Glycerol models of the ciliated epithelium of bronchial mucosa and their use in the diagnosis of chronic nonspecific lung diseases

The method of extraction of ciliated epithelium from biopsy samples of human bronchial mucosa with glycerol is suggested. Permeabilized cilia of glycerol-extracted cells can be easily reactivated by exogenous ATP. This method was used for the study of ciliary dyskinesia in patients with chronic lung diseases. It was shown that in patients with Kartagener's syndrome neither freshly-isolated, nor glycerol-extracted ATP-treated cilia were motile. On the other hand, in some patients with bronchial asthma ATP reactivated glycerol-extracted cilia, while cilia of freshly-isolated cells remained immotile. The study shows that glycerol permeabilization and reactivation by ATP can be used for the analysis of cilial contractile apparatus in patients with chronic lung disease.     

A simple non-enzymatic method for the isolation of high yields of functional rat hepatocytes

A method for isolation of rat hepatocytes using liver perfusion by ethylenediamine tetra-acetic acid (EDTA)-containing sucrose solution and mechanical tissue disaggregation by controlled vibration (MVD) is described. The yields of hepatocytes produced by this method were similar to those obtained using collagenase perfusion. The cells had well preserved membrane integrity as judged by the trypan blue staining test (91 +/- 4%), ATP contents, rates of endogenous respiration and enzyme leakage that indicated they were functional cells. There was little evidence of expression of latent damage when the cells were stored either at 37 degrees C (by pre-incubation) or at 4 degrees C. This method can be used to isolate high yields of functional cells from rat liver if the collagenase perfusion technique is not available.     

Dynamics of the changes in homogenate respiratory activity after rat whole liver storage at 4 degrees C

Homogenate respiratory activity was studied after different storage terms of the whole rat liver at 4 degrees C in sucrose-based solution and following normothermic reperfusion. Preservation of homogenate respiratory activity in all metabolic states after normothermic reperfusion of the control liver (60 min, 4 degrees C) is shown. Further storage (6 and 24 hrs) of isolated liver under the mentioned above conditions strengthens the substrate respiration of homogenate both after storage and after normothermic reperfusion. At the same time oxidative phosphorylation does not practically change. No change was noted in respiratory activity in the states 3, 4ADP and 3DNP after 24 hrs of liver storage in respect of a previous term. Following normothermic liver reperfusion contributes to a statistically true reduction of mentioned parameters of respiration, that correlates with a decrease in the degree of respiration and phosphorylation coupled of the studied system.     

Virions and the Coat Protein of the Potato Virus X Interact with Microtubules and Induce Tubulin Polymerization In Vitro

A study was made of the in vitro interactions of virions and the coat protein (CP) of the potato virus X (PVX) with microtubules (MT). Both virions and CP cosedimented with taxol-stabilized MT. In the presence of PVX CP, tubulin polymerized to produce structures resistant to chilling. Electron microscopy revealed the aberrant character of the resulting tubulin polymers (protofilaments and their sheets), which differed from MT assembled in the presence of cell MAP2. In contrast, PVX virions induced the assembly of morphologically normal MT sensitive to chilling. Virions were shown to compete with MAP2 for MT binding, suggesting an overlap for the MT sites interacting with MAP2 and with PVX virions. It was assumed that PVX virions interact with MT in vivo and that, consequently, cytoskeleton elements participate in intracellular compartmentalization of the PVX genome.     

