Antibody-mediated delivery of IL-10 inhibits the progression of established collagen-induced arthritis

The antibody-mediated targeted delivery of cytokines to sites of disease is a promising avenue for cancer therapy, but it is largely unexplored for the treatment of chronic inflammatory conditions. Using both radioactive and fluorescent techniques, the human monoclonal antibodies L19 and G11 (specific to two markers of angiogenesis that are virtually undetectable in normal adult tissues) were found to selectively localize at arthritic sites in the murine collagen-induced model of rheumatoid arthritis following intravenous (i.v.) administration. The same animal model was used to study the therapeutic action of the L19 antibody fused to the cytokines IL-2, tumour necrosis factor (TNF) and IL-10. Whereas L19–IL-2 and L19–TNF treatment led to increased arthritic scores and paw swellings, the fusion protein L19–IL-10 displayed a therapeutic activity, which was superior to the activity of IL-10 fused to an antibody of irrelevant specificity in the mouse. The anti-inflammatory cytokine IL-10 has been investigated for the treatment of patients with rheumatoid arthritis, but clinical development plans have been discontinued because of a lack of efficacy. Because the antigen recognised by L19 is strongly expressed at sites of arthritis in humans and identical in both mice and humans, it suggests that the fusion protein L19–IL-10 might help overcome some of the clinical limitations of IL-10 and provide a therapeutic benefit to patients with chronic inflammatory disorders, including arthritis.     

Two new species of Edmockfordia García Aldrete (Psocodea, ‘Psocoptera’, Epipsocidae), from Valle del Cauca,Colombia, and description of the female E. chiquibulensis García Aldrete

Two new species of Edmockfordia García Aldrete, from Valle del Cauca, Colombia, and the female of Edmockfordia chiquibulensis García Aldrete, are described and illustrated. A key to the species of Edmockfordia is included; the genus was previously known only from Belize. The genus is re-diagnosed to include female characters. The distribution of the genus is considerably widened, from Belize to northeastern South America.     

Influence of Water Activity and System Mobility on Peroxidase Activity in Maltodextrin Solutions

Maltodextrins influenced the enzymatic activity in aqueous solutions by affecting the water activity (a_w) and mobility as described by viscosity and T’g. In diluted solutions, viscosity being equal, (1) maltodextrin with dextrose equivalents (DE) of 33 was more effective than glucose in limiting horseradish peroxidase (HRP) activity; (2) an increase in the maltodextrin chain length from DE 33 to DE 8.7 did not further limited enzymatic activity; (3) the maltodextrin with the highest chain length (DE 2.5) determined the highest enzyme inhibition. In general, the increase of molecular weight negatively affected the HRP activity by increasing the viscosity and T’g (decreasing molecular mobility) but it positively affected the a_w and, in some cases, this compensated the HRP activity inhibition. In concentrated solutions (apparent viscosity ≈ 40 mPa s) the HRP activity decreased with the increase of the maltodextrin molecular weight, and it showed a dependence on T’g which could be described by a William, Landel and Ferry (WLF)-type equation. On the contrary, in the solution added with the maltodextrin with the highest chain length (DE 2.5) the HRP activity was much higher than that predicted by the WLF-type equation. The maltodextrin with DE 2.5 contains intact starch fragments and in water forms a suspension. In such a discontinuous system, the viscosity in the vicinity of the protein is lower than the bulk viscosity, and thus, the enzyme activity is higher than expected. Moreover, since T’g is a property of the soluble phase, it does not explain the mobility in discontinuous systems.     

