Targeting fibroblast-like synovial cells at sites of inflammation with peptide targeted liposomes results in inhibition of experimental arthritis.

In this study we examined a synovium-specific targeted liposomal drug delivery system for its ability to localize and release its drug cargo to inflamed joints. Targeted liposomes were tested in vitro for binding to synovial fibroblast like (FLS) and endothelial cells using flow cytometry and in vivo for localization to joints using a rat model of adjuvant induced arthritis (AIA). Targeted liposomes were then loaded with anti-arthritic medications and examined for clinical efficacy in AIA. Targeted liposomes specifically bound to rabbit FLS and human FLS and showed a 7-10 fold increase in vivo localization in affected joints compared to unaffected joints. Histological sections from rats treated with prednisone and a new immunosuppressive peptide CP showed minimal inflammation. This report substantiates the ability of the novel FLS sequence to target liposomal drug delivery and offers an alternative therapeutic approach for the treatment of arthritis.

Postnatal development of cannabinoid receptor type 1 expression in rodent somatosensory cortex.

Endocannabinoids are powerful modulators of synaptic transmission that act on presynaptic cannabinoid receptors. Cannabinoid receptor type 1 (CB1) is the dominant receptor in the CNS, and is present in many brain regions, including sensory cortex. To investigate the potential role of CB1 receptors in cortical development, we examined the developmental expression of CB1 in rodent primary somatosensory (barrel) cortex, using immunohistochemistry with a CB1-specific antibody. We found that before postnatal day (P) 6, CB1 receptor staining was present exclusively in the cortical white matter, and that CB1 staining appeared in the gray matter between P6 and P20 in a specific laminar pattern. CB1 staining was confined to axons, and was most prominent in cortical layers 2/3, 5a, and 6. CB1 null (-/-) mice showed altered anatomical barrel maps in layer 4, with enlarged inter-barrel septa, but normal barrel size. These results indicate that CB1 receptors are present in early postnatal development and influence development of sensory maps.

The structural and functional polarity of the hepatic human/rat hybrid WIF-B is a stable and dominant trait.

WIF-B cells were generated previously to obtain a good in vitro model expressing the structural and functional polarity of hepatocytes. Here we tested the stability and the strength of the WIF-B polarized phenotype. WIF-B cells stayed polarized and formed functional bile canaliculi even after 3 months in culture or after injection in nude mice and culture of the resulting tumors. WIF-B was subcloned and 10,000 colonies were examined; all (except for 3) were composed of bile canaliculi forming cells. Some subclones were characterized; the polarized ones presented the same properties and karyotype as the WIF-B cells; the 3 unpolarized subclones had a lower level of E-cadherin and different karyotypes. WIF-B cells were fused with their nonpolarized hepatic parental cells. The polarity state of the resulting FWIF hybrids was studied from day 11 to day 38 after fusion, by immunolocalization of hepatocyte domain-specific plasma membrane proteins. Most FWIF colonies (>80%) were composed of polarized cells. Soon after fusion these cells were exclusively polarized as simple epithelial cells. The percent of colonies containing cells expressing the typical hepatocyte polarity increased with time and reached 80% at day 38. This result confirms the two-step polarization process previously described for WIF-B. Chromosomally complete FWIF hybrids were examined several months after fusion. As shown by the study of bile acid transport and by confocal analysis of the localization of membrane domain markers, FWIF cells expressed a functional and fully polarized hepatic phenotype. In conclusion, polarity is a stable and dominant trait of WIF-B.

A transfection compound series based on a versatile Tris linkage.

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.

Expression and localization of hepatocyte domain-specific plasma membrane proteins in hepatoma x fibroblast hybrids and in hepatoma dedifferentiated variants.

