design of therapeutic agents aimed at reducing Abeta for the treatment of Alzheimer's disease. Toward this ultimate goal, we have expressed a recombinant, truncated human BACE in a Drosophila melanogaster S2 cell expression system to generate high levels of secreted BACE protein. The protein was convenient to purify and was enzymatically active and specific for cleaving the beta-secretase site of human APP, as demonstrated with soluble APP as the substrate in novel sandwich enzyme-linked immunosorbent assay and Western blot assays. Further kinetic analysis revealed no catalytic differences between this recombinant, secreted BACE, and brain BACE. Both showed a strong preference for substrates that contained the Swedish mutation, where NL is substituted for KM immediately upstream of the cleavage site, relative to the wild-type sequence, and both showed the same extent of inhibition by a peptide-based inhibitor. The capability to produce large quantities of BACE enzyme will facilitate protein structure determination and inhibitor development efforts that may lead to the evolution of useful Alzheimer's disease treatments.

A series of isoflavone and tyrphostin compounds were found to inhibit the degradation of cAMP by several cyclic nucleotide phosphodiesterase (PDE) isozymes. Specific hydroxyl groups on the isoflavone structure were critical for PDE isozyme-selective inhibition. Replacement of the C-7 hydroxyl group of the isoflavone with a methoxy group raised the IC(50) for PDE1, PDE3, and PDE4. The absence of the C-5 hydroxyl group raised the IC(50) from 5 to >100 microM for PDE4, but actually lowered the IC(50) for PDE3 and PDE1. Replacement of the C-4' hydroxyl group with a methoxy group raised the IC(50) for PDE3 and PDE1, yet only slightly changed the IC(50) for PDE4. Various tyrphostins were also potent inhibitors of PDE1, PDE3, and PDE4. The four-carbon side chained tyrphostins were much less potent; however, a very interesting pattern was observed in which removal of phenolic hydroxyls on the tyrphostin structure increased the potency for PDE1 and PDE3, but not PDE4. These results may help to explain some of the therapeutic and intracellular signaling effects of isoflavones and tyrphostins. Moreover, the isozyme selectivity demonstrated by the isoflavones and tyrphostins can serve as a pharmacophore for the design of specific PDE inhibitors.

All healthcare providers have an obligation to promote smoking cessation to their patients who smoke. While patients are advised to stop smoking, the use of specific smoking cessation strategies is rarely addressed by primary care providers. This article discusses smoking cessation for two specifically vulnerable groups: women and African-Americans. Both groups have been identified as having increased rates of nicotine metabolism and may be less likely to respond effectively to nicotine replacement for smoking cessation. Increased nicotine metabolism results in greatest difficulty with successful smoking cessation and creates an increased vulnerability to a wide range of cardiovascular and respiratory health problems associated with smoking. Nicotine replacement therapy for smoking cessation has been the most common treatment available; however, many patients have not been successful with that method, and a majority (75%) of smokers who attempt to stop smoking return to smoking. The use of bupropion hydrochloride (Zyban) as a nonnicotine replacement therapy for smoking cessation is discussed. Relapses in smoking cessation are due to nicotine craving and the attempt to alleviate symptoms of nicotine withdrawal. Bupropion hydrochloride, an oral antidepressant, is believed to work by elimination of nicotine cravings and to decrease the physiologic and psychologic symptoms associated with nicotine withdrawal. Patients who have not been successful in smoking cessation may benefit from bupropion hydrochloride therapy in conjunction with counseling and behavior modification.

The phosphodiesterase activity in the HT4.7 neural cell line was pharmacologically characterized, and phosphodiesterase isozyme 4 (PDE4) was found to be the predominant isozyme. The Km for cAMP was 1-2 microM, indicative of a "low Km" phosphodiesterase, and the activity was inhibited by PDE4-selective inhibitors rolipram and Ro20-1724, but not PDE3- or PDE2-selective inhibitors. Calcium, calmodulin, and cGMP, regulators of PDE1, PDE2, and PDE3, had no effect on cAMP hydrolysis. The protein tyrosine kinase inhibitor, genistein, inhibited HT4.7 cAMP phosphodiesterase activity by 85-95% with an IC50 of 4 microM; whereas daidzein, an inactive structural analog of genistein, had little effect on phosphodiesterase activity. This is a common pharmacological criterion used to implicate the regulation by a tyrosine kinase. However, genistein still inhibited phosphodiesterase activity with a mixed pattern of inhibition even when ion-exchange chromatography was used to partially purify phosphodiesterase away from the tyrosine kinase activity. Moreover, tyrphostin 51, another tyrosine kinase inhibitor, was found to also inhibit partially purified phosphodiesterase activity noncompetitively. These data suggest that HT4.7 phosphodiesterase activity is dominated by PDE4 and can be regulated by genistein and tyrphostin 51 by a tyrosine kinase-independent mechanism., (Copyright 1999 Academic Press.)

Cationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a "T-shape" configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 microgram of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTR gene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTR in CF lungs is a viable and promising approach for treatment of the disease.