Chloroquine enhances the antimycobacterial activity of isoniazid and pyrazinamide by reversing inflammation-induced macrophage efflux.

Mycobacterium tuberculosis (MTB) is notorious for persisting within host macrophages. Efflux pumps decrease intracellular drug levels, thus fostering persistence of MTB during therapy. Isoniazid (INH) and pyrazinamide (PZA) are substrates of the efflux pump breast cancer resistance protein-1 (BCRP-1), which is inhibited by chloroquine (CQ). In this study, BCRP-1 was found to be expressed on macrophages of human origin and on foamy giant cells at the site of MTB infection. In the current in vitro study, interferon-gamma (IFNγ) increased the expression of BCRP-1 in macrophages derived from the human monocytic leukaemia cell line THP-1. Using a BCRP-1-specific fluorescent dye and radioactively labelled INH, it was demonstrated that efflux from macrophages increased upon activation with IFNγ. CQ was able to inhibit active efflux and augmented the intracellular concentrations both of INH and the dye. In agreement, CQ and specific inhibition of BCRP-1 increased the antimycobacterial activity of INH against intracellular MTB. Although PZA behaved differently, CQ had comparable advantageous effects on the intracellular pharmacokinetics and activity of PZA. The adjunctive effects of CQ on intracellular killing of MTB were measurable at concentrations achievable in humans at approved therapeutic doses. Therefore, CQ, a widely used and worldwide available drug, may potentiate the efficacy of standard MTB therapy against bacteria in the intracellular compartment.

Calibrated nanoscale capacitance measurements using a scanning microwave microscope.

A scanning microwave microscope (SMM) for spatially resolved capacitance measurements in the attofarad-to-femtofarad regime is presented. The system is based on the combination of an atomic force microscope (AFM) and a performance network analyzer (PNA). For the determination of absolute capacitance values from PNA reflection amplitudes, a calibration sample of conductive gold pads of various sizes on a SiO(2) staircase structure was used. The thickness of the dielectric SiO(2) staircase ranged from 10 to 200 nm. The quantitative capacitance values determined from the PNA reflection amplitude were compared to control measurements using an external capacitance bridge. Depending on the area of the gold top electrode and the SiO(2) step height, the corresponding capacitance values, as measured with the SMM, ranged from 0.1 to 22 fF at a noise level of ~2 aF and a relative accuracy of 20%. The sample capacitance could be modeled to a good degree as idealized parallel plates with the SiO(2) dielectric sandwiched in between. The cantilever/sample stray capacitance was measured by lifting the tip away from the surface. By bringing the AFM tip into direct contact with the SiO(2) staircase structure, the electrical footprint of the tip was determined, resulting in an effective tip radius of ~60 nm and a tip-sample capacitance of ~20 aF at the smallest dielectric thickness.

Atomic force microscopy imaging of the human trigeminal ganglion.

This paper describes an investigation of gangliocytes via imaging semithin sections of two human trigeminal ganglia with an atomic force microscope (AFM). Whereas semithin sections are usually employed for transmission electron microscopy, we adopted this special type of sample preparation for our AFM studies to extract topographical data from the gangliocyte itself and from the nucleus, the nucleolus, the crystal-arranged lipofuscin granules, and the cell-surrounding mantle cells; simultaneously we characterized the samples with error signal mode. This AFM-related technique revealed no information concerning friction force and elasticity due to the presence of the embedding material (epoxy), but it gave additional topographical contrast. These are the first images of the human trigeminal ganglion by AFM.

A non-invasive method for the tight anchoring of cells for scanning force microscopy.

Use of scanning force microscopy (SFM) for high resolution imaging of cell surfaces requires the cells to be tightly attached to substrates. Imaging of loosely adhered RBL-2H3 cells enabled determination of the cell size and investigation of larger structures and pseudopodia but failed in resolving more detail. Immobilization under non-invasive conditions via flexible crosslinkers containing a hydrophobic anchoring group enhanced resolution enormously. The cells were tightly attached to the substrates and were not removed by shear forces up to 80 nN as determined in a flow through apparatus. Morphological structures and dynamic processes on cell surfaces were observed as well as structural changes after cell stimulation upon ionomycin treatment. Molecular or atomic resolution, however, was not attainable which is attributed to the displacement of the flexible cell surface due to shear forces arising from the scanning tip during contact mode.

Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.

A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands. For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN. Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule. The two Fab fragments of the antibody were found to bind independently and with equal probability. The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.

Scanning force microscopy studies of the S-layers from Bacillus coagulans E38-66, Bacillus sphaericus CCM2177 and of an antibody binding process.

In many prokaryotic cells (eubacteria and archaebacteria) the outermost cell envelope component is composed of a regularly structured protein surface layer (S-layer). The two-dimensional S-layer from Bacillus coagulans E38-66 and Bacillus sphaericus CCM2177 has been investigated by SFM at molecular resolution under physiological conditions (i.e., in buffer solution). We find the E38-66 S-layer lattice to be oblique with lattice parameters of a = 9-10 nm, b = 7-8 nm and gamma = 80 degrees -90 degrees (E38-66). The CCM2177 lattice is square with a = 12-14 nm, in good agreement with TEM data. We have used the unique possibility of the SFM to study the kinematics of biological processes and have performed experiments on the adhesion of polyclonal antibodies to the recrystallized E38-66 protein layer on a time scale of about two to ten seconds per image frame. This represents a first step in directly visualizing molecular recognition reactions.