Colony stimulating factor-1 producing endothelial cells and mesenchymal stromal cells maintain monocytes within a perivascular bone marrow niche.

Macrophage colony stimulating factor-1 (CSF-1) plays a critical role in maintaining myeloid lineage cells. However, congenital global deficiency of CSF-1 (Csf1op/op) causes severe musculoskeletal defects that may indirectly affect hematopoiesis. Indeed, we show here that osteolineage-derived Csf1 prevented developmental abnormalities but had no effect on monopoiesis in adulthood. However, ubiquitous deletion of Csf1 conditionally in adulthood decreased monocyte survival, differentiation, and migration, independent of its effects on bone development. Bone histology revealed that monocytes reside near sinusoidal endothelial cells (ECs) and leptin receptor (Lepr)-expressing perivascular mesenchymal stromal cells (MSCs). Targeted deletion of Csf1 from sinusoidal ECs selectively reduced Ly6C- monocytes, whereas combined depletion of Csf1 from ECs and MSCs further decreased Ly6Chi cells. Moreover, EC-derived CSF-1 facilitated recovery of Ly6C- monocytes and protected mice from weight loss following induction of polymicrobial sepsis. Thus, monocytes are supported by distinct cellular sources of CSF-1 within a perivascular BM niche.

Adventitial recruitment of Lyve-1- macrophages drives aortic aneurysm in an angiotensin-2-based murine model.

OBJECTIVE: Aortic macrophage accumulation is characteristic of the pathogenesis of abdominal aortic aneurysm (AAA) but the mechanisms of macrophage accumulation and their phenotype are poorly understood. Lymphatic vessel endothelial receptor-1 (Lyve-1+) resident aortic macrophages independently self-renew and are functionally distinct from monocyte-derived macrophages recruited during inflammation. We hypothesized that Lyve-1+ and Lyve-1- macrophages differentially contribute to aortic aneurysm. Approach and results: Angiotensin-2 and ß-aminopropionitrile (AT2/BAPN) were administered to induce AAA in C57BL/6J mice. Using immunohistochemistry (IHC), we demonstrated primarily adventitial accumulation of aortic macrophages, and in association with areas of elastin fragmentation and aortic dissection. Compared with controls, AAA was associated with a relative percent depletion of Lyve-1+ resident aortic macrophages and accumulation of Lyve-1- macrophages. Using CD45.1/CD45.2 parabiosis, we demonstrated aortic macrophage recruitment in AAA. Depletion of aortic macrophages in CCR2-/- mice was associated with reduced aortic dilatation indicating the functional role of recruitment from the bone marrow. Depletion of aortic macrophages using anti-macrophage colony-stimulating factor 1 receptor (MCSF1R)-neutralizing antibody (Ab) reduced the incidence of AAA. Conditional depletion of Lyve-1+ aortic macrophages was achieved by generating Lyve-1wt/cre Csf1rfl/fl mice. Selective depletion of Lyve-1+ aortic macrophages had no protective effects following AT2/BAPN administration and resulted in increased aortic dilatation in the suprarenal aorta. CONCLUSIONS: Aortic macrophage accumulation in AAA derives from adventitial recruitment of Lyve-1- macrophages, with relative percent depletion of Lyve-1+ macrophages. Selective targeting of macrophage subtypes represents a potential novel therapeutic avenue for the medical treatment of AAA.

c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells.

Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease.

Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid.

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic properties allow them to dissolve hydrophobic compounds in the aqueous environment of the human body. However, self-assembling peptide solutions have poor blood compatibility (e.g., low osmolarity), hindering their clinical application through intravenous administrations. We have recently developed a generalized platform for hydrophobic drug delivery, which combines SAPs with amino acid solutions (SAP-AA) to enhance drug solubility and increase formulation osmolarity to reach the requirements for clinical uses. This formulation strategy was thoroughly tested in the context of three structurally different hydrophobic compounds - PP2, rottlerin, and curcumin - in order to demonstrate its versatility. Furthermore, we examined effects of changing formulation components by analyzing 6 different SAPs, 20 naturally existing amino acids at low and high concentrations, and two different co-solvents dimethyl sulfoxide (DMSO) and ethanol. Our strategy proved to be effective in optimizing components for a given hydrophobic drug, and therapeutic function of the formulated inhibitor, PP2, was observed both in vitro and in vivo. This manuscript outlines our generalized formulation method using SAP-AA combinations for hydrophobic compounds, and analysis of solubility as a first step towards potential use of these formulations in more functional studies. We include representative solubility results for formulation of the hydrophobic compound, curcumin, and discuss how our methodology serves as a platform for future biological studies and disease models.

