Effect of the Anti‐C1s Humanized Antibody TNT009 and Its Parental Mouse Variant TNT003 on HLA Antibody–Induced Complement Activation—A Preclinical In Vitro Study

The classic pathway (CP) of complement is believed to significantly contribute to alloantibody‐mediated transplant injury, and targeted complement inhibition is currently considered to be a promising approach for preventing rejection. Here, we investigated the mode of action and efficacy of the humanized anti‐C1s monoclonal antibody TNT009 and its parental mouse variant, TNT003, in preclinical in vitro models of HLA antibody–triggered CP activation. In flow cytometric assays, we measured the attachment of C1 subcomponents and C4/C3 split products (C4b/d, C3b/d) to HLA antigen–coated flow beads or HLA‐mismatched aortic endothelial cells and splenic lymphocytes. Anti‐C1s antibodies profoundly inhibited C3 activation at concentrations >20 μg/mL, in both solid phase and cellular assays. While C4 activation was also prevented, this was not the case for C1 subcomponent attachment. Analysis of serum samples obtained from 68 sensitized transplant candidates revealed that the potency of inhibition was related to the extent of baseline CP activation. This study demonstrates that anti‐C1s antibodies TNT009 and TNT003 are highly effective in blocking HLA antibody–triggered complement activation downstream of C1. Our results provide the foundation for clinical studies designed to investigate the potential of TNT009 in the treatment or prevention of complement‐mediated tissue injury in sensitized transplant recipients.     

CXCR4 blockade augments bone marrow progenitor cell recruitment to the neovasculature and reduces mortality after myocardial infarction

We hypothesized that a small molecule CXCR4 antagonist, AMD3100 (AMD), could augment the mobilization of bone marrow (BM)-derived endothelial progenitor cells (EPCs), thereby enhancing neovascularization and functional recovery after myocardial infarction. Single-dose AMD injection administered after the onset of myocardial infarction increased circulating EPC counts and myocardial vascularity, reduced fibrosis, and improved cardiac function and survival. In mice transplanted with traceable BM cells, AMD increased BM-derived cell incorporation in the ischemic border zone. In contrast, continuous infusion of AMD, although increasing EPCs in the circulation, worsened outcome by blocking EPC incorporation. In addition to its effects as a CXCR4 antagonist, AMD also up-regulated VEGF and matrix metalloproteinase 9 (MMP-9) expression, and the benefits of AMD were not observed in the absence of MMP-9 expression in the BM. These findings suggest that AMD3100 preserves cardiac function after myocardial infarction by enhancing BM-EPC–mediated neovascularization, and that these benefits require MMP-9 expression in the BM, but not in the ischemic region. Our results indicate that AMD3100 could be a potentially useful therapy for the treatment of myocardial infarction.     

Synthesis of Sorption Materials from Low Grade Titanium Raw Materials

In this paper, a universal technology is proposed for processing low-titanium mineral raw material—apatite-nepheline ore waste, including its treatment with sulfuric or hydrochloric acid in a two-stage mode with a sequential increase in the concentration. This technique allowed us to remove nepheline and apatite in the first stage and achieve a titanium mineral content of TiO₂ of more than 30%; in the second stage, we were able to convert the titanium into its precursors—titanyl sulfate monohydrate TiOSO₄·H₂O and a hybrid rutile-silica composition. The key stage in the sorbent synthesis is the reaction of the precursor with a phosphoric acid solution. The preferred sequence of operations begins with the mechanical activation of the precursor, causing morphological changes in it, and subsequent treatment with phosphoric acid at different concentrations under atmospheric conditions and in an autoclave, accompanied by phase transformations. Conditions for the chemical reactions which regulated the composition and structure of the final product and, accordingly, its sorption activity were found. With the help of XFA, the phase compositions of the sorbents were identified, including the individual crystalline phase α-TiP obtained from TS or the crystalline phase αTi(HPO₄)₂∙H₂O, which is in an amorphous silica matrix obtained from a rutile–silica composition.     

The Influence of the Elemental Composition,Crystal Structure,and Grain Structure of the Ferro-Piezoceramics of Various Degrees of the Ferro-Hardness on the Stability of the Polarized State

Ferro-piezoceramic materials (FPCM) with different degrees of ferrohardness were fabricated by double solid-phase synthesis followed by the sintering technique using hot pressing method. The X-ray studies carried out in a wide temperature range showed that with increasing temperature, each of the studied FPCM undergoes a series of phase transformations, accompanied by a change in the symmetry of the unit cell. In this case, near the phase transition to the nonpolar cubic phase, in each of the FPCM, the formation of a fuzzy symmetry region is observed, which is characterized by weak distortions and temperature–time instability of the crystal structure. The study of the piezoelectric modulus d₃₃ in the quasi-static regime as a function of temperature made it possible to reveal the different nature of its behavior in materials of various degrees of ferrohardness. It was shown that the conservation of the state in ferrosoft materials above the Curie temperature is associated with the relaxation nature of the change in their properties, the existence of a region of fuzzy symmetry (noncubic phase) in them above the Curie temperature, and increased inertia of the system. The expediency of taking into account the presented results in the development of electromechanical converters based on FPCM of various degrees of ferrohardness, operated under temperature effects, including cyclic ones, was shown.     

