The Strong In Vivo Anti-Tumor Effect of the UIC2 Monoclonal Antibody Is the Combined Result of Pgp Inhibition and Antibody Dependent Cell-Mediated Cytotoxicity

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10–40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC₅₀ of doxorubicin (DOX) in KB-V1 (Pgp⁺) cells in vitro almost to the level of KB-3-1 (Pgp^-) cells. At the same time, UIC2 alone did not affect the EC₅₀ value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp⁺ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp⁺ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased ¹⁸FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).     

Novel Method to Load Multiple Genes onto a Mammalian Artificial Chromosome

Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe’s disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.     

New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) II: Albumin Suppresses Angiotensin Converting Enzyme (ACE) Activity in Human

About 8% of the adult population is taking angiotensin-converting enzyme (ACE) inhibitors to treat cardiovascular disease including hypertension, myocardial infarction and heart failure. These drugs decrease mortality by up to one-fifth in these patients. We and others have reported previously that endogenous inhibitory substances suppress serum ACE activity, in vivo, similarly to the ACE inhibitor drugs. Here we have made an effort to identify this endogenous ACE inhibitor substance. ACE was crosslinked with interacting proteins in human sera. The crosslinked products were immunoprecipitated and subjected to Western blot. One of the crosslinked products was recognized by both anti-ACE and anti-HSA (human serum albumin) antibodies. Direct ACE-HSA interaction was confirmed by binding assays using purified ACE and HSA. HSA inhibited human purified (circulating) and human recombinant ACE with potencies (IC₅₀) of 5.7±0.7 and 9.5±1.1 mg/mL, respectively. Effects of HSA on the tissue bound native ACE were tested on human saphenous vein samples. Angiotensin I evoked vasoconstriction was inhibited by HSA in this vascular tissue (maximal force with HSA: 6.14±1.34 mN, without HSA: 13.54±2.63 mN), while HSA was without effects on angiotensin II mediated constrictions (maximal force with HSA: 18.73±2.17 mN, without HSA: 19.22±3.50 mN). The main finding of this study is that HSA was identified as a potent physiological inhibitor of the ACE. The enzymatic activity of ACE appears to be almost completely suppressed by HSA when it is present in its physiological concentration. These data suggest that angiotensin I conversion is limited by low physiological ACE activities, in vivo.     

Identification and quantification of neuropeptides in naïve mouse spinal cord using mass spectrometry reveals [des‐Ser1]‐cerebellin as a novel modulator of nociception

Neuropeptide transmitters involved in nociceptive processes are more likely to be expressed in the dorsal than the ventral horn of the spinal cord. This study was designed to examine the relative distribution of neuropeptides between the dorsal and ventral spinal cord in naïve mice using liquid chromatography, high‐resolution mass spectrometry. We identified and relatively quantified 36 well‐characterized full‐length neuropeptides and an additional 168 not previously characterized peptides. By extraction with organic solvents we identified seven additional full‐length neuropeptides. The peptide [des‐Ser1]‐cerebellin (desCER), originating from cerebellin precursor protein 1 (CBLN1), was predominantly expressed in the dorsal horn. Immunohistochemistry showed the presence of CBLN1 immunoreactivity with a punctate cytoplasmic pattern in neuronal cell bodies throughout the spinal gray matter. The signal was stronger in the dorsal compared to the ventral horn, with most CBLN1 positive cells present in outer laminae II/III, colocalizing with calbindin, a marker for excitatory interneurons. Intrathecal injection of desCER induced a dose‐dependent mechanical hypersensitivity but not heat or cold hypersensitivity. This study provides evidence for involvement of desCER in nociception and provides a platform for continued exploration of involvement of novel neuropeptides in the regulation of nociceptive transmission.

     

Nicotinic acid suppresses sebaceous lipogenesis of human sebocytes via activating hydroxycarboxylic acid receptor 2 (HCA2)

Nicotinic acid (NA) activates hydroxycarboxylic acid receptor 2 (HCA₂), and it is widely used in treating dyslipidaemias. Since its side effects include skin dryness, whereas its deficiency can be accompanied by dyssebacia, characterized by sebaceous gland enlargement, we asked if HCA₂ is expressed on human sebocytes, and if NA influences sebocyte functions. By using human immortalized SZ95 sebocytes, we found that non‐cytotoxic (≤100 μmol/L; MTT‐assay) concentrations of NA had no effect on the homeostatic sebaceous lipogenesis (SLG; Nile Red), but normalized excessive, acne‐mimicking SLG induced by several lipogenic agents (arachidonic acid, anandamide, linoleic acid + testosterone; Nile Red; 48‐hr treatments). Moreover, it exerted significant anti‐proliferative actions (CyQUANT‐assay), and increased [Ca²⁺]_IC (Fluo‐4 AM‐based Ca²⁺‐measurement). Although NA did not prevent the lipopolysaccharide‐induced pro‐inflammatory response (up‐regulation [Q‐PCR] and release [ELISA] of several pro‐inflammatory cytokines) of the sebocytes, collectively, these data support the concept that NA may be effective in suppressing sebum production in vivo. While exploring the mechanism of the sebostatic actions, we found that sebocytes express HCA₂ (Q‐PCR, immunofluorescent labelling), siRNA‐mediated silencing of which prevented the NA‐induced Ca²⁺‐signal and the lipostatic action. Collectively, our data introduce NA, and HCA₂ activators in general, as novel, potent and most likely safe sebostatic agents, with possible anti‐acne potential.     