We have studied two aspects of the plasma membrane of hepatocytes, highly differentiated epithelial cells that exhibit a particular and complex polarity. Using a genetic approach, we have distinguished between the expression/regulation of proteins specific for all three hepatocyte membrane domains and their organization into discrete domains. For this analysis we used a panel of previously isolated cell clones, derived from the differentiated rat hepatoma line H4IIEC3, and that present different expression patterns for liver-specific genes. This panel was composed of (1) differentiated clones, (2) chromosomally reduced hepatoma-fibroblast hybrids characterized by a pleiotropic extinction/reexpression of liver-specific genes and (3) dedifferentiated variant and revertant clones. The expression of 16 hepatocyte membrane polarity markers was studied by western blotting and immunolocalization. Even though cells of differentiated clones express all of these polarity markers, they are not polarized, and are therefore suitable for studying the regulation of plasma membrane protein expression, and for identifying gene products implicated in the establishment of membrane polarity. In hepatoma-fibroblast hybrids the expression of four markers, three apical (dipeptidylpeptidase IV, alkaline phosphodiesterase B10 and polymeric IgA receptor) and one lateral (E-cadherin), is down-regulated in extinguished clones and restored in reexpressing subclones, as previously reported for liver-specific functions. The dipeptidylpeptidase IV mRNA was undetectable or strongly reduced in extinguished hybrids, but expressed at a robust level in some of the reexpressing clones. Concerning the dedifferentiated variants, each has its own pattern of membrane marker expression (loss of expression of three to six markers), that differs from that of extinguished hybrids. Revertant cells express all of the membrane markers examined. Among all of these hepatoma derivatives, only cells of reexpressing hybrids are polarized, and form bile canaliculi-like structures, with spherical and even, for one clone, long tubular and branched forms. All apical markers examined are confined in these canalicular structures, whereas the other markers are excluded from them, and present on the rest of the membrane (basolateral markers) or at the cell-cell contacts (lateral markers). Cells of reexpressing hybrids also express simple epithelial polarity. Thus the expression of only a few hepatocyte-domain-specific plasma membrane proteins is subject to down-regulation, as is the case for liver-specific genes so far studied, and the expression of polarity markers and the formation of poles are dissociable events.

Phenotypic effects of the forced expression of HNF4 and HNF1alpha are conditioned by properties of the recipient cell.

Tagged versions of HNF4 or HNF1alpha cDNAs in expression vectors have been introduced by transient and stable transfection into three cell lines of hepatic origin that all fail to express these two liver-enriched transcription factors and hepatic functions. C2 and H5 cells are dedifferentiated rat hepatoma variants and WIF12-E cells are human fibroblast-rat hepatoma hybrids with a reduced complement of human chromosomes. Transfectants were analyzed for the expression state of the endogenous genes coding for these transcription factors and for hepatic functions. Each cell line showed a different response to the forced expression of the transcription factors. In C2 cells, no measurable effect was observed, either upon transitory or stable expression. H5 cells reexpressed the endogenous HNF4 gene only upon transient HNF1alpha transfection, and the endogenous HNF1alpha gene only in stable HNF4 transfectants. WIF12-E cells responded to the forced transient or stable expression of either HNF1alpha or HNF4 by cross-activation of the corresponding endogenous gene. In addition, the stable transfectants reexpress HNF3alpha and C/EBPalpha, as well as all of the hepatic functions examined. Hybrid cells similar to WIF12-E had previously been observed to show pleiotropic reexpression of the hepatic phenotype in parallel with loss of human chromosome 2. For the stable WIF12-E transfectants, it was verified that reexpression of the hepatic phenotype was not due to loss of human chromosome 2. The demonstration of reciprocal cross-regulation between HNF4 and HNF1alpha in transient as well as stable transfectants implies that direct effects are involved.

Efficient in vitro vectorial transport of a fluorescent conjugated bile acid analogue by polarized hepatic hybrid WIF-B and WIF-B9 cells.

Efficient transport of bile acids, a typical characteristic of hepatocytes, is partially lost in most hepatoma cell lines and in normal hepatocytes after some days in culture. We have tested whether the polarized rat hepatoma-human fibroblast hybrid WIF (hybrids between W138 and Fao cells) cells previously obtained by our group were able to perform vectorial transport of the fluorescent bile acid derivative cholylglycylamidofluorescein (CGamF) towards the bile canaliculi (BC). Four different WIF clones were analyzed. All were well polarized, as shown by the formation of spherical and even tubular BC-like structures and by the restricted localization at the BC, visualized by immunofluorescence, of the apical membrane marker HA4, a possible bile acid carrier. WIF-B and its subclone WIF-B9 were found to accumulate CGamF in 65% to 75% of their BC. This transport was time, temperature, and partly sodium dependent and was inhibited by coincubation with the parental natural bile salt cholylglycine. Dinitrophenyl glutathione, a substrate of the canalicular multispecific organic anion transporter, did not inhibit CGamF canalicular secretion, whereas it greatly impaired the canalicular secretion of a non-bile acid organic anion, fluorescein, generated intracellularly from fluorescein diacetate. Confocal microscopy confirmed the presence of CGamF in the cytoplasm, supporting a transcellular route from medium to BC. In contrast, two other polarized clones exhibited a poor ability (WIF 12-6) or no ability (WIF12-1 TGdelta) to vectorially transport CGamE In conclusion, WIF-B and WIF-B9 exhibit not only structural but also functional polarity, at least as far as vectorial organic anion transport is concerned.

T-cell antigen receptor transmembrane peptides modulate T-cell function and T cell-mediated disease.

