Comparison of a Prototype SARS-CoV-2 Lateral Flow IMMUNOASSAY with the BinaxNOWTM COVID-19 Antigen CARD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the COVID-19 pandemic. From the onset of the pandemic, rapid antigen tests have quickly proved themselves to be an accurate and accessible diagnostic platform. The initial (and still most commonly used antigen tests) for COVID-19 diagnosis were constructed using monoclonal antibodies (mAbs) specific to severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP). These mAbs are able to bind SARS-CoV-2 NP due to high homology between the two viruses. However, since first being identified in 2019, SARS-CoV-2 has continuously mutated, and a multitude of variants have appeared. These mutations have an elevated risk of leading to possible diagnostic escape when using tests produced with SARS-CoV-derived mAbs. Here, we established a library of 18 mAbs specific to SARS-CoV-2 NP and used two of these mAbs (1CV7 and 1CV14) to generate a prototype antigen-detection lateral flow immunoassay (LFI). A side-by-side analysis of the 1CV7/1CV14 LFI and the commercially available BinaxNOWTM COVID-19 Antigen CARD was performed. Results indicated the 1CV7/1CV14 LFI outperformed the BinaxNOWTM test in the detection of BA.2, BA.2.12.1, and BA.5 Omicron sub-variants when testing remnant RT-PCR positive patient nasopharyngeal swabs diluted in viral transport media.

Development of a dual antigen lateral flow immunoassay for detecting Yersinia pestis.

BACKGROUND: Yersinia pestis is the causative agent of plague, a zoonosis associated with small mammals. Plague is a severe disease, especially in the pneumonic and septicemic forms, where fatality rates approach 100% if left untreated. The bacterium is primarily transmitted via flea bite or through direct contact with an infected host. The 2017 plague outbreak in Madagascar resulted in more than 2,400 cases and was highlighted by an increased number of pneumonic infections. Standard diagnostics for plague include laboratory-based assays such as bacterial culture and serology, which are inadequate for administering immediate patient care for pneumonic and septicemic plague. PRINCIPAL FINDINGS: The goal of this study was to develop a sensitive rapid plague prototype that can detect all virulent strains of Y. pestis. Monoclonal antibodies (mAbs) were produced against two Y. pestis antigens, low-calcium response V (LcrV) and capsular fraction-1 (F1), and prototype lateral flow immunoassays (LFI) and enzyme-linked immunosorbent assays (ELISA) were constructed. The LFIs developed for the detection of LcrV and F1 had limits of detection (LOD) of roughly 1-2 ng/mL in surrogate clinical samples (antigens spiked into normal human sera). The optimized antigen-capture ELISAs produced LODs of 74 pg/mL for LcrV and 61 pg/mL for F1 when these antigens were spiked into buffer. A dual antigen LFI prototype comprised of two test lines was evaluated for the detection of both antigens in Y. pestis lysates. The dual format was also evaluated for specificity using a small panel of clinical near-neighbors and other Tier 1 bacterial Select Agents. CONCLUSIONS: LcrV is expressed by all virulent Y. pestis strains, but homologs produced by other Yersinia species can confound assay specificity. F1 is specific to Y. pestis but is not expressed by all virulent strains. Utilizing highly reactive mAbs, a dual-antigen detection (multiplexed) LFI was developed to capitalize on the diagnostic strengths of each target.

Monoclonal Antibodies Counteract Opioid-Induced Behavioral and Toxic Effects in Mice and Rats.

Monoclonal antibodies (mAbs) and vaccines have been proposed as medical countermeasures to treat opioid use disorder (OUD) and prevent opioid overdose. In contrast to current pharmacotherapies (e.g., methadone, buprenorphine, naltrexone, and naloxone) for OUD and overdose, which target brain opioid receptors, mAbs and vaccine-generated polyclonal antibodies sequester the target opioid in the serum and reduce drug distribution to the brain. Furthermore, mAbs offer several potential clinical benefits over approved medications, such as longer serum half-life, higher selectivity, reduced side effects, and no abuse liability. Using magnetic enrichment to isolate opioid-specific B cell lymphocytes prior to fusion with myeloma partners, this study identified a series of murine hybridoma cell lines expressing mAbs with high affinity for opioids of clinical interest, including oxycodone, heroin and its active metabolites, and fentanyl. In mice, passive immunization with lead mAbs against oxycodone, heroin, and fentanyl reduced drug-induced antinociception and the distribution of the target opioid to the brain. In mice and rats, mAb pretreatment reduced fentanyl-induced respiratory depression and bradycardia, two risk factors for opioid-related overdose fatality. Overall, these results support use of mAbs to counteract toxic effects of opioids and other chemical threats. SIGNIFICANCE STATEMENT: The incidence of fatal overdoses due to the widespread access to heroin, prescription opioids, and fentanyl suggests that current Food and Drug Administration-approved countermeasures are not sufficient to mitigate the opioid epidemic. Monoclonal antibodies (mAbs) may provide acute protection from overdose by binding to circulating opioids in serum. Use of mAbs prophylactically, or after exposure in combination with naloxone, may reduce hospitalization and increase survival.

Wbox2: A clathrin terminal domain-derived peptide inhibitor of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) occurs via the formation of clathrin-coated vesicles from clathrin-coated pits (CCPs). Clathrin is recruited to CCPs through interactions between the AP2 complex and its N-terminal domain, which in turn recruits endocytic accessory proteins. Inhibitors of CME that interfere with clathrin function have been described, but their specificity and mechanisms of action are unclear. Here we show that overexpression of the N-terminal domain with (TDD) or without (TD) the distal leg inhibits CME and CCP dynamics by perturbing clathrin interactions with AP2 and SNX9. TDD overexpression does not affect clathrin-independent endocytosis or, surprisingly, AP1-dependent lysosomal trafficking from the Golgi. We designed small membrane-permeant peptides that encode key functional residues within the four known binding sites on the TD. One peptide, Wbox2, encoding residues along the W-box motif binding surface, binds to SNX9 and AP2 and potently and acutely inhibits CME.

The American Nutrition Association®: Championing the Science and Practice of Personalized Nutrition.

Poor nutrition is the leading cause of our chronic disease and obesity crisis. To unleash the potential of personalized nutrition to reverse this crisis, five leading nutrition organizations have affiliated together as the American Nutrition Association® (ANA®). The ANA envisions a society of Healthy People Powered by Nutrition. The ANA brings that vision to life through its mission to Champion the Science and Practice of Personalized Nutrition and its strategy to Educate, Certify, Advocate, and Connect.

Toward the Definition of Personalized Nutrition: A Proposal by The American Nutrition Association.

Personalized nutrition holds tremendous potential to improve human health. Despite exponential growth, the field has yet to be clearly delineated and a consensus definition of the term "personalized nutrition" (PN) has not been developed. Defining and delineating the field will foster standardization and scalability in research, data, training, products, services, and clinical practice; and assist in driving favorable policy. Building on the seminal work of pioneering thought leaders across disciplines, we propose that personalized nutrition be defined as: a field that leverages human individuality to drive nutrition strategies that prevent, manage, and treat disease and optimize health, and be delineated by three synergistic elements: PN science and data, PN professional education and training, and PN guidance and therapeutics. Herein we describe the application of PN in these areas and discuss challenges and solutions that the field faces as it evolves. This and future work will contribute to the continued refinement and growth of the field of PN.Teaching pointsPN approaches can be most effective when there is consensus regarding its definition and applications.PN can be delineated into three main areas of application: PN science and data, PN education and training, PN guidance and therapeutics.PN science and data foster understanding about the impact of genetic, phenotypic, biochemical and nutritional inputs on an individual's health.PN education and training equip a variety of healthcare professionals to apply PN strategies in many healthcare settings.PN professionals have greater ability to tailor interventions via PN guidance and therapeutics.Favorable policy allows PN to be more fully integrated into the healthcare system.

Enhanced Dendritic Actin Network Formation in Extended Lamellipodia Drives Proliferation in Growth-Challenged Rac1P29S Melanoma Cells.

Actin assembly supplies the structural framework for cell morphology and migration. Beyond structure, this actin framework can also be engaged to drive biochemical signaling programs. Here, we describe how the hyperactivation of Rac1 via the P29S mutation (Rac1P29S) in melanoma hijacks branched actin network assembly to coordinate proliferative cues that facilitate metastasis and drug resistance. Upon growth challenge, Rac1P29S-harboring melanoma cells massively upregulate lamellipodia formation by dendritic actin polymerization. These extended lamellipodia form a signaling microdomain that sequesters and phospho-inactivates the tumor suppressor NF2/Merlin, driving Rac1P29S cell proliferation in growth suppressive conditions. These biochemically active lamellipodia require cell-substrate attachment but not focal adhesion assembly and drive proliferation independently of the ERK/MAPK pathway. These data suggest a critical link between cell morphology and cell signaling and reconcile the dichotomy of Rac1's regulation of both proliferation and actin assembly by revealing a mutual signaling axis wherein actin assembly drives proliferation in melanoma.

Immunoglobulin G subclass switching impacts sensitivity of an immunoassay targeting Francisella tularensis lipopolysaccharide.

The CDC Tier 1 select agent Francisella tularensis is a small, Gram-negative bacterium and the causative agent of tularemia, a potentially life-threatening infection endemic in the United States, Europe and Asia. Currently, there is no licensed vaccine or rapid point-of-care diagnostic test for tularemia. The purpose of this research was to develop monoclonal antibodies (mAbs) specific to the F. tularensis surface-expressed lipopolysaccharide (LPS) for a potential use in a rapid diagnostic test. Our initial antigen capture ELISA was developed using murine IgG3 mAb 1A4. Due to the low sensitivity of the initial assay, IgG subclass switching, which is known to have an effect on the functional affinity of a mAb, was exploited for the purpose of enhancing assay sensitivity. The ELISA developed using the IgG1 or IgG2b mAbs from the subclass-switch family of 1A4 IgG3 yielded improved assay sensitivity. However, surface plasmon resonance (SPR) demonstrated that the functional affinity was decreased as a result of subclass switching. Further investigation using direct ELISA revealed the potential self-association of 1A4 IgG3, which could explain the higher functional affinity and higher assay background seen with this mAb. Additionally, the higher assay background was found to negatively affect assay sensitivity. Thus, enhancement of the assay sensitivity by subclass switching is likely due to the decrease in assay background, simply by avoiding the self-association of IgG3.

Postoperative Pain Management following Thoracic Outlet Decompression.

BACKGROUND: Thoracic outlet decompression (TOD) is associated with significant postoperative pain often leading to hospital length of stay out of proportion to the risk profile of the operation. We seek to describe the improvement in hospital length of stay and patient pain control with an improved multiagent pain management regimen. METHODS: We retrospectively reviewed the hospital length of stay, medication regimen/usage, operative details, and operative indications for all patients undergoing TOD from January 2012 through June 2015. During early experience, single-agent narcotic therapy was the mainstay of postoperatively pain control. Since 2014, we have adopted a regimen consisting of narcotic patient controlled analgesia, oral narcotics, and scheduled ibuprofen and valium, which is transitioned to oral narcotics/valium upon discharge. Operative approach (supraclavicular, infraclavicular, transaxial, or paraclavicular) was determined by patient anatomy and indication for procedure (neurogenic/arterial thoracic outlet syndrome or arteriovenous access dysfunction). RESULTS: Seventy-four patients were treated with TOD over the study period: 36 (49.3%) for neurogenic thoracic outlet syndrome, 23 (31.5%) for venous thoracic outlet syndrome, and 15 (19.2%) for arteriovenous access dysfunction. Prior to 2014, the mean length of stay was 4 days with a median pain score of 6. Since 2014, the mean length of stay was 2.6 (P = 0.04) with a median pain score of 4 (P = 0.005). There was no statistically significant difference in the indication for operation or operative approach between the two periods. CONCLUSIONS: Since adoption of a multiagent pain management regimen to include scheduled NSAIDs and benzodiazepines, we have reduced the mean pain score experienced by our patients as well as the hospital length of stay.

Regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of AP2.

The critical initiation phase of clathrin-mediated endocytosis (CME) determines where and when endocytosis occurs. Heterotetrameric adaptor protein 2 (AP2) complexes, which initiate clathrin-coated pit (CCP) assembly, are activated by conformational changes in response to phosphatidylinositol-4,5-bisphosphate (PIP2) and cargo binding at multiple sites. However, the functional hierarchy of interactions and how these conformational changes relate to distinct steps in CCP formation in living cells remains unknown. We used quantitative live-cell analyses to measure discrete early stages of CME and show how sequential, allosterically regulated conformational changes activate AP2 to drive both nucleation and subsequent stabilization of nascent CCPs. Our data establish that cargoes containing Yxxφ motif, but not dileucine motif, play a critical role in the earliest stages of AP2 activation and CCP nucleation. Interestingly, these cargo and PIP2 interactions are not conserved in yeast. Thus, we speculate that AP2 has evolved as a key regulatory node to coordinate CCP formation and cargo sorting and ensure high spatial and temporal regulation of CME.

Ikarugamycin: A Natural Product Inhibitor of Clathrin-Mediated Endocytosis.

Ikarugamycin (IKA) is a previously discovered antibiotic, which has been shown to inhibit the uptake of oxidized low-density lipoproteins in macrophages. Furthermore, several groups have previously used IKA to inhibit clathrin-mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA has yet to be performed. Consequently, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We show that IKA has an IC50 of 2.7 µm in H1299 cells and acutely inhibits CME, but not other endocytic pathways, in a panel of cell lines. Although long-term incubation with IKA has cytotoxic effects, the short-term inhibitory effects on CME are reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking.

Potential confounders of bisphenol-a analysis in dental materials.

OBJECTIVES: In the published literature, a variety of analytical methods have been used to quantify and report bisphenol A (BPA) release from dental resins. The objective of this study was to compare results obtained for quantification of BPA in dental resin extracts using an LC/UV analytical method and an LC/MS/MS method. METHODS: A cured Bis-GMA-based resin representative of commercial dental products was extracted according to ISO 10993 guidelines for medical devices. d16BPA was included as an internal standard. Sample processing followed expert recommendations for minimizing BPA sample contamination. Extracts were separated using HPLC methods and analyzed for BPA using LC/UV and LC/MS/MS detection methods. RESULTS: The reported BPA concentrations were about 30-fold higher using LC/UV vs. LC/MS/MS. Full scan LC/MS/MS in both positive and negative modes showed that the apparent high BPA values seen with LC/UV were caused by co-elution of a previously unidentified chemical, thought to arise from one of the polymerization initiators. SIGNIFICANCE: These results emphasize the potential difficulties in obtaining accurate analyses of BPA in complex mixtures such as dental resins and their extracts. Both good separation methodology and a detection method with high specificity and sensitivity are important to avoid incorrect identification of extractables, and consequent incorrect quantitative assignments for species of interest. Reliable methods are essential for accurate estimation of patient exposure to BPA and development of meaningful health risk assessments.

Contribution of murine IgG Fc regions to antibody binding to the capsule of Burkholderia pseudomallei.

Immunoglobulin G3 (IgG3) is the predominant IgG subclass elicited in response to polysaccharide antigens in mice. This specific subclass has been shown to crosslink its fragment crystallizable (Fc) regions following binding to multivalent polysaccharides. Crosslinking leads to increased affinity through avidity, which theoretically should lead to more effective protection against bacteria and yeast displaying capsular polysaccharides on their surface. To investigate this further we have analyzed the binding characteristics of 2 IgG monoclonal antibody (mAb) subclass families that bind to the capsular polysaccharide (CPS) of Burkholderia pseudomallei. The first subclass family originated from an IgG3 hybridoma cell line (3C5); the second family was generated from an IgG1 cell line (2A5). When the Fc region of the 3C5 IgG3 is removed by proteolytic cleavage, the resulting F(ab')2 fragments exhibit decreased affinity compared to the full-length mAb. Similarly, when the parent IgG3 mAb is subclass-switched to IgG1, IgG2b, and IgG2a, all of these subclasses exhibit decreased affinity. This decrease in affinity is not seen when the 2A5 IgG1 mAb is switched to an IgG2b or IgG2a, strongly suggesting the drop in affinity is related to the IgG3 Fc region.

Identification and function of conformational dynamics in the multidomain GTPase dynamin.

Vesicle release upon endocytosis requires membrane fission, catalyzed by the large GTPase dynamin. Dynamin contains five domains that together orchestrate its mechanochemical activity. Hydrogen-deuterium exchange coupled with mass spectrometry revealed global nucleotide- and membrane-binding-dependent conformational changes, as well as the existence of an allosteric relay element in the α2(S) helix of the dynamin stalk domain. As predicted from structural studies, FRET analyses detect large movements of the pleckstrin homology domain (PHD) from a 'closed' conformation docked near the stalk to an 'open' conformation able to interact with membranes. We engineered dynamin constructs locked in either the closed or open state by chemical cross-linking or deletion mutagenesis and showed that PHD movements function as a conformational switch to regulate dynamin self-assembly, membrane binding, and fission. This PHD conformational switch is impaired by a centronuclear myopathy-causing disease mutation, S619L, highlighting the physiological significance of its role in regulating dynamin function. Together, these data provide new insight into coordinated conformational changes that regulate dynamin function and couple membrane binding, oligomerization, and GTPase activity during dynamin-catalyzed membrane fission.

Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis.

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.

Unraveling the interaction between the LPS O-antigen of Burkholderia anthina and the 5D8 monoclonal antibody by using a multidisciplinary chemical approach, with synthesis, NMR, and molecular modeling methods.

The interaction between the O-chain from the lipopolysaccharide from Burkholderia anthina and a lipopolysaccharide-specific monoclonal antibody (5D8) has been studied at high resolution by NMR spectroscopy. In particular, the 5D8-bound epitope of the saccharide entity has been unraveled by a combination of saturation transfer difference (STD) and transferred NOESY (tr-NOESY) experiments performed on the 5D8/polysaccharide complex. To dissect the fine details of the molecular recognition events, further experiments with simpler carbohydrate ligands were carried out. Thus, experiments were also performed with ad hoc synthesized trisaccharide and hexasaccharide O-antigen repeating units. By using this multidisciplinary approach (chemical synthesis, NMR spectroscopy and molecular dynamics simulation), determination of the binding epitope and the contribution to the binding of the sugar units composing the O-chain have been determined.

IgG subclass and heavy chain domains contribute to binding and protection by mAbs to the poly γ-D-glutamic acid capsular antigen of Bacillus anthracis.

Bacterial capsules are common targets for antibody-mediated immunity. The capsule of Bacillus anthracis is unusual among capsules because it is composed of a polymer of poly-γ-d-glutamic acid (γdPGA). We previously generated murine IgG3 monoclonal antibodies (mAbs) to γdPGA that were protective in a murine model of pulmonary anthrax. IgG3 antibodies are characteristic of the murine response to polysaccharide antigens. The goal of the present study was to produce subclass switch variants of the γdPGA mAbs (IgG3 → IgG1 → IgG2b → IgG2a) and assess the contribution of subclass to antibody affinity and protection. Subclass switch antibodies had identical variable regions but differed in their heavy chains. The results showed that a switch from the protective IgG3 to IgG1, IgG2b or IgG2a was accompanied by i) a loss of protective activity ii) a change in mAb binding to the capsular matrix, and iii) a loss of affinity. These results identify a role for the heavy chain constant region in mAb binding. Hybrid mAbs were constructed in which the CH1, CH2 or CH3 heavy chain constant domains from a non-protective, low binding IgG2b mAb were swapped into the protective IgG3 mAb. The IgG3 mAb that contained the CH1 domain from IgG2b showed no loss of affinity or protection. In contrast, swapping the CH2 or CH3 domains from IgG2b into IgG3 produced a reduction in affinity and a loss of protection. These studies identify a role for the constant region of IgG heavy chains in affinity and protection against an encapsulated bacterial pathogen.

Intramolecular gas-phase reactions of synthetic nonheme oxoiron(IV) ions: proximity and spin-state reactivity rules.

The intramolecular gas-phase reactivity of four oxoiron(IV) complexes supported by tetradentate N(4) ligands (L) has been studied by means of tandem mass spectrometry measurements in which the gas-phase ions [Fe(IV)(O)(L)(OTf)](+) (OTf = trifluoromethanesulfonate) and [Fe(IV) (O)(L)](2+) were isolated and then allowed to fragment by collision-induced decay (CID). CID fragmentation of cations derived from oxoiron(IV) complexes of 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane (tmc) and N,N'-bis(2-pyridylmethyl)-1,5-diazacyclooctane (L(8)Py(2)) afforded the same predominant products irrespective of whether they were hexacoordinate or pentacoordinate. These products resulted from the loss of water by dehydrogenation of ethylene or propylene linkers on the tetradentate ligand. In contrast, CID fragmentation of ions derived from oxoiron(IV) complexes of linear tetradentate ligands N,N'-bis(2-pyridylmethyl)-1,2-diaminoethane (bpmen) and N,N'-bis(2-pyridylmethyl)-1,3-diaminopropane (bpmpn) showed predominant oxidative N-dealkylation for the hexacoordinate [Fe(IV)(O)(L)(OTf)](+) cations and predominant dehydrogenation of the diaminoethane/propane backbone for the pentacoordinate [Fe(IV)(O)(L)](2+) cations. DFT calculations on [Fe(IV)(O)(bpmen)] ions showed that the experimentally observed preference for oxidative N-dealkylation versus dehydrogenation of the diaminoethane linker for the hexa- and pentacoordinate ions, respectively, is dictated by the proximity of the target C-H bond to the oxoiron(IV) moiety and the reactive spin state. Therefore, there must be a difference in ligand topology between the two ions. More importantly, despite the constraints on the geometries of the TS that prohibit the usual upright σ trajectory and prevent optimal σ(CH)-σ*(z2) overlap, all the reactions still proceed preferentially on the quintet (S = 2) state surface, which increases the number of exchange interactions in the d block of iron and leads thereby to exchange enhanced reactivity (EER). As such, EER is responsible for the dominance of the S = 2 reactions for both hexa- and pentacoordinate complexes.

Polysaccharide specific monoclonal antibodies provide passive protection against intranasal challenge with Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative bacillus that is the causative agent of melioidosis. The bacterium is inherently resistant to many antibiotics and mortality rates remain high in endemic areas. The lipopolysaccharide (LPS) and capsular polysaccharide (CPS) are two surface-associated antigens that contribute to pathogenesis. We previously developed two monoclonal antibodies (mAbs) specific to the CPS and LPS; the CPS mAb was shown to identify antigen in serum and urine from melioidosis patients. The goal of this study was to determine if passive immunization with CPS and LPS mAbs alone and in combination would protect mice from a lethal challenge with B. pseudomallei. Intranasal (i.n.) challenge experiments were performed with B. pseudomallei strains 1026b and K96423. Both mAbs provided significant protection when administered alone. A combination of mAbs was protective when low doses were administered. In addition, combination therapy provided a significant reduction in spleen colony forming units (cfu) compared to results when either the CPS or LPS mAbs were administered alone.

Amyloid-beta(1-42) rapidly forms protofibrils and oligomers by distinct pathways in low concentrations of sodium dodecylsulfate.

Alzheimer's disease (AD) is characterized by large numbers of senile plaques in the brain that consist of fibrillar aggregates of 40- and 42-residue amyloid-beta (Abeta) peptides. However, the degree of dementia in AD correlates better with the concentration of soluble Abeta species assayed biochemically than with histologically determined plaque counts, and several investigators now propose that soluble aggregates of Abeta are the neurotoxic agents that cause memory deficits and neuronal loss. These endogenous aggregates are minor components in brain extracts from AD patients and transgenic mice that express human Abeta, but several species have been detected by gel electrophoresis in sodium dodecylsulfate (SDS) and isolated by size exclusion chromatography (SEC). Endogenous Abeta aggregation is stimulated at cellular interfaces rich in lipid rafts, and anionic micelles that promote Abeta aggregation in vitro may be good models of these interfaces. We previously found that micelles formed in dilute SDS (2 mM) promote Abeta(1-40) fiber formation by supporting peptide interaction on the surface of a single micelle complex. In contrast, here we report that monomeric Abeta(1-42) undergoes an immediate conversion to a predominant beta-structured conformation in 2 mM SDS which does not proceed to amyloid fibrils. The conformational change is instead rapidly followed by the near quantitative conversion of the 4 kDa monomer SDS gel band to 8-14 kDa bands consistent with dimers through tetramers. Removal of SDS by dialysis gave a shift in the predominant SDS gel bands to 30-60 kDa. While these oligomers resemble the endogenous aggregates, they are less stable. In particular, they do not elute as discrete species on SEC, and they are completed disaggregated by boiling in 1% SDS. It appears that endogenous oligomeric Abeta aggregates are stabilized by undefined processes that have not yet been incorporated into in vitro Abeta aggregation procedures.