In vivo mucosal delivery of bioactive human interleukin 1 receptor antagonist produced by Streptococcus gordonii

Background

Interleukin-1 (IL-1) is a cytokine involved in the initiation and amplification of the defence response in infectious and inflammatory diseases. IL-1 receptor antagonist (IL-1ra) is an inactive member of the IL-1 family and represents one of the most potent mechanisms for controlling IL-1-dependent inflammation. IL-1ra has proven effective in the therapy of acute and chronic inflammatory diseases in experimental animal models and also in preliminary clinical trials. However, optimisation of therapeutic schedules is still needed. For instance, the use of drug delivery systems targeting specific mucosal sites may be useful to improve topical bioavailability and avoid side effects associated with systemic administration.

Results

In order to develop systems for the delivery of IL-1ra to mucosal target sites, a Streptococcus gordonii strain secreting human IL-1ra was constructed. The recombinant IL-1ra produced by S. gordonii was composed of the four amino acid residues RVFP of the fusion partner at the N-terminus, followed by the mature human IL-1ra protein. RFVP/IL-1ra displayed full biological activity in vitro in assays of inhibition of IL-1β-induced lymphocyte proliferation and was released by recombinant S. gordonii in vivo both at the vaginal and the gastrointestinal mucosa of mice. RFVP/IL-1ra appeared beneficial in the model of ulcerative colitis represented by IL-2^-/- mice (knock-out for the interleukin-2 gene), as shown by the body weight increase of IL-2^-/- mice locally treated with S. gordonii producing RFVP/IL-1ra.

Conclusions

These results indicate that recombinant S. gordonii can be successfully used as a delivery system for the selective targeting of mucosal surfaces with therapeutic proteins.     

Unilamellar vesicles as potential capreomycin sulfate carriers: Preparation and physicochemical characterization

The aim of this work was to evaluate unilamellar liposomes as new potential capreomycin sulfate (CS) delivery systems for future pulmonary targeting by aerosol administration. Dipalmitoylphosphatidylcholine, hydrogenated phosphatidylcholine, and distearoylphosphatidylcholine were used for liposome preparation. Peptide-membrane interaction was investigated by differential scanning calorimetry (DSC) and attenuated total internal reflection Fourier-transform infrared spectroscopy (ATIR-FTIR). Peptide entrapment, size, and morphology were evaluated by UV spectrophotometry, photocorrelation spectroscopy, and transmission electron microscopy, respectively. Interaction between CS and the outer region of the bilayer was revealed by DSC and ATIR-FTIR. DSPC liposomes showed enhanced interdigitation when the CS molar fraction was increased. Formation of a second phase on the bilayer surface was observed. From kinetic and permeability studies, CS loaded DSPC liposomes resulted more stable if compared to DPPC and HPC over the period of time investigated. The amount of entrapped peptide oscillated between 10% and 13%. Vesicles showed a narrow size distribution, from 138 to 166 nm, and a good morphology. These systems, in particular DSPC liposomes, could represent promising carriers for this peptide.     

Proline-glutamate chimeras in isopeptides. Synthesis and biological evaluation of conformationally restricted glutathione analogues

The two novel diastereoisomeric glutathione analogues 1 and 2 have been designed and synthesized by replacing the native gamma-glutamylic moiety with the conformational rigid skeleton of cis- or trans-4-carboxy-L-proline residue. Both analogues have been obtained by following the solution phase peptide chemistry methodologies and final reduction of the corresponding disulfide forms 13 and 14. The two analogues 1 and 2 have been tested towards gamma-glutamyltranspeptidase (gamma-GT) and human glutathione S-transferase (hGST P1-1). Both analogues 1 and 2 are completely resistant to enzymatic degradation by gamma-GT. The S-transferase utilizes the analogue 2 as a good substrate while is unable to bind the analogue 1.     

A unified model for apical caspase activation

Apoptosis is orchestrated by the concerted action of caspases, activated in a minimal two-step proteolytic cascade. Existing data suggests that apical caspases are activated by adaptor-mediated clustering of inactive zymogens. However, the mechanism by which apical caspases achieve catalytic competence in their recruitment/activation complexes remains unresolved. We explain that proximity-induced activation of apical caspases is attributable to dimerization. Internal proteolysis does not activate these apical caspases but is a secondary event resulting in partial stabilization of activated dimers. Activation of caspases-8 and -9 occurs by dimerization that is fully recapitulated in vitro by kosmotropes, salts with the ability to stabilize the structure of proteins. Further, single amino acid substitutions at the dimer interface abrogate the activity of caspases-8 and -9 introduced into recipient mammalian cells. We propose a unified caspase activation hypothesis whereby apical caspases are activated by dimerization of monomeric zymogens.     

On the analysis of hand synergies during grasping in weightlessness

The aim of this paper was to analyse how the strategies implemented by the Central Nervous System to control the hand during grasping are modified under microgravity conditions. Two right-handed subjects carried out simple grasping tasks during parabolic flights. The trajectories of the fingers of the hand were recorded using a sensorised glove and processed in order to extract a variable (here indicated as K) which can indicated the degree of synergies existing among the fingers. The results showed that K was quite small during the trial at 1g while becoming significantly greater than 1 during the first parabolas. Then, the value k decreased to the values at 1 g after some parabolas. These results suggested a possible adaptation process of the manipulation abilities during the permanence at 0g conditions. Future extensive trials will be performed in order to confirm these preliminary results.     

Brown fat thermogenesis in cold-acclimated rats is not abolished by the suppression of thyroid function

The effects of long-term cold exposure on brown adipose tissue (BAT) thermogenesis in hypothyroid rats have been examined. Thyroid ablation was performed in normal rats after 2 mo of exposure to 4 degrees C, when BAT hypertrophy and thermogenic activity were maximal. After ablation, hypothyroid and normal controls remained in the cold for 2 additional months. At the end of the 4-mo cold exposure, all untreated hypothyroid rats were alive, had normal body temperature, and had gained an average 12.8% more weight than normal controls. Long-term cold exposure of hypothyroid rats markedly increased BAT weight, mitochondrial proteins, uncoupling protein (UCP)-1, mRNA for UCP-1, and oxygen consumption to levels similar to those seen in cold-exposed normal rats. The results indicate that thyroid hormones are required for increased thermogenic capacity to occur as an adaptation to long-term cold exposure. However, cold adaptation can be maintained in the absence of thyroid hormone.     

Microtensile measurements of single trabeculae stiffness in human femur

In this paper, the authors perform microtensile tests of single trabeculae excised from a human femur head. One of the main issues of this work is to establish some experimental procedures for preparing and testing the specimens. The use of a well-characterized microtensile apparatus allows for a low intraspecimen dispersion of the measured stiffness. Tensile/compressive tests were chosen because they appear less sensitive to errors in the cross-sectional area measurements with respect to bending tests. By these considerations, some tensile/compressive tests of plate-like trabecular specimens have been carried out. Typical stiffness values are 74.2+/-0.7Nmm(-1) for tensile tests, and 58.9+/-0.6Nmm(-1) for compressive test. Another compressive test performed on a shorter specimen yielded a stiffness value of 148.3+/-5.3Nmm(-1). The maximum applied load was about 0.5N. Rough measurements of specimens sizes yielded a Young's modulus value ranging from 1.41 to 1.89GPa.