Liposomes as sustained release system for human interferon-gamma: biopharmaceutical aspects

Interferon-gamma (IFNgamma) has proven to be a promising adjuvant in vaccines against cancer and infectious diseases. However, due to its rapid biodegradation and clearance, its efficacy is severely reduced. Liposomal association might prolong the residence time of IFNgamma, but no efforts have been made to optimize the biopharmaceutical characteristics of liposomal IFNgamma for its application in therapy or as vaccine immunoadjuvant. In the present study, various liposomal formulations of recombinant human IFNgamma (hIFNgamma), differing in lipid composition, were prepared via the film hydration method and characterized in vitro regarding association efficiency and bioactivity, and in vivo regarding cytokine release kinetics after subcutaneous (s.c.) administration into mice. Human IFNgamma can be formulated in large, multilamellar liposomes with high association efficiency (>80%) and preservation of bioactivity. A critical parameter is the inclusion of negatively charged phospholipids to obtain a high liposome association efficiency, which is dominated by electrostatic interactions. The fraction of externally adsorbed protein compared to the total associated protein can be minimized from 74+/-9% to 8+/-3% by increasing the ionic strength of the dispersion medium. After injection of free (125)I-hIFNgamma, the radiolabel was detectable up to 48 h at the injection site. Liposomal encapsulation of (125)I-hIFNgamma increased the local area under the curve 4-fold, and the presence of the radiolabeled hIFNgamma at the injection site was prolonged to 7 days. The release kinetics and overall residence time of the cytokine at the s.c. administration site was influenced by depletion of the externally adsorbed IFNgamma, reducing the initial burst release. Increasing the rigidity of the liposome bilayer also resulted in a more pronounced reduction of the burst release and a 19-fold increase in the residence time of the protein at the s.c. administration site, compared to the free cytokine. As adjuvanticity of liposomal IFNgamma may strongly depend on the release kinetics of cytokines in vivo, the findings in this paper may contribute to a rational design of liposomal-cytokine adjuvants in vaccines against cancer and infectious diseases.     

SEGMENT: identifying compositional domains in DNA sequences

MOTIVATION: DNA sequences are formed by patches or domains of different nucleotide composition. In a few simple sequences, domains can simply be identified by eye; however, most DNA sequences show a complex compositional heterogeneity (fractal structure), which cannot be properly detected by current methods. Recently, a computationally efficient segmentation method to analyse such nonstationary sequence structures, based on the Jensen-Shannon entropic divergence, has been described. Specific algorithms implementing this method are now needed. RESULTS: Here we describe a heuristic segmentation algorithm for DNA sequences, which was implemented on a Windows program (SEGMENT). The program divides a DNA sequence into compositionally homogeneous domains by iterating a local optimization procedure at a given statistical significance. Once a sequence is partitioned into domains, a global measure of sequence compositional complexity (SCC), accounting for both the sizes and compositional biases of all the domains in the sequence, is derived. SEGMENT computes SCC as a function of the significance level, which provides a multiscale view of sequence complexity.     

Oligodendroglial Response to the Immune Cytokine Interferon Gamma

In the human demyelinating disorder multiple sclerosis, and its animal model experimental allergic encephalomyelitis, there is a breakdown of the blood-brain barrier and an infiltration of immune cells into the CNS. Infiltrating T lymphocytes and macrophages are believed to be key mediators of the disease process. Considerable circumstantial and experimental evidence has suggested that the pleiotropic cytokine interferon gamma (IFN-), which is exclusively expressed by T cells and natural killer cells, is a deleterious component of the immune response in these disorders. When experimentally introduced into the CNS IFN- promotes many of the pathological changes that occur in immune-mediated demyelinating disorders. In vitro, this cytokine elicits a number of effects on oligodendrocytes, including cell death. The harmful actions of IFN- on CNS myelin are likely mediated through direct effects on the myelinating cells, as well as through the activation of macrophages and microglia. In this review we summarize relevant studies concerning the action of IFN- in demyelinating disorders and discuss possible mechanisms for the observed effects.     

Development of a composite map in Vicia faba,breeding applications and future prospects

A composite map of the Vicia faba genome based on morphological markers, isozymes, RAPDs, seed protein genes and microsatellites was constructed. The map incorporates data from 11 F₂ families for a total of 654 individuals all sharing the common female parent Vf 6. The integrated map is arranged in 14 major linkage groups (five of which were located in specific chromosomes). These linkage groups include 192 loci and cover 1,559 cM with an overall average marker interval of 8 cM. By joining data of a new F₂ population segregating for resistance to ascochyta, broomrape and others traits of agronomic interest, have been saturated new areas of the genome. The combination of trisomic segregation, linkage analysis among loci from different families with a recurrent parent, and the analysis of new physically located markers, has allowed the establishment of the present status of the V. faba map with a wide coverage. This map provides an efficient tool in breeding applications such as disease-resistance mapping, QTL analyses and marker-assisted selection.Communicated by J.W. Snape     

Carnitine: transport and physiological functions in the brain

Carnitine (4-N-trimethylammonium-3-hydroxybutyric acid), a compound necessary for a transfer of fatty acids for their oxidation within the cell, accumulates in brain although β-oxidation of fatty acids is very low in neurons. Carnitine accumulates to lower extent in the brain than in peripheral tissues and the mechanism of its transport through the blood–brain barrier is discussed, with the involvement of two transporters, OCTN2 and B^0,+ being presented. A limitation by the blood–brain barrier of carnitine supply for the brain and the mechanism of its transport to neural cells by a protein belonging to neurotransmitters' transporters superfamily is further discussed.

Due to the beneficial effects of administration of acetylcarnitine in case of patients with dementia, the role of this acylcarnitine is presented in the context of neuronal cell metabolism and the role of acetylcarnitine in the synthesis of acetylcholine. The roles of long-chain acyl derivatives of carnitine, in particular palmitoylcarnitine, responsible for interaction with the membranes, lipids acylation and specific interactions with proteins have been summarized. Stimulation of protein palmitoylation and a possibility of changing the acylation status of G proteins is described, as well as interaction of palmitoylcarnitine with protein kinase C. Diminished interaction of the isoform δ of this kinase with GAP-43 (B-50, neuromodulin), whose expression increases upon accumulation of either carnitine or palmitoylcarnitine points to a possible regulation of differentiation by these compounds and their role in neuroregeneration.     

Automated systems for protein crystallization

Growth of high quality crystals is often the most difficult step in the determination of protein structures by X-ray diffraction. Automation can improve the success of this process both by reducing the amount of protein required for each screen and by relieving the tedium of setting up crystallization experiments by hand. We have been using an automated system for the design and execution of hanging drop crystallization experiments for the last two years. The system includes robots for the preparation of solutions, setup of hanging drops, and automated imaging, as well as a new software package (RoCKS) for managing all phases of the crystallization process. Here, we review the fundamentals of automated protein crystallization and present results from our comparisons of various approaches to screening.     

The RhoA effector mDia is induced during T cell activation and regulates actin polymerization and cell migration in T lymphocytes

Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.     

Understanding resistance to beta-lactams and beta-lactamase inhibitors in the SHV beta-lactamase: lessons from the mutagenesis of SER-130

Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.