High resolution, high speed, long working distance, large field of view confocal fluorescence microscope.

Confocal fluorescence microscopy is often used in brain imaging experiments, however conventional confocal microscopes are limited in their field of view, working distance, and speed for high resolution imaging. We report here the development of a novel high resolution, high speed, long working distance, and large field of view confocal fluorescence microscope (H2L2-CFM) with the capability of multi-region and multifocal imaging. To demonstrate the concept, a 0.5 numerical aperture (NA) confocal fluorescence microscope is prototyped with a 3 mm × 3 mm field of view and 12 mm working distance, an array of 9 beams is scanned over the field of view in 9 different regions to speed up the acquisition time by a factor of 9. We test this custom designed confocal fluorescence microscope for future use with brain clarification methods to image large volumes of the brain at subcellular resolution. This multi-region and multi-spot imaging method can be used in other imaging modalities, such as multiphoton microscopes, and the field of view can be extended well beyond 12 mm × 12 mm.

Snapshot linear-Stokes imaging spectropolarimeter using division-of-focal-plane polarimetry and integral field spectroscopy.

In this paper, the design and experimental demonstration of a snapshot linear-Stokes imaging spectropolarimeter (SLSIS) is presented. The SLSIS, which is based on division-of-focal-plane polarimetry with four parallel linear polarization channels and integral field spectroscopy with numerous slit dispersive paths, has no moving parts and provides video-rate Stokes-vector hyperspectral datacubes. It does not need any scanning in the spectral, spatial or polarization dimension and offers significant advantages of rapid reconstruction without heavy computation during post-processing. The principle and the experimental setup of the SLSIS are described in detail. The image registration, Stokes spectral reconstruction and calibration procedures are included, and the system is validated using measurements of tungsten light and a static scene. The SLSIS's snapshot ability to resolve polarization spectral signatures is demonstrated using measurements of a dynamic scene.

Formulation of hydrophobic therapeutics with self-assembling peptide and amino acid: A new platform for intravenous drug delivery.

Clinical application of hydrophobic therapeutics is restricted by lack of an efficient vehicle which permits their solubility in aqueous environments. We have previously developed a novel formulation strategy to deliver a hydrophobic Src inhibitor, PP2, involving combinations of one self-assembling peptide (SAP) and one of 4 selected amino acids (AAs). The present study aims to develop a generalized drug delivery platform for intravenous application of hydrophobic drugs by combining self-assembling peptide, amino acid and low concentration of co-solvent. A multi-step screening pipeline is established which includes assessment of drug solubility and physicochemical characteristics, as well as functional efficacy and safety in vitro and in vivo. Using PP2 as an exemplary hydrophobic compound, 480 different combinations of 6 SAPs, 20 naturally existing AAs at 2 concentrations, and 2 co-solvents were evaluated. Among the combinations, 60 formulae dissolved PP2; 10 of which significantly reduced thrombin-induced IL-8 production, a sign of inflammatory response, in normal human lung epithelial BEAS2B cells. These formulations did not show cytotoxicity alone, but 2 reduced cell viability with presence of thrombin. We then performed a double-blinded test in a rat model of pulmonary ischemia-reperfusion. PP2 formulated with EAK16-I peptide plus methionine and 2% ethanol were administrated intravenously, significantly reducing severity of lung injury. The SAP-AA formulation strategy was also successfully applied to other hydrophobic compounds, suggesting this strategy could be applicable to other hydrophobics for a variety of clinical applications.

Reflective Fourier ptychography.

The Fourier ptychography technique in reflection mode has great potential applications in tissue imaging and optical inspection, but the current configuration either has a limitation on cut-off frequency or is not practical. By placing the imaging aperture stop outside the illumination path, the illumination numerical aperture (NA) can be greater than the imaging NA of the objective lens. Thus, the cut-off frequency achieved in the proposed optical system is greater than twice the objective's NA divided by the wavelength (2NAobj/λ ), which is the diffraction limit for the cut-off frequency in an incoherent epi-illumination configuration. We experimentally demonstrated that the synthesized NA is increased by a factor of 4.5 using the proposed optical concept. The key advantage of the proposed system is that it can achieve high-resolution imaging over a large field of view with a simple objective. It will have a great potential for applications in endoscopy, biomedical imaging, surface metrology, and industrial inspection.

Generation of a controllable multifocal array from a modulated azimuthally polarized beam.

In this Letter, the focal spot areas of an azimuthally polarized beam modulated with a vortex-0-2π-phase plate or a π-phase-step plate are numerically found to be smaller than a radially polarized beam for three pupil functions with uniform, Gaussian, and Bessel-Gauss profiles. Several types of multizone phase plates are theoretically designed and numerically simulated for generating tight multifocal arrays from the azimuthally polarized beams for what we believe is the first time. The positions and polarization states of the multifocal arrays can be controlled simply by varying the pattern of the multizone plates. The produced multifocal array with controllable position and polarization is beneficial to parallel optical recording and parallel optical imaging.