Cerebellar granule cells show age‐dependent migratory differences in vitro

Developmental differences between cerebellar granule cells during their migratory period were revealed using dissociated granule cell cultures isolated from 4, 7, or 10 days old (P4, P7, P10) mice. Under all culture conditions, the great majority of cultivated cell populations consisted of those granule cells that had not reach their final destination in the internal granule cell layer (IGL) by the age of isolation. In vitro morphological development and the expression of migratory markers (TAG‐1, astrotactin, or EphB2) showed similar characteristics between the cultures. The migration of 1008 granule cells isolated from P4, P7, and P10 cerebella and cultivated under identical conditions were analyzed using statistical methods. In vitro time‐lapse videomicroscopy revealed that P4 cells possessed the fastest migratory speed while P10 granule cells retained their migratory activity for the longest time in culture. Cultures obtained from younger postnatal ages showed more random migratory trajectories than P10 cultures. Our observations indicate that despite similar morphological and molecular properties, migratory differences exist in granule cell cultures isolated from different postnatal ages. Therefore, the age of investigation can substantially influence experimental results on the regulation of cell migration. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Assessment of synthetic methods for the preparation of N-beta-d-glucopyranosyl-N'-substituted ureas, -thioureas and related compounds

Preparation of O-peracetylated N-beta-d-glucopyranosyl-N'-acyl urea derivatives resulted in the formation of anomeric mixtures under the following conditions: acylation of O-peracetylated beta-d-glucopyranosyl urea by acyl chlorides in the presence of ZnCl(2) in refluxing CHCl(3); addition of O-peracetylated beta-d-glucopyranosylamine to acyl isocyanates in acetonitrile at rt; addition of carboxamides to in situ prepared O-peracetylated beta-d-glucopyranosyl isocyanate in refluxing toluene. Deprotection of O-peracetylated N-beta-d-glucopyranosyl-N'-acyl ureas either under base (NaOMe in MeOH at or below rt) or under acid (KHSO(4) or AcCl in MeOH at rt) catalyzed transesterification conditions resulted in unavoidable partial cleavage of the N'-acyl moieties. Reaction of beta-d-glucopyranosylammonium carbamate with an isocyanate, isothiocyanate or isoselenocyanate in dry pyridine at rt appears as a general method for the preparation of the corresponding beta-d-glucopyranosyl ureas, -thio- and -selenoureas, respectively, inclusive N'-acyl derivatives.     

Genetic determination of the effect of post-translational modification on the innate immune response to the 19 kDa lipoprotein of Mycobacterium tuberculosis

Background  

The 19 kDa lipoprotein of Mycobacterium tuberculosis (MTB) is an important target of the innate immune response. To investigate the effect of post-translation modification of this protein on innate recognition in the context of the whole bacillus, we derived a recombinant M. tuberculosis H37Rv that lacked the 19 kDa gene (Δ19) and complemented this strain by reintroduction of the 19 kDa gene into the chromosome as a single copy to produce Δ19::19. We also reintroduced the 19 kDa gene in two modified forms that lacked motifs for acylation (Δ19::19NA) and O-glycosylation (Δ19::19NOG).     

TSG-6 Protein Is Crucial for the Development of Pulmonary Hyaluronan Deposition,Eosinophilia, and Airway Hyperresponsiveness in a Murine Model of Asthma

Hyaluronan (HA) deposition is often correlated with mucosal inflammatory responses, where HA mediates both protective and pathological responses. By modifying the HA matrix, Tnfip6 (TNF-α-induced protein-6; also known as TSG-6 (TNF-stimulated gene-6)) is thought to potentiate anti-inflammatory and anti-plasmin effects that are inhibitory to leukocyte extravasation. In this study, we examined the role of endogenous TSG-6 in the pathophysiological responses associated with acute allergic pulmonary inflammation. Compared with wild-type littermate controls, TSG-6^−/− mice exhibited attenuated inflammation marked by a significant decrease in pulmonary HA concentrations measured in the bronchoalveolar lavage and lung tissue. Interestingly, despite the equivalent induction of both humoral and cellular Th2 immunity and the comparable levels of cytokines and chemokines typically associated with eosinophilic pulmonary inflammation, airway eosinophilia was significantly decreased in TSG-6^−/− mice. Most importantly, contrary to their counterpart wild-type littermates, TSG-6^−/− mice were resistant to the induction of airway hyperresponsiveness and manifested improved lung mechanics in response to methacholine challenge. Our study demonstrates that endogenous TSG-6 is dispensable for the induction of Th2 immunity but is essential for the robust increase in pulmonary HA deposition, propagation of acute eosinophilic pulmonary inflammation, and development of airway hyperresponsiveness. Thus, TSG-6 is implicated in the experimental murine model of allergic pulmonary inflammation and is likely to contribute to the pathogenesis of asthma.