This study describes a novel method of inhibiting T-cell function by the use of peptides rationally designed from the T-cell antigen receptor (TCR) alpha-chain transmembrane sequence involved with TCR receptor assembly. The most effective peptide (core peptide, CP) modulating in vitro and in vivo T-cell function contained nine amino acids two of which, lysine and arginine, were hydrophilic and separated by four hydrophobic amino acids. CP without chemical modification or conjugation was able to enter non-T and T cells. Conjugation of CP at the carboxyl terminus with palmitic acid resulted in a greater inhibition of T-cell interleukin-2 (IL-2) production in vitro than peptide alone. When examined for effects in vivo, CP reduced clinical signs of inflammation in three T cell-mediated disease models including adjuvant-induced arthritis, experimental allergic neuritis, and cyclophosphamide-induced diabetes in NOD/Lt(F) mice. This peptide or its analogues has potential as a therapeutic agent in human inflammatory and autoimmune disorders.

A gentle method for linking Tris to amino acids and peptides.

A gentle method for the addition of Tris to the carboxyl group of amino acids or peptides for the purpose of altering their solubility and/or for providing sites for further derivatization is described. Under mild alkaline conditions and in high concentrations of aqueous dimethylformamide, the amino group of Tris cleaves simple esters of N-protected amino acids or peptides to form an amino acid-Tris linkage. The effects of pH, temperature and dimethylformamide concentration on the rate and the efficiency of the reaction of Tris with benzyloxycarbonyl-alanine methyl ester were examined and the general applicability of the method demonstrated on a range of amino acid and small peptide substrates. Side-chain protection was not required and the degree of racemization was found to be lower than with conventional chemical coupling.

Identification of reactive synthetic gliadin peptides specific for coeliac disease.

Gluten intolerance (coeliac disease) is characterised by the development of a small intestinal lesion following exposure to the gliadin fraction after consumption of wheat and related cereals. Cellular immune mechanisms are thought to be responsible for gliadin toxicity, but the toxic sequence/s within gliadin have not been clearly established. A panel of synthetic gliadin peptides was tested using peripheral blood mononuclear cells from coeliac patients and two assays for cell-mediated immunity. Using the indirect leucocyte migration inhibition factor and the macrophage procoagulant activity assays, gliadin peptides which were located in the aminoterminal or the proline-rich domain of the alpha/beta gliadin molecule were coeliac-active. Peptides predicted by T cell algorithms or on the basis of homology to adenovirus Ad12 Elb protein and which were located in the proline-poor gliadin domains were inactive. Protein sequence studies which indicate significant homology in the proline-poor gliadin domains with a number of non-coeliac-toxic seed proteins also supported the hypothesis that the proline-rich domains may be more important in the pathogenesis of coeliac disease.

Antigenic structure of bovine growth hormone: location of a growth enhancing region.

Site-directed antisera generated by peptide immunization have been used to study the antigenicity of bovine growth hormone (bGH). Prediction of sequential antigenic sites has been performed using secondary structure information derived from the 'Protean' prediction routine. The structures predicted by this programme agree closely with the corresponding structure of GH recently derived from crystallographic studies. We have previously shown that the binding of monoclonal antibodies of particular epitope specificity to human or bovine GH results in significant enhancement of hormonal activity in vivo; however, the sites recognized by these antibodies were not known. Here we identify a sequence region, corresponding to a loop structure joining helices 3 and 4, which, is associated with the growth enhancement phenomenon. Antisera raised to either of two overlapping peptides (residues 120-140 and 134-154) significantly increase the biological activity of GH in vivo. Antisera directed to other regions on the GH molecule failed to demonstrate this property. Coincidentally, the sites recognized by the growth-enhancing anti-peptide antisera overlap with the site on GH which is highly susceptible to proteolytic cleavage; such cleavage has been shown in some cases to result in hormone enhancement.

New functions of epidermal growth factor: stimulation of capillary endothelial cell migration and matrix dependent proliferation.

The proliferative response of bovine retinal capillary endothelial cells to EGF is dependent upon attaching the cells to a matrix of fibronectin. Bovine capillary endothelial cells are also stimulated to actively migrate when exposed to EGF in vitro. These activities provide an explanation for the angiogenic properties of EGF in vivo. Capillary cell migration and proliferation are proposed as sensitive quantifiable bioassays to explore the functional domains of the EGF molecule. Studies on the inactivation of these properties of EGF by specific cleavage of the molecule with CNBr or proteases suggest that an intact loop composed in part by amino acid residues 20 to 31 is essential for at least some functions.

[Lactate concentration in the ventricular fluid and blood plasma following severe craniocerebral trauma]. / Laktatkonzentration im Ventrikelliquor und Blutplasma nach schwerem Schädel-Hirn-Trauma.

