Ehrlich ascites cells activate the alternative pathway of the human complement system

Incubation of Ehrlich ascites cells with normal or C1q or C2 deficient human sera results in killing of the cells. Killing occurred also in the absence of free Ca++, which supported by the fact that factor B and C3 were cleaved, leads to the conclusion that the alternative pathway of the complement system is activated on the surface of the Ehrlich ascites cells.     

Estimating the relative frequency of leukodystrophies and recommendations for carrier screening in the era of next‐generation sequencing

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A‐related leukodystrophy, Peroxisomal biogenesis disorders, POLR3‐related Leukodystrophy, Vanishing White Matter, and Pelizaeus‐Merzbacher Disease. Despite the relative frequency of these conditions, carrier‐screening laboratories regularly test only 20 of the 55 leukodystrophy‐related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.     

High-Throughput Generation of P. falciparum Functional Molecules by Recombinational Cloning

Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression. We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes. Master clones and their ORFs were transferred en masse to multiple expression vectors. Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure. Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency. The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors. These transfers yielded a 100% success rate without any observed recombination based on single colony screening. The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite. From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages. We have overcome the potential limitation of producing large P. falciparum clone sets in multiple expression vectors. This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P. falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.     

Detection of fish antigens aerosolized during fish processing using newly developed immunoassays

BACKGROUND: Aerosolization of fish proteins during seafood processing has been identified as a potential route for allergic sensitization and occupational asthma among workers involved in high-risk activities. The aim of this study was to develop immunological assays for the quantification of aerosolized fish antigens in a fish-processing factory. METHODS: Polyclonal antibodies to the main fish species processed in the factory (anchovy and pilchard) were generated in rabbits and compared by ELISA inhibition assay and immunoblotting. These antisera were utilized to develop ELISA assays for the detection of fish antigens. The ELISA inhibition assays were evaluated by analyzing environmental air samples collected from three areas in a fish-processing factory: pilchard canning, fish meal production and lobster processing. RESULTS: By immunoblotting, the rabbit polyclonal antibodies demonstrated IgG antibody binding patterns comparable with IgE antibodies of fish-sensitized patients, particularly in regard to the major fish allergens parvalbumins. The sensitivity of the fish-specific ELISA assays developed was 0.5 microg/ml. The ELISA inhibition assays were able to differentiate between the two different fish species of interest but did not recognize a crustacean species. Notable differences in exposure levels to canned pilchard and anchovy antigens were demonstrated in the three different working areas of the factory, with assays having a detection limit as low as 105 ng/m(3). CONCLUSION: These ELISA-based assays are sensitive and specific to quantify differential exposure levels to fish antigens produced during fish processing, making it possible to investigate exposure-disease response relationships among workers in this industry.     

High-throughput genetic screening using matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a powerful and widespread analytical tool in all fields of life science. Compared with other techniques, its high accuracy and sensitivity makes it a superior method, especially for the analysis of nucleic acids. Recent problems in the analysis of nucleic acids by MALDI-TOF MS can be solved using an automated MALDI-compatible sample-preparation system. Together with the reliable minisequencing assay, high-throughput genotyping of single nucleotide polymorphisms by MALDI-TOF MS is able to become a routine method in research, clinical genetics and diagnostics.     

Characterization of a population of small macrophages in induced sputum of patients with chronic obstructive pulmonary disease and healthy volunteers

The inflammatory process in chronic obstructive pulmonary disease (COPD) is active mainly in the airways, but little is known about the properties of the inflammatory cells in this compartment. We have studied leucocytes in induced sputum of COPD patients compared to controls in order to uncover what types of macrophages might be involved in the disease. Sputum induction was performed by inhalation of nebulized sodium chloride solution. Leucocytes were isolated and stained with specific monoclonal antibodies for analysis in flow cytometry. Flow cytometry analysis revealed that a major portion of CD14+ macrophages in COPD has lower forward scatter, i.e. they are small macrophages. While in control donors these small macrophages accounted for 6.9% of all macrophages, the percentage of these cells in COPD was 45.7%. CD14 and HLA-DR expression was high on these small sputum macrophages while the large sputum macrophages expressed only low levels of these surface molecules, both in control donors and COPD patients. Small sputum macrophages of both control donors and COPD patients showed higher levels of constitutive tumour necrosis factor (TNF) compared to the large macrophages. TNF was inducible by lipopolysaccharide (LPS) preferentially in the small sputum macrophages in the control donors but there was no further induction in COPD patients. These data show that the small sputum macrophages are a major macrophage population in COPD and that these cells exhibit features of highly active inflammatory cells and may therefore be instrumental in airway inflammation in COPD.     

B cell early response gene expression coupled to B cell receptor,CD40 and interleukin-4 receptor co-stimulation: evidence for a role of the egr-2/krox20 transcription factor in B cell proliferation

B lymphocytes are activated following antigen stimulation of the B cell receptor but require co-stimulation with accessory molecules provided by interleukin (IL)-4/CD40 ligand for cell cycle progression and proliferation. By analyzing a panel of 11 early response genes induced by cross-linking of surface immunoglobulin, we show that CD40 signaling alone induces only 2 genes, c-myc together with an anonymous gene, 3L3, and that these are distinct from the set of genes induced in response to IL-4. Co-stimulation with the proliferative combination of anti-μ, IL-4 + CD40 signaling led to a fourfold enhancement of egr-2/krox20 expression over that seen with anti-μ alone. Egr-2 expression/activity was selectively inhibited by the immunosuppressive drug cyclosporin A, and antisense oligonucleotide blockade of Egr-2 activity elicited a dose-dependent inhibition of B cell proliferation. Taken together, these observations show that the early gene regulatory programs coupled to different surface receptors on B cells are largely distinct from each other, but that certain genes, exemplified by egr-2, may represent a point of convergence in the integration of different signaling pathways into the B cell proliferative response.     

A novel inhibitor of the alternative complement pathway prevents antiphospholipid antibody-induced pregnancy loss in mice

Studies in gene-targeted mice have demonstrated that factor B of the alternative complement pathway plays an important role in several disease models, but an exogenous inhibitor of factor B has not previously been available. We have developed an inhibitory monoclonal antibody directed against a critical epitope on mouse factor B and have tested it in a model of antiphospholipid (aPL) antibody (Ab)-induced fetal loss. Gene-targeted factor B-deficient mice (fB-/-) were injected with a fusion protein comprised of the second and third short consensus repeat (SCR) domains of mouse factor B linked to a mouse IgG1 Fc domain. Hybridomas were made from splenocytes of the immunized mouse. One mAb, designated 1379, produced an IgG1 antibody that inhibited alternative pathway activation in vitro and in vivo by preventing formation of the C3bBb complex. Strikingly, this mAb inhibited alternative pathway activation in serum from mice, rats, humans, monkeys, pigs and horses. Fab fragments made from this mAb also inhibited alternative pathway activation. Epitope mapping demonstrated that this antibody binds to factor B within the third SCR domain. When mAb 1379 was administered to mice that also received human IgG containing antiphospholipid antibodies, it provided significant protection from antiphospholipid antibody-induced complement activation and fetal loss. Thus, this mAb to factor B has broad species reactivity and effectively inhibits alternative pathway activation. The mAb protects mice in an in vivo model of antiphospholipid antibody syndrome, demonstrating the therapeutic potential for the inhibition of factor B in this disease.