A novel synthetic na?ve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin

Human monoclonal antibodies (mAbs) can routinely be isolated from phage display libraries against virtually any protein available in sufficient purity and quantity, but library design can influence epitope coverage on the target antigen. Here we describe the construction of a novel synthetic human antibody phage display library that incorporates hydrophilic or charged residues at position 52 of the CDR2 loop of the variable heavy chain domain, instead of the serine residue found in the corresponding germline gene. The novel library was used to isolate human mAbs to various antigens, including the alternatively-spliced EDA domain of fibronectin, a marker of tumor angiogenesis. In particular, the mAb 2H7 was proven to bind to a novel epitope on EDA, which does not overlap with the one recognized by the clinical-stage F8 antibody. F8 and 2H7 were used for the construction of chelating recombinant antibodies (CRAbs), whose tumor-targeting properties were assessed in vivo in biodistribution studies in mice bearing F9 teratocarcinoma, revealing a preferential accumulation at the tumor site.Key words: human antibody library, phage display, oncofetal fibronectin, vascular tumor targeting, scFv antibody fragments, chelating recombinant antibody (CRAb)     

Copper exposure effects on yeast mitochondrial proteome

Mitochondria play an important role on the entire cellular copper homeostatic mechanisms. Alteration of cellular copper levels may thus influence mitochondrial proteome and its investigation represents an important contribution to the general understanding of copper-related cellular effects. In these study we have performed an organelle targeted proteomic investigation focusing our attention on the effect of non-lethal 1mM copper concentration on Saccharomyces cerevisiae mitochondrial proteome. Functional copper effects on yeast mitochondrial proteome were evaluated by using both 2D electrophoresis (2-DE) and liquid chromatography coupled with tandem mass spectrometry. Proteomic data have been then analyzed by different unsupervised meta-analysis approaches that highlight the impairment of mitochondrial functions and the activation of oxidative stress response. Interestingly, our data have shown that stress response generated by 1mM copper treatment determines the activation of S. cerevisiae survival pathway. To investigate these findings we have treated yeast cells responsiveness to copper with hydrogen peroxide and observed a protective role of this metal. These results are suggestive of a copper role in the protection from oxidative stress possibly due to the activation of mechanisms involved in cellular survival and growth.     

Genitourinary anomalies in Mowat-Wilson syndrome with deletion/mutation in the zinc finger homeo box 1B gene (ZFHX1B). Report of three Italian cases with hypospadias and review

Hypospadias, when the urethra opens on the ventral side of the penis, is a common malformation seen in about 3 per 1,000 male births. It is a complex disorder associated with genetic and environmental factors and can be part of genetic syndromes. Mowat-Wilson syndrome (MWS) is a multiple congenital anomaly syndrome characterized by a distinct facial phenotype, Hirschsprung disease, microcephaly and mental retardation. It is caused by mutations in the zinc finger homeo box 1B gene, ZFHX1B (SIP1). To date, 68 deletion/mutation-positive cases have been reported. Genitourinary anomalies are common in MWS. Here we report that hypospadias is common in males with this syndrome. In 39 patients where this information was available, hypospadias was present in 46% of patients (18/39). In the 3 Italian male cases reported here, hypospadias was always present. MWS should be considered by endocrinologists in patients with hypospadias associated with developmental delays/mental retardation, in particular in the presence of a distinct facial phenotype.     

Pleural malignant mesothelioma, genetic susceptibility and asbestos exposure

Pleural malignant mesothelioma (MM) is a rare but extremely aggressive cancer. The limited impact of standard therapeutic treatments on survival rates makes the identification of factors that increase the individual risk a leading priority. The high proportion of cases explained by exposure to asbestos has guided intervention policies to an effective ban of this compound from our environment. However, MM cannot be solely attributed to this agent, and the role of predisposing factors and their interaction with asbestos exposure is increasingly studied. The role of mEH, GSTM1, GSTT1, NAT2, and CYP1A1 genotypes in modulating susceptibility to MM was examined in a case-control study of 80 subjects with a confirmed diagnosis of MM and 255 controls. Subjects with low mEH activity showed a significantly increased risk of MM (OR, 2.51; 95% CI, 1.11-5.68). The association was stronger in the group with low asbestos exposure (OR, 7.83; 95% CI, 0.98-62.60). A significant increased risk of MM was also found in NAT2 fast acetylators (OR, 1.74; 95% CI, 1.02-2.96). The presence of synergisms between genotypes, i.e., mEH and NAT2 (LRT for heterogeneity p<0.023), mEH and GSTM1 (LRT p<0.061), and NAT2 and GSTM1 (LRT p<0.049), combined with the interaction observed with exposure to asbestos, suggests the presence of gene-environment and gene-gene interactions in the development of MM, although the size of the study group does not allow to draw clearcut conclusions. Since genetic polymorphisms can also modify the extent of genetic damage occurring in subjects exposed to carcinogens, we measured the frequency of micronuclei in peripheral blood lymphocytes of a subgroup of MM cases. The limited number of cases (28) did not allow to observe significant effects. In conclusion, these results strengthen the hypothesis that individual susceptibility to MM can be modulated by the interaction between polymorphic genes involved in the metabolism and the intensity of asbestos exposure.     