Cerebral ischemia/hypoxia is of central importance in the sequence of pathogenetic processes after severe head injuries. Investigations in 72 patients showed that after severe head injury the changes of lactate concentration in blood plasma do not allow any statement with respect to cerebral ischaemia/hypoxia. However in ventricular liquor the amount and duration of increase in lactate concentration is significantly different in nonsurvivors in comparison to survivors. In lethal courses after 3rd day after head injury the increase of lactate concentration in ventricular liquor points to secondary cerebral ischemia/hypoxia.

Molecular species of epidermal growth factor carrying immunosuppressive activity.

The suppression of antibody formation to sheep red cells in mice by partially purified fractions of mouse submaxillary gland was shown to be caused by epidermal growth factor (EGF). Purification of EGF by the method of Savage and Cohen resolved three components referred to as EGF a, EGF b, and EGF c. All three induced premature eye opening in neonatal mice, but only EGF a (identified as EGF 1-53) had full immunosuppressive activity. EGF c was shown by micropeptide mapping of chymotryptic and thermolytic digests and amino-terminal analysis to differ from EGF a only by the presence of beta-aspartyl instead of an asparaginyl residue. EGF b differed from EGF a in that it lacked the N-terminal asparagine. EGF shortened enzymatically at its carboxy terminal by two or five amino acids did not have any immunosuppressive activity. These findings suggest that, in contrast to some other biological effects of EGF, intact amino and carboxy terminals are required for the expression of immunosuppressive activity.

Severe head trauma and the changes of concentration of neuron-specific enolase in plasma and in cerebrospinal fluid.

In nine patients with severe head trauma, the concentration of neuron-specific enolase in cerebrospinal fluid and in plasma was determined and compared with the activity of creatine kinase and alpha-hydroxybutyrate dehydrogenase, and with the concentration of lactate. In patients who died of the head trauma, a concentration of neuron-specific enolase of 6.8-64 micrograms/l in the plasma (reference range: 3.0-6.0 micrograms/l) and of 2.2-9.0 micrograms/l in the cerebrospinal fluid (reference range: 0.5-2.0 micrograms/l) was detected. Investigations of three patients showed that the changes of the concentration of neuron-specific enolase in plasma and in cerebrospinal fluid were independent of each other. Furthermore, the initial concentration of neuron-specific enolase in the plasma after the accident and the dynamics of its changes during the disease show a close relationship to the outcome.

Antigenic drift in the hemagglutinin of the Hong Kong influenza subtype: correlation of amino acid changes with alterations in viral antigenicity.

The nucleotide sequence of the gene coding for the large subunit of influenza virus hemagglutinin (HA1) was determined for strains A/NT/60/68, A/Eng/878/69, and A/Qu/7/70, three early isolates of the Hong Kong subtype. Sequences were obtained by the dideoxy chain termination method, using reverse transcriptase to synthesize partial DNA copies of the RNA gene. HA1 amino acid sequences predicted from the gene sequences were compared with published data for strains A/Aichi/2/68 and A/Vic/3/75. Compared with earlier strains, the HA1s of A/eng/878/69 and A/Qu/7/70 each contained three amino acid changes. Some of these were also found in A/Vic/3/75, but some were unique to the particular strain. When all of the strains were titrated with a panel of monoclonal antibodies directed against A/NT/60/68, alterations in viral antigenicity could be correlated with particular amino acid changes. The existence of multiple pathways for viral evolution during antigenic drift is discussed.

Experimental investigations in adult respiratory distress syndrome (ARDS). Repeated pulmonary lavage in LEWE-mini-pigs. I. Pulmonary mechanics, gas exchange, and pulmonary hemodynamics.

Disturbed water balance at the alveolo-capillary membrane resulting in decreased pulmonary compliance is one of the functional characteristics in the Adult Respiratory Distress Syndrome (ARDS). In artificially ventilated LEWE Mini Pigs repeated pulmonary lavage was performed. The aim was to produce a state like ARDS by affecting the lung alveolar surfactant system which is included into the sum of factors which are responsible for the normal water balance of the alveolo-capillary membrane. This condition was supposed to be maximized if the lung compliance reached its lowest value after pulmonary lavage. Regarding this the pathophysiologic changes in this ARDS model were characterized by increased airway resistance and dead space ventilation as well as decreased arterial oxygenation. Severe ventilation perfusion abnormality was represented hemodynamically by the increased pulmonary vascular resistance and the decreased pulmonary non-shunt blood flow. The morphological (macroscopic, light-and electronmicroscopic) changes indicate strongly that in this type of ARDS model all functional compartments of the alveolocapillary membrane are severely affected. Decreased colloid osmotic pressure of blood plasma samples underlines the severity of changes in the water balance at the alveolo-capillary membrane in this type of lung injury.