Inhibition of Autoimmune Diabetes in NOD Mice by miRNA Therapy

Autoimmune destruction of the pancreatic islets in Type 1 diabetes is mediated by both increased proinflammatory (Teff) and decreased regulatory (Treg) T lymphocytes resulting in a significant decrease in the Treg:Teff ratio. The non-obese diabetic (NOD) mouse is an excellent in vivo model for testing potential therapeutics for attenuating the decrease in the Treg:Teff ratio and inhibiting disease pathogenesis. Here we show for the first time that a bioreactor manufactured therapeutic consisting of a complex of miRNA species (denoted as TA1) can effectively reset the NOD immune system from a proinflammatory to a tolerogenic state thus preventing or delaying autoimmune diabetes. Treatment of NOD mice with TA1 resulted in a systemic broad-spectrum upregulation of tolerogenic T cell subsets with a parallel downregulation of Teff subsets yielding a dramatic increase in the Treg:Teff ratio. Moreover, the murine-derived TA1 was highly effective in the inhibition of allorecognition of HLA-disparate human PBMC. TA1 demonstrated dose-responsiveness and exhibited equivalent or better inhibition of allorecognition driven proliferation than etanercept (a soluble TNF receptor). These findings demonstrate that miRNA-based therapeutics can effectively attenuate or arrest autoimmune disease processes and may be of significant utility in a broad range of autoimmune diseases including Type 1 diabetes.     

High molecular weight protein detected in higher plant cells by antibodies against dynein is associated with vesicular organelles including Golgi apparatus

The cytoplasmic dynein is a multisubunit complex driving organelles along microtubules to their minus-end. We used antibodies against two functional domains (motor and microtubule-binding) of one of principal components of the complex--dynein heavy chain of slime mould Dictyostelium discoideum--to test root meristem cells of wheat Triticum aestivum. The antibodies reacted with a high molecular weight protein (> 500 kDa) in the total cell extract and the band recognized by the antibodies in plant extracts had a lower electrophoretic mobility than the high molecular weight band of mammalian dynein. Antibodies coupled to protein A-Sepharose precipitated the high molecular weight protein from the purified cell extracts. Immunocytochemical analysis demonstrated that the antigen recognized by antibodies against dynein heavy chains is associated with the vesicles whose localization depends on the cell cycle stage. The antigen-positive vesicles were localized to the perinuclear region in interphase and early prophase, to the spindle periphery and to spindle pole region during mitosis, and to the interzonal region in the period of fragmoplast and cell plate formation. Some antigen-positive vesicles also reacted with antibodies against Golgi protein markers. The obtained data indicate that higher plant cells contain a high molecular weight protein interacting with antibodies against the motor and microtubules-binding domains of Dictyostelium dynein heavy chain. The revealed antigen was associated with the vesicular structures in the cytoplasm including the Golgi apparatus.     

Development of an HIV-1 Specific Microbicide Using Caulobacter crescentus S-Layer Mediated Display of CD4 and MIP1α

The development of alternative strategies to prevent HIV infection is a global public health priority. Initial efforts in anti-HIV microbicide development have met with poor success as the strategies have relied on a non-specific mechanism of action. Here, we report the development of a microbicide aimed at specifically blocking HIV entry by displaying molecular components of the HIV/host cell attachment complex on the surface of Caulobacter crescentus, a harmless aquatic bacterium. This bacterium can be readily manipulated to present heterologous proteins at high density on its surface by genetic insertion into its crystalline surface layer protein [1], [2]. In separate constructions, we generated bacteria displaying domain 1 of CD4 and MIP1α. Each moiety reacted with specific antibodies by Western immunoblot and immuno-fluorescence microscopy. Microbicide functionality was assessed using an HIV pseudotype virus assay system representing Clade B subtypes. Bacteria displaying MIP1α reduced infectivity by 35–78% depending on the specific subtype while CD4 display reduced infection by as much as 56%. Combinations of both constructs reduced infectivity by nearly 98%. We demonstrated that HIV infection could be inhibited using a strategy aimed at HIV-specific molecular interactions with Caulobacter surface protein display, and that sufficient protein folding and conformation could be mimicked to bind and block entry. Further, this is the first demonstration that Caulobacter surface protein display may be a useful approach to preventing HIV infection or other viruses as a microbicide. We propose that this harmless bacterium, which is inexpensive to produce and formulate, might be suitable for topical applications as a viable alternative in the search for effective microbicides to counteract the world wide incidence of HIV infection.