Transfer function analysis in epi-illumination Fourier ptychography.

This Letter explores Fourier ptychography (FP) using epi-illumination. The approach effectively modifies the FP transfer function to be coherent-like out to the incoherent limit of twice the numerical aperture over the wavelength 2NA/λ. Images reconstructed using this approach are shown to have higher contrast at finer details compared with images using incoherent illumination, indicating that the FP transfer function is superior in high spatial frequency regions.

Analysis of grating doublets for achromatic beam-splitting.

Achromatic beam-splitting grating doublets are designed for both continuous phase and binary phase gratings. By analyzing the sensitivity to lateral shifts between the two grating layers, it is shown that continuous-profile grating doublets are extremely difficult to fabricate. Achromatic grating doublets that have profiles with a constant first spatial derivative are significantly more resistant to lateral shifts between grating layers, where one design case showed a 17 times improvement in performance. Therefore, binary phase, multi-level phase, and blazed grating doublets perform significantly better than continuous phase grating doublets in the presence of a lateral shift between two grating layers. By studying the sensitivity to fabrication errors in the height of both grating layers, one grating layer height can be adjusted to maintain excellent performance over a large wavelength range if the other grating layer is fabricated incorrectly. It is shown in one design case that the performance of an achromatic Dammann grating doublet can be improved by a factor of 215 if the heights of the grating layers are chosen to minimize the performance change in the presence of fabrication errors.

Effective delivery of a rationally designed intracellular peptide drug with gold nanoparticle-peptide hybrids.

A novel gold nanoparticle-peptide hybrid strategy was developed to intracellularly deliver a potent PKCδ inhibitor peptide for the treatment of acute lung injury. The gold nanoparticle-peptide hybrids showed good stability with high uptake, and demonstrated in vitro and in vivo efficacy. Our formulation strategy shows great promise in intracellular delivery of peptides.

Sensitivity analysis and optimization method for the fabrication of one-dimensional beam-splitting phase gratings.

A method to design one-dimensional beam-spitting phase gratings with low sensitivity to fabrication errors is described. The method optimizes the phase function of a grating by minimizing the integrated variance of the energy of each output beam over a range of fabrication errors. Numerical results for three 1x9 beam splitting phase gratings are given. Two optimized gratings with low sensitivity to fabrication errors were compared with a grating designed for optimal efficiency. These three gratings were fabricated using gray-scale photolithography. The standard deviation of the 9 outgoing beam energies in the optimized gratings were 2.3 and 3.4 times lower than the optimal efficiency grating.

Impact of CCD camera SNR on polarimetric accuracy.

A comprehensive charge-coupled device (CCD) camera noise model is employed to study the impact of CCD camera signal-to-noise ratio (SNR) on polarimetric accuracy. The study shows that the standard deviations of the measured degree of linear polarization (DoLP) and angle of linear polarization (AoLP) are mainly dependent on the camera SNR. With increase in the camera SNR, both the measurement errors and the standard deviations caused by the CCD camera noise decrease. When the DoLP of the incident light is smaller than 0.1, the camera SNR should be at least 75 to achieve a measurement error of less than 0.01. When the input DoLP is larger than 0.5, a SNR of 15 is sufficient to achieve the same measurement accuracy. An experiment is carried out to verify the simulation results.

Single camera imaging system for color and near-infrared fluorescence image guided surgery.

Near-infrared (NIR) fluorescence imaging systems have been developed for image guided surgery in recent years. However, current systems are typically bulky and work only when surgical light in the operating room (OR) is off. We propose a single camera imaging system that is capable of capturing NIR fluorescence and color images under normal surgical lighting illumination. Using a new RGB-NIR sensor and synchronized NIR excitation illumination, we have demonstrated that the system can acquire both color information and fluorescence signal with high sensitivity under normal surgical lighting illumination. The experimental results show that ICG sample with concentration of 0.13 µM can be detected when the excitation irradiance is 3.92 mW/cm(2) at an exposure time of 10 ms.

Snapshot, reconfigurable multispectral and multi-polarization telecentric imaging system.

A reconfigurable telecentric imaging system that can simultaneously capture multispectral and multi-polarization information in a single snapshot is demonstrated. The proposed design utilizes a telecentric imaging objective and a light pipe for image multiplexing. An array of filters is used to filter each of the multiplexed images at an intermediate image plane. In this paper, we will discuss the system configuration and present the experimental results.