Biocatalysed synthesis of beta-O-glucosides from 9-fluorenon-2-carbohydroxyesters. Part 3: IFN-inducing and anti-HSV-2 properties

In pursuing research on the antiviral, interferon (IFN)-inducing tilorone congeners, a new series of fluoren-carboxyhydroxyesters has been prepared and biologically explored. These esters have subsequently been used as sugar acceptors in the enzymatic transglycosylation reaction using the 'retaining' beta-glycosidase from the archaeon Sulfolobus solfataricus (Ssbeta-Gly). Both aglycones (1-6) and corresponding beta-glucosides (beta-glu 1-beta-glu 6) have been screened for cytotoxicity, interferon-stimulating and antiviral properties against HSV-2. It was found that the addition of compounds beta-glu 5, beta-glu 6 and beta-glu 4 to HSV-2 infected U937 cells downregulates viral replication and triggers cells to release IFN-alpha/beta. Taken together, the results showed improved pharmacological profiles as a consequence of glycosylation. A molecular modelling study carried out on this series of compounds completed the structural characterisation of the novel compounds.     

In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature

We describe a new methodology, based on terminal perfusion of rodents with a reactive ester derivative of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ extracts can be purified on streptavidin resin in the presence of strong detergents, digested on the resin and subjected to liquid chromatography-tandem mass spectrometry for identification. In the present study, in vivo biotinylation procedure led to the identification of hundreds of proteins in different mouse organs, including some showing a restricted pattern of expression in certain body tissues. Furthermore, biotinylation of mice with F9 subcutaneous tumors or orthotopic kidney tumors revealed both quantitative and qualitative differences in the recovery of biotinylated proteins, as compared to normal tissues. This technology is applicable to proteomic investigations of the differential expression of accessible proteins in physiological and pathological processes in animal models, and to human surgical specimens using ex vivo perfusion procedures.     

Texture of locomotor path: a replicable characterization of a complex behavioral phenotype

A database of mouse locomotor path in spatial tests can be used to search in silico for behavioral measures that better discriminate between genotypes and are more replicable across laboratories. In this study, software for the exploration of exploration (SEE) was used to search a large database for a novel behavioral measure that would characterize complex movement paths. The database included mouse open-field behavior assessed in 3 laboratories, 7 inbred strains, several pharmacological treatments and hundreds of animals. The new behavioral measure, "path texture", was characterized using the local curvature of the path (the change of direction per unit distance, in degrees/cm) across several spatial scales, starting from scales smaller than the animal's body length and up to the scale of the arena size. Path texture analysis differs from fractal dimension analysis in that it does not assume self-similarity across scales. Path texture was found to discriminate inbred strains with relatively high broad-sense heritability (43%-71%) and high replicability across laboratories. Even genotypes that had similar path curvatures in some scales usually differed in other scales, and self-similarity across scales was not displayed by all genotypes. Amphetamine decreased the path curvature of C57BL/6 mice in small and medium scales, while having no effect on DBA/2J mice. Diazepam dose-dependently decreased the curvature of C57BL/6 mice across all scales, while 2 anxiogenic drugs, FG-7142 and pentylenetetrazole, increased it. Path texture thus has high potential for behavioral phenotyping and the study of drug effects in the mouse.