Approximations of the target-mediated drug disposition model and identifiability of model parameters

Models for drugs exhibiting target-mediated drug disposition (TMDD) play an important role in the investigation of biological products (Mager and Jusko 2001). These models are often overparameterized and difficult to converge. A simpler quasi-equilibrium (QE) approximation of the general model has been suggested (Mager and Krzyzanski 2005), but even this simpler form can be overparameterized when, for example, drug target level is not available. This work (a) introduces quasi-steady-state (QSS) and Michaelis-Menten (MM) approximations of the TMDD model, (b) derives the relationships between the parameters of the TMDD, QE, QSS and MM models, (c) investigates the parameter ranges where the simplified approximations are equivalent to the TMDD model, (d) proposes an algorithm for establishing identifiability of these models, and (e) tests this algorithm on simulated datasets. The proposed QSS approximation is more general than the QE approximation: it degenerates into the QE approximation when the internalization rate of the drug-target complex is much smaller than its dissociation rate. The proposed identifiability analysis algorithm may be applied to provide justification for use of simplified approximations, avoiding use of incorrect parameter estimates of over-parameterized TMDD models while simultaneously saving time and resources required for the pharmacokinetics analysis of drugs with TMDD. The utility of the derived approximations and of the identifiability algorithm was demonstrated on the examples of the simulated data sets. The simulation examples indicated that the QSS model may be preferable to the QE model when the internalization rate of the drug-target complex significantly exceeds its dissociation rate. The MM approximation may be adequate when the drug concentration significantly exceeds the target concentrations or when the target occupancy is close to 100%.     

Molecular follow-up of preneoplastic lesions in bronchial epithelium of former Chernobyl clean-up workers

Ionizing radiation is a potent lung carcinogen, but the precise molecular damage associated with it is still unknown. In this study we investigated cancer-related molecular abnormalities including K-ras (codon 12) mutation, p16(INK4A) promoter hypermethylation and microsatellite alterations at seven chromosomal regions in successive biopsies obtained from former Chernobyl cleanup workers in comparison with smokers and nonsmokers who have never had radiation exposure. Our results indicate that prolonged persistence of inhaled radioactive particles is associated with appearance of allelic loss at 3p12, 3p14.2 (FHIT), 3p21, 3p22-24 (hMLH1) and 9p21 (p16INK4A) in bronchial epithelium of former Chernobyl clean-up workers. The prevalence of 3p14.2 allelic loss was associated with decreased expression of the FHIT mRNA in their bronchial epithelium in comparison with control group of smokers. During several years of our monitoring samples of epithelium were collected from the same area of bronchial tree. In epithelium exposed to carcinogens (tobacco smoke and/or radioactivity) the total number of molecular abnormalities was significantly higher in dysplasia and in morphologically normal foci progressed later to dysplasia than in these samples which never showed evidence of such progression. Our findings indicate that extensive cancer-related molecular abnormalities sequentially occur in radiation damaged bronchial epithelium of former Chernobyl clean-up workers.     

Antitumor effect of valorphin in vitro and in vivo: combined action with cytostatic drugs

The action of the cytostatic drugs (epirubicin and vincristine) in combination with the endogenous antiproliferative beta-hemoglobin fragment (33-39), valorphin, was studied in tumor (L929 and A549) cell cultures, primary culture of murine bone marrow cells and in murine model of breast carcinoma in vivo. Simultaneous application of 1 microM valorphin and 1 microM epirubicin, in vitro, did not result in an additive suppressive effect on cell culture growth. Additive effects were achieved with alternating applications of the peptide and the drugs, namely, 0.5 microM (but not 1 microM) epirubicin added 24 h prior to 1 microM valorphin; 1 microM valorphin added 48 h prior to 0.1 microM epirubicin, or 0.1 microM vincristine, or 0.05 microM vincristine, which resulted in 100% cell death in the both series with vincristine and up to 78% cell biomass reduction in the experiments with epirubicin. In the in vivo model (female BLRB mice with subcutaneously inoculated syngeneic mammary carcinoma), simultaneous treatment with 25 mg/m(2) epirubicin and 1 mg/kg valorphin resulted in 42% of tumor growth inhibition, as compared with the negative control group and 22% inhibition as compared with the epirubcin-treated group (at 20th day of treatment). Survival was significantly improved (69% compared to 39% in the group treated with epirubicin only) at day 26 after the treatment beginning.     

Detection of apoptosis and drug resistance of human breast cancer cells to taxane treatments using quartz crystal microbalance biosensor technology

Taxanes are used for the treatment of many human cancers, as first- and second-line chemotherapeutics. In the course of treatment many patients develop resistance or hypersensitivity to one form of taxane and require a different taxane to rescue the therapeutic benefit of the drug. There is currently no method to reliably predict tumor responses to taxanes prior to therapy or when resistance or hypersensitivity develops. We adapted the quartz crystal microbalance (QCM) biosensor technique to study responses of human mammary epithelial tumor cells to taxanes. Studies indicate that stable frequency and resistance levels are reached at 24 h. Cells in the QCM can then be treated with taxanes and responses monitored in real time via frequency and resistance changes reflecting alterations of cell mass distribution and viscoelastic properties. Distinct shifts in frequency and resistance accurately predicted apoptosis or resistance to treatment, as determined in parallel convention assays. QCM analysis accurately predicted docetaxel was more effective than paclitaxel and MCF-7 cells were more resistant to taxanes compared to MDA-MB-231 cells. These studies suggest "signature" patterns for taxane responsivity could be compared to those of patient biopsy samples to predict therapy outcome prior to treatment for initial therapy or to rescue therapy efficacy.