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Immune checkpoints are the crucial regulators of immune system and play essential roles in maintaining self-tolerance, preventing autoimmune responses, and minimizing tissue damage by regulating the duration and intensity of the immune response. Furthermore, immune checkpoints are usually overexpressed in cancer cells or noninvasive cells in tumor tissues and are capable of suppressing the antitumor response. Based on substantial physiological analyses as well as preclinical and clinical studies, checkpoint molecules have been evaