Mechanisms of Allergen-Antibody Interaction of Cockroach Allergen Bla g 2 with Monoclonal Antibodies That Inhibit IgE Antibody Binding

BACKGROUND: Cockroach allergy is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two specific monoclonal antibodies (mAb) that interfere with IgE antibody binding were identified by X-ray crystallography on opposite sites of the quasi-symmetrical cockroach allergen Bla g 2. METHODOLOGY/PRINCIPAL FINDINGS: Mutational analysis of selected residues in both epitopes was performed based on the X-ray crystal structures of the allergen with mAb Fab/Fab' fragments, to investigate the structural basis of allergen-antibody interactions. The epitopes of Bla g 2 for the mAb 7C11 or 4C3 were mutated, and the mutants were analyzed by SDS-PAGE, circular dichroism, and/or mass spectrometry. Mutants were tested for mAb and IgE antibody binding by ELISA and fluorescent multiplex array. Single or multiple mutations of five residues from both epitopes resulted in almost complete loss of mAb binding, without affecting the overall folding of the allergen. Preventing glycosylation by mutation N268Q reduced IgE binding, indicating a role of carbohydrates in the interaction. Cation-π interactions, as well as electrostatic and hydrophobic interactions, were important for mAb and IgE antibody binding. Quantitative differences in the effects of mutations on IgE antibody binding were observed, suggesting heterogeneity in epitope recognition among cockroach allergic patients. CONCLUSIONS/SIGNIFICANCE: Analysis by site-directed mutagenesis of epitopes identified by X-ray crystallography revealed an overlap between monoclonal and IgE antibody binding sites and provided insight into the B cell repertoire to Bla g 2 and the mechanisms of allergen-antibody recognition, including involvement of carbohydrates

A sensitive flow cytometric methodology for studying the binding of L. chagasi to canine peritoneal macrophages

BACKGROUND: The Leishmania promastigote-macrophage interaction occurs through the association of multiple receptors on the biological membrane surfaces. The success of the parasite infection is dramatically dependent on this early interaction in the vertebrate host, which permits or not the development of the disease. In this study we propose a novel methodology using flow cytometry to study this interaction, and compare it with a previously described "in vitro" binding assay. METHODS: To study parasite-macrophage interaction, peritoneal macrophages were obtained from 4 dogs and adjusted to 3 × 10(6 )cells/mL. Leishmania (Leishmania) chagasi parasites (stationary-phase) were adjusted to 5 × 10(7 )cells/mL. The interaction between CFSE-stained Leishmania chagasi and canine peritoneal macrophages was performed in polypropylene tubes to avoid macrophage adhesion. We carried out assays in the presence or absence of normal serum or in the presence of a final concentration of 5% of C5 deficient (serum from AKR/J mice) mouse serum. Then, the number of infected macrophages was counted in an optical microscope, as well as by flow citometry. Macrophages obtained were stained with anti-CR3 (CD11b/CD18) antibodies and analyzed by flow citometry. RESULTS: Our results have shown that the interaction between Leishmania and macrophages can be measured by flow cytometry using the fluorescent dye CFSE to identify the Leishmania, and measuring simultaneously the expression of an important integrin involved in this interaction: the CD11b/CD18 (CR3 or Mac-1) β2 integrin. CONCLUSION: Flow cytometry offers rapid, reliable and sensitive measurements of single cell interactions with Leishmania in unstained or phenotypically defined cell populations following staining with one or more fluorochromes

Acupuncture and chiropractic care for chronic pain in an integrated health plan: a mixed methods study

<p>Abstract</p> <p>Background</p> <p>Substantial recent research examines the efficacy of many types of complementary and alternative (CAM) therapies. However, outcomes associated with the "real-world" use of CAM has been largely overlooked, despite calls for CAM therapies to be studied in the manner in which they are practiced. Americans seek CAM treatments far more often for chronic musculoskeletal pain (CMP) than for any other condition. Among CAM treatments for CMP, acupuncture and chiropractic (A/C) care are among those with the highest acceptance by physician groups and the best evidence to support their use. Further, recent alarming increases in delivery of opioid treatment and surgical interventions for chronic pain--despite their high costs, potential adverse effects, and modest efficacy--suggests the need to evaluate real world outcomes associated with promising non-pharmacological/non-surgical CAM treatments for CMP, which are often well accepted by patients and increasingly used in the community.</p> <p>Methods/Design</p> <p>This multi-phase, mixed methods study will: (1) conduct a retrospective study using information from electronic medical records (EMRs) of a large HMO to identify unique clusters of patients with CMP (e.g., those with differing demographics, histories of pain condition, use of allopathic and CAM health services, and comorbidity profiles) that may be associated with different propensities for A/C utilization and/or differential outcomes associated with such care; (2) use qualitative interviews to explore allopathic providers' recommendations for A/C and patients' decisions to pursue and retain CAM care; and (3) prospectively evaluate health services/costs and broader clinical and functional outcomes associated with the receipt of A/C relative to carefully matched comparison participants receiving traditional CMP services. Sensitivity analyses will compare methods relying solely on EMR-derived data versus analyses supplementing EMR data with conventionally collected patient and clinician data.</p> <p>Discussion</p> <p>Successful completion of these aggregate aims will provide an evaluation of outcomes associated with the real-world use of A/C services. The trio of retrospective, qualitative, and prospective study will also provide a clearer understanding of the decision-making processes behind the use of A/C for CMP and a transportable methodology that can be applied to other health care settings, CAM treatments, and clinical populations.</p> <p>Trial registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01345409">NCT01345409</a></p

DDX5 plays essential transcriptional and post-transcriptional roles in the maintenance and function of spermatogonia

Mammalian spermatogenesis is sustained by mitotic germ cells with self-renewal potential known as undifferentiated spermatogonia. Maintenance of undifferentiated spermatogonia and spermatogenesis is dependent on tightly co-ordinated transcriptional and post-transcriptional mechanisms. The RNA helicase DDX5 is expressed by spermatogonia but roles in spermatogenesis are unexplored. Using an inducible knockout mouse model, we characterise an essential role for DDX5 in spermatogonial maintenance and show that Ddx5 is indispensable for male fertility. We demonstrate that DDX5 regulates appropriate splicing of key genes necessary for spermatogenesis. Moreover, DDX5 regulates expression of cell cycle genes in undifferentiated spermatogonia post-transcriptionally and is required for cell proliferation and survival. DDX5 can also act as a transcriptional co-activator and we demonstrate that DDX5 interacts with PLZF, a transcription factor required for germline maintenance, to co-regulate select target genes. Combined, our data reveal a critical multifunctional role for DDX5 in regulating gene expression programmes and activity of undifferentiated spermatogonia

Reversible Disruption of Pre-Pulse Inhibition in Hypomorphic-Inducible and Reversible CB1-/- Mice

Although several genes are implicated in the pathogenesis of schizophrenia, in animal models for such a severe mental illness only some aspects of the pathology can be represented (endophenotypes). Genetically modified mice are currently being used to obtain or characterize such endophenotypes. Since its cloning and characterization CB1 receptor has increasingly become of significant physiological, pharmacological and clinical interest. Recently, its involvement in schizophrenia has been reported. Among the different approaches employed, gene targeting permits to study the multiple roles of the endocannabinoid system using knockout (-/-) mice represent a powerful model but with some limitations due to compensation. To overcome such a limitation, we have generated an inducible and reversible tet-off dependent tissue-specific CB1-/- mice where the CB1R is re-expressed exclusively in the forebrain at a hypomorphic level due to a mutation (IRh-CB1-/-) only in absence of doxycycline (Dox). In such mice, under Dox+ or vehicle, as well as in wild-type (WT) and CB1-/-, two endophenotypes motor activity (increased in animal models of schizophrenia) and pre-pulse inhibition (PPI) of startle reflex (disrupted in schizophrenia) were analyzed. Both CB1-/- and IRh-CB1-/- showed increased motor activity when compared to WT animals. The PPI response, unaltered in WT and CB1-/- animals, was on the contrary highly and significantly disrupted only in Dox+ IRh-CB1-/- mice. Such a response was easily reverted after either withdrawal from Dox or haloperidol treatment. This is the first Inducible and Reversible CB1-/- mice model to be described in the literature. It is noteworthy that the PPI disruption is not present either in classical full CB1-/- mice or following acute administration of rimonabant. Such a hypomorphic model may provide a new tool for additional in vivo and in vitro studies of the physiological and pathological roles of cannabinoid system in schizophrenia and in other psychiatric disorders

The ELBA Force Field for Coarse-Grain Modeling of Lipid Membranes

A new coarse-grain model for molecular dynamics simulation of lipid membranes is presented. Following a simple and conventional approach, lipid molecules are modeled by spherical sites, each representing a group of several atoms. In contrast to common coarse-grain methods, two original (interdependent) features are here adopted. First, the main electrostatics are modeled explicitly by charges and dipoles, which interact realistically through a relative dielectric constant of unity (). Second, water molecules are represented individually through a new parametrization of the simple Stockmayer potential for polar fluids; each water molecule is therefore described by a single spherical site embedded with a point dipole. The force field is shown to accurately reproduce the main physical properties of single-species phospholipid bilayers comprising dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE) in the liquid crystal phase, as well as distearoylphosphatidylcholine (DSPC) in the liquid crystal and gel phases. Insights are presented into fundamental properties and phenomena that can be difficult or impossible to study with alternative computational or experimental methods. For example, we investigate the internal pressure distribution, dipole potential, lipid diffusion, and spontaneous self-assembly. Simulations lasting up to 1.5 microseconds were conducted for systems of different sizes (128, 512 and 1058 lipids); this also allowed us to identify size-dependent artifacts that are expected to affect membrane simulations in general. Future extensions and applications are discussed, particularly in relation to the methodology's inherent multiscale capabilities

The macrophage in HIV-1 infection: From activation to deactivation?

Macrophages play a crucial role in innate and adaptative immunity in response to microorganisms and are an important cellular target during HIV-1 infection. Recently, the heterogeneity of the macrophage population has been highlighted. Classically activated or type 1 macrophages (M1) induced in particular by IFN-γ display a pro-inflammatory profile. The alternatively activated or type 2 macrophages (M2) induced by Th-2 cytokines, such as IL-4 and IL-13 express anti-inflammatory and tissue repair properties. Finally IL-10 has been described as the prototypic cytokine involved in the deactivation of macrophages (dM). Since the capacity of macrophages to support productive HIV-1 infection is known to be modulated by cytokines, this review shows how modulation of macrophage activation by cytokines impacts the capacity to support productive HIV-1 infection. Based on the activation status of macrophages we propose a model starting with M1 classically activated macrophages with accelerated formation of viral reservoirs in a context of Th1 and proinflammatory cytokines. Then IL-4/IL-13 alternatively activated M2 macrophages will enter into the game that will stop the expansion of the HIV-1 reservoir. Finally IL-10 deactivation of macrophages will lead to immune failure observed at the very late stages of the HIV-1 disease