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Single-wall carbon nanotubes present unique opportunities for drug delivery, but have not advanced into the clinic. Differential nanotube accretion and clearance from critical organs have been observed, but the mechanism not fully elucidated. The liver has a complex cellular composition that regulates a range of metabolic functions and coincidently accumulates most particulate drugs. Here we provide the unexpected details of hepatic processing of covalently functionalized nanotubes including receptor-mediated endocytosis, cellular trafficking and biliary elimination. Ammonium-functionalized fibrillar nanocarbon is found to preferentially localize in the fenestrated sinusoidal endothelium of the liver but not resident macrophages. Stabilin receptors mediate the endocytic clearance of nanotubes. Biocompatibility is evidenced by the absence of cell death and no immune cell infiltration. Towards clinical application of this platform, nanotubes were evaluated for the first time in non-human primates. The pharmacologic profile in cynomolgus monkeys is equivalent to what was reported in mice and suggests that nanotubes should behave similarly in humans.

Automated detection of mRNAs and proteins in the same tissue sections is not a routine procedure. Successful experiment depends on the preparation of the tissue, the detection procedure, as well as the quality of the probes and antibodies. The multiplexed detections require experimental conditions, preserving the state of the molecular targets of interest and providing expression pattern of each target the same as in a single detection. Here we describe in detail the automated protocols used to detect mouse Lgr5 mRNA by in situ hybridization and immunofluorescence detection of lysozyme in the same mouse intestinal sections. Both the in situ hybridization and the protein detection were performed with an automated staining processor and provided strong and reproducible results.

Finding a valid antibody to detect mouse programmed death ligand 1 (PDL-1) by immunohistochemistry or immunofluorescence staining has been notoriously difficult. Successful validation of an antibody requires the use of multiple detection methods with the ability to compare appropriate positive and negative controls. Here, we describe in detail the protocols used to validate a mouse-specific PDL-1 antibody used in immunohistochemistry staining with an mRNA in situ hybridization on adjacent sections of mouse B16 tumor. This validation is supported by immunohistochemistry staining of PDL-1 on B16 cell pellets either treated or not treated with IFN-gamma.

The natural antibodies against glucosaminylmuramyl dipeptide (GMDP), the epitope of peptidoglycan of bacterial cell wall, isolated from human serum by thermal extraction possess a capability to cross-react with determinant of glycan chain - tetrasaccharide consisting of N-acetylglucosamine and N-acetylmuramic acid. The intensity of interaction of natural anti-GMDP-antibodies with specific ligand is significantly higher than with tetrasaccharide. The natural antibodies against tetrasaccharide carry properties of heteroclitic antibodies, i.e. the intensity of their interaction with heterologous ligand, GMDP, is significantly higher than with homologous one, tetrasaccharide GMGM. GMDP is supposed to be specific antigenic peptidoglycan determinant against which the antibodies reacting with various intensity to homologous (GMDP) and relative (tetrasaccharide) hapten are formed in the process of natural immunization.

We studied affinity of natural antibodies of human serum against glucosaminylmuramyl dipeptide (GMDP), the epitope peptidoglycan bacterial cell wall component which carries adjuvant activity. Antibodies against GMDP were isolated from the blood sera of healthy donors using thermal extraction of antibodies from specific ligand on plastic. Determination of the dissociation constant (K(d)) showed equal K(d) in the serum and affinity-purified anti-GMDP-antibodies, i.e. extraction by this method led to the isolation of all subpopulations of antibodies in spite of their affinity. K(d) of serum, affinity-purified and monoclonal anti-GMDP-antibodies proved of low value - 10(-6) M, and according to this index anti-GMDP-antibodies may be classified between anti-protein and anti-carbohydrate antibodies.