Particulate allergens potentiate allergic asthma in mice through sustained IgE-mediated mast cell activation.

Allergic asthma is characterized by airway hyperresponsiveness, inflammation, and a cellular infiltrate dominated by eosinophils. Numerous epidemiological studies have related the exacerbation of allergic asthma with an increase in ambient inhalable particulate matter from air pollutants. This is because inhalable particles efficiently deliver airborne allergens deep into the airways, where they can aggravate allergic asthma symptoms. However, the cellular mechanisms by which inhalable particulate allergens (pAgs) potentiate asthmatic symptoms remain unknown, in part because most in vivo and in vitro studies exploring the pathogenesis of allergic asthma use soluble allergens (sAgs). Using a mouse model of allergic asthma, we found that, compared with their sAg counterparts, pAgs triggered markedly heightened airway hyperresponsiveness and pulmonary eosinophilia in allergen-sensitized mice. Mast cells (MCs) were implicated in this divergent response, as the differences in airway inflammatory responses provoked by the physical nature of the allergens were attenuated in MC-deficient mice. The pAgs were found to mediate MC-dependent responses by enhancing retention of pAg/IgE/FcεRI complexes within lipid raft–enriched, CD63(+) endocytic compartments, which prolonged IgE/FcεRI-initiated signaling and resulted in heightened cytokine responses. These results reveal how the physical attributes of allergens can co-opt MC endocytic circuitry and signaling responses to aggravate pathological responses of allergic asthma in mice.

Walking in old age and development of metabolic syndrome: the health, aging, and body composition study.

BACKGROUND: The specific health benefits of meeting physical activity guidelines are unclear in older adults. We examined the association between meeting, not meeting, or change in status of meeting physical activity guidelines through walking and the 5-year incidence of metabolic syndrome in older adults. METHODS: A total of 1,863 Health, Aging, and Body Composition (Health ABC) Study participants aged 70-79 were followed for 5 years (1997-1998 to 2002-2003). Four walking groups were created based on self-report during years 1 and 6: Sustained low (Year 1, <150 min/week, and year 6, <150 min/week), decreased (year 1, >150 min/week, and year 6, <150 min/week), increased (year 1, <150 min/week, and year 6, >150 min/week), and sustained high (year 1, >150 min/week, and year 6, >150 min/week). Based on the Adult Treatment Panel III (ATP III) panel guidelines, the metabolic syndrome criterion was having three of five factors: Large waist circumference, elevated blood pressure, triglycerides, blood glucose, and low high-density lipoprotein (HDL) levels. RESULTS: Compared to the sustained low group, the sustained high group had a 39% reduction in odds of incident metabolic syndrome [adjusted odds ratio (OR) = 0.61; 95% confidence interval (CI), 0.40-0.93], and a significantly lower likelihood of developing the number of metabolic syndrome risk factors that the sustained low group developed over 5 years (beta = -0.16, P = 0.04). CONCLUSIONS: Meeting or exceeding the physical activity guidelines via walking significantly reduced the odds of incident metabolic syndrome and onset of new metabolic syndrome components in older adults. This protective association was found only in individuals who sustained high levels of walking for physical activity.

Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.

Described here is a mass spectrometry-based screening assay for the detection of protein-ligand binding interactions in multicomponent protein mixtures. The assay utilizes an oxidation labeling protocol that involves using hydrogen peroxide to selectively oxidize methionine residues in proteins in order to probe the solvent accessibility of these residues as a function of temperature. The extent to which methionine residues in a protein are oxidized after specified reaction times at a range of temperatures is determined in a MALDI analysis of the intact proteins and/or an LC-MS analysis of tryptic peptide fragments generated after the oxidation reaction is quenched. Ultimately, the mass spectral data is used to construct thermal denaturation curves for the detected proteins. In this proof-of-principle work, the protocol is applied to a four-protein model mixture comprised of ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). The new protocol's ability to detect protein-ligand binding interactions by comparing thermal denaturation data obtained in the absence and in the presence of ligand is demonstrated using cyclosporin A (CsA) as a test ligand. The known binding interaction between CsA and CypA was detected using both the MALDI- and LC-MS-based readouts described here.

Hand-held spectroscopic device for in vivo and intraoperative tumor detection: contrast enhancement, detection sensitivity, and tissue penetration.

Surgery is one of the most effective and widely used procedures in treating human cancers, but a major problem is that the surgeon often fails to remove the entire tumor, leaving behind tumor-positive margins, metastatic lymph nodes, and/or satellite tumor nodules. Here we report the use of a hand-held spectroscopic pen device (termed SpectroPen) and near-infrared contrast agents for intraoperative detection of malignant tumors, based on wavelength-resolved measurements of fluorescence and surface-enhanced Raman scattering (SERS) signals. The SpectroPen utilizes a near-infrared diode laser (emitting at 785 nm) coupled to a compact head unit for light excitation and collection. This pen-shaped device effectively removes silica Raman peaks from the fiber optics and attenuates the reflected excitation light, allowing sensitive analysis of both fluorescence and Raman signals. Its overall performance has been evaluated by using a fluorescent contrast agent (indocyanine green, or ICG) as well as a surface-enhanced Raman scattering (SERS) contrast agent (pegylated colloidal gold). Under in vitro conditions, the detection limits are approximately 2-5 × 10(-11) M for the indocyanine dye and 0.5-1 × 10(-13) M for the SERS contrast agent. Ex vivo tissue penetration data show attenuated but resolvable fluorescence and Raman signals when the contrast agents are buried 5-10 mm deep in fresh animal tissues. In vivo studies using mice bearing bioluminescent 4T1 breast tumors further demonstrate that the tumor borders can be precisely detected preoperatively and intraoperatively, and that the contrast signals are strongly correlated with tumor bioluminescence. After surgery, the SpectroPen device permits further evaluation of both positive and negative tumor margins around the surgical cavity, raising new possibilities for real-time tumor detection and image-guided surgery.

Thermodynamic analysis of a molecular chaperone binding to unfolded protein substrates.

Molecular chaperones are a highly diverse group of proteins that recognize and bind unfolded proteins to facilitate protein folding and prevent nonspecific protein aggregation. The mechanisms by which chaperones bind their protein substrates have been studied for decades. However, there are few reports about the affinity of molecular chaperones for their unfolded protein substrates. Thus, little is known about the relative binding affinities of different chaperones and about the relative binding affinities of chaperones for different unfolded protein substrates. Here we describe the application of SUPREX (stability of unpurified proteins from rates of H-D exchange), an H-D exchange and MALDI-based technique, in studying the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates, including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of our studies suggest that the cooperativity of the Hsp33 folding-unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX-derived K(d) values for Hsp33 complexes with four different substrates were all found to be within the range of 3-300 nM.

Measurements of nonlinear refractive index in scattering media.

We have recently developed a spectral re-shaping technique to simultaneously measure nonlinear refractive index and nonlinear absorption. In this technique, the information about the nonlinearities is encoded in the frequency domain, rather than in the spatial domain as in the conventional Z-scan method. Here we show that frequency encoding is much more robust with respect to scattering. We compare spectral re-shaping and Z-scan measurements in a highly scattering environment and show that reliable spectral re-shaping measurements can be performed even in a regime that precludes standard Z-scans.

Comparison of a reduced carbohydrate and reduced fat diet for LDL, HDL, and VLDL subclasses during 9-months of weight maintenance subsequent to weight loss.

OBJECTIVES: This study compared LDL, HDL, and VLDL subclasses in overweight or obese adults consuming either a reduced carbohydrate (RC) or reduced fat (RF) weight maintenance diet for 9 months following significant weight loss. METHODS: Thirty-five (21 RC; 14 RF) overweight or obese middle-aged adults completed a 1-year weight management clinic. Participants met weekly for the first six months and bi-weekly thereafter. Meetings included instruction for diet, physical activity, and behavior change related to weight management. Additionally, participants followed a liquid very low-energy diet of approximately 2092 kJ per day for the first three months of the study. Subsequently, participants followed a dietary plan for nine months that targeted a reduced percentage of carbohydrate (approximately 20%) or fat (approximately 30%) intake and an energy intake level calculated to maintain weight loss. Lipid subclasses using NMR spectroscopy were analyzed prior to weight loss and at multiple intervals during weight maintenance. RESULTS: Body weight change was not significantly different within or between groups during weight maintenance (p>0.05). The RC group showed significant increases in mean LDL size, large LDL, total HDL, large and small HDL, mean VLDL size, and large VLDL during weight maintenance while the RF group showed increases in total HDL, large and small HDL, total VLDL, and large, medium, and small VLDL (p<0.05). Group*time interactions were significant for large and medium VLDL (p>0.05). CONCLUSION: Some individual lipid subclasses improved in both dietary groups. Large and medium VLDL subclasses increased to a greater extent across weight maintenance in the RF group.

Boosting high-intensity focused ultrasound-induced anti-tumor immunity using a sparse-scan strategy that can more effectively promote dendritic cell maturation.

BACKGROUND: The conventional treatment protocol in high-intensity focused ultrasound (HIFU) therapy utilizes a dense-scan strategy to produce closely packed thermal lesions aiming at eradicating as much tumor mass as possible. However, this strategy is not most effective in terms of inducing a systemic anti-tumor immunity so that it cannot provide efficient micro-metastatic control and long-term tumor resistance. We have previously provided evidence that HIFU may enhance systemic anti-tumor immunity by in situ activation of dendritic cells (DCs) inside HIFU-treated tumor tissue. The present study was conducted to test the feasibility of a sparse-scan strategy to boost HIFU-induced anti-tumor immune response by more effectively promoting DC maturation. METHODS: An experimental HIFU system was set up to perform tumor ablation experiments in subcutaneous implanted MC-38 and B16 tumor with dense- or sparse-scan strategy to produce closely-packed or separated thermal lesions. DCs infiltration into HIFU-treated tumor tissues was detected by immunohistochemistry and flow cytometry. DCs maturation was evaluated by IL-12/IL-10 production and CD80/CD86 expression after co-culture with tumor cells treated with different HIFU. HIFU-induced anti-tumor immune response was evaluated by detecting growth-retarding effects on distant re-challenged tumor and tumor-specific IFN-gamma-secreting cells in HIFU-treated mice. RESULTS: HIFU exposure raised temperature up to 80 degrees centigrade at beam focus within 4 s in experimental tumors and led to formation of a well-defined thermal lesion. The infiltrated DCs were recruited to the periphery of lesion, where the peak temperature was only 55 degrees centigrade during HIFU exposure. Tumor cells heated to 55 degrees centigrade in 4-s HIFU exposure were more effective to stimulate co-cultured DCs to mature. Sparse-scan HIFU, which can reserve 55 degrees-heated tumor cells surrounding the separated lesions, elicited an enhanced anti-tumor immune response than dense-scan HIFU, while their suppressive effects on the treated primary tumor were maintained at the same level. Flow cytometry analysis showed that sparse-scan HIFU was more effective than dense-scan HIFU in enhancing DC infiltration into tumor tissues and promoting their maturation in situ. CONCLUSION: Optimizing scan strategy is a feasible way to boost HIFU-induced anti-tumor immunity by more effectively promoting DC maturation.

Study protocol: home-based telehealth stroke care: a randomized trial for veterans.

BACKGROUND: Stroke is one of the most disabling and costly impairments of adulthood in the United States. Stroke patients clearly benefit from intensive inpatient care, but due to the high cost, there is considerable interest in implementing interventions to reduce hospital lengths of stay. Early discharge rehabilitation programs require coordinated, well-organized home-based rehabilitation, yet lack of sufficient information about the home setting impedes successful rehabilitation. This trial examines a multifaceted telerehabilitation (TR) intervention that uses telehealth technology to simultaneously evaluate the home environment, assess the patient's mobility skills, initiate rehabilitative treatment, prescribe exercises tailored for stroke patients and provide periodic goal oriented reassessment, feedback and encouragement. METHODS: We describe an ongoing Phase II, 2-arm, 3-site randomized controlled trial (RCT) that determines primarily the effect of TR on physical function and secondarily the effect on disability, falls-related self-efficacy, and patient satisfaction. Fifty participants with a diagnosis of ischemic or hemorrhagic stroke will be randomly assigned to one of two groups: (a) TR; or (b) Usual Care. The TR intervention uses a combination of three videotaped visits and five telephone calls, an in-home messaging device, and additional telephonic contact as needed over a 3-month study period, to provide a progressive rehabilitative intervention with a treatment goal of safe functional mobility of the individual within an accessible home environment. Dependent variables will be measured at baseline, 3-, and 6-months and analyzed with a linear mixed-effects model across all time points. DISCUSSION: For patients recovering from stroke, the use of TR to provide home assessments and follow-up training in prescribed equipment has the potential to effectively supplement existing home health services, assist transition to home and increase efficiency. This may be particularly relevant when patients live in remote locations, as is the case for many veterans. TRIAL REGISTRATION: Clinical Trials.gov Identifier: NCT00384748.

Risk of ovarian cancer and inherited variants in relapse-associated genes.

BACKGROUND: We previously identified a panel of genes associated with outcome of ovarian cancer. The purpose of the current study was to assess whether variants in these genes correlated with ovarian cancer risk. METHODS AND FINDINGS: Women with and without invasive ovarian cancer (749 cases, 1,041 controls) were genotyped at 136 single nucleotide polymorphisms (SNPs) within 13 candidate genes. Risk was estimated for each SNP and for overall variation within each gene. At the gene-level, variation within MSL1 (male-specific lethal-1 homolog) was associated with risk of serous cancer (p = 0.03); haplotypes within PRPF31 (PRP31 pre-mRNA processing factor 31 homolog) were associated with risk of invasive disease (p = 0.03). MSL1 rs7211770 was associated with decreased risk of serous disease (OR 0.81, 95% CI 0.66-0.98; p = 0.03). SNPs in MFSD7, BTN3A3, ZNF200, PTPRS, and CCND1A were inversely associated with risk (p<0.05), and there was increased risk at HEXIM1 rs1053578 (p = 0.04, OR 1.40, 95% CI 1.02-1.91). CONCLUSIONS: Tumor studies can reveal novel genes worthy of follow-up for cancer susceptibility. Here, we found that inherited markers in the gene encoding MSL1, part of a complex that modifies the histone H4, may decrease risk of invasive serous ovarian cancer.

R5 clade C SHIV strains with tier 1 or 2 neutralization sensitivity: tools to dissect env evolution and to develop AIDS vaccines in primate models.

BACKGROUND: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an "early," recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a "late" form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to "late" SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4+ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. CONCLUSIONS/SIGNIFICANCE: SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.

Immunization with cocktail of HIV-derived peptides in montanide ISA-51 is immunogenic, but causes sterile abscesses and unacceptable reactogenicity.

BACKGROUND: A peptide vaccine was produced containing B and T cell epitopes from the V3 and C4 Envelope domains of 4 subtype B HIV-1 isolates (MN, RF, CanO, & Ev91). The peptide mixture was formulated as an emulsion in incomplete Freund's adjuvant (IFA). METHODS: Low-risk, healthy adult subjects were enrolled in a randomized, placebo-controlled dose-escalation study, and selected using criteria specifying that 50% in each study group would be HLA-B7+. Immunizations were scheduled at 0, 1, and 6 months using a total peptide dose of 1 or 4 mg. Adaptive immune responses in16 vaccine recipients and two placebo recipients after the 2nd immunization were evaluated using neutralization assays of sera, as well as ELISpot and ICS assays of cryopreserved PBMCs to assess CD4 and CD8 T-cell responses. In addition, (51)Cr release assays were performed on fresh PBMCs following 14-day stimulation with individual vaccine peptide antigens. RESULTS: 24 subjects were enrolled; 18 completed 2 injections. The study was prematurely terminated because 4 vaccinees developed prolonged pain and sterile abscess formation at the injection site-2 after dose 1, and 2 after dose 2. Two other subjects experienced severe systemic reactions consisting of headache, chills, nausea, and myalgia. Both reactions occurred after the second 4 mg dose. The immunogenicity assessments showed that 6/8 vaccinees at each dose level had detectable MN-specific neutralizing (NT) activity, and 2/7 HLA-B7+ vaccinees had classical CD8 CTL activity detected. However, using both ELISpot and ICS, 8/16 vaccinees (5/7 HLA-B7+) and 0/2 controls had detectable vaccine-specific CD8 T-cell responses. Subjects with moderate or severe systemic or local reactions tended to have more frequent T cell responses and higher antibody responses than those with mild or no reactions. CONCLUSIONS: The severity of local responses related to the formulation of these four peptides in IFA is clinically unacceptable for continued development. Both HIV-specific antibody and T cell responses were induced and the magnitude of response correlated with the severity of local and systemic reactions. If potent adjuvants are necessary for subunit vaccines to induce broad and durable immune responses, careful, incremental clinical evaluation is warranted to minimize the risk of adverse events. TRIAL REGISTRATION: ClinicalTrials.gov NCT00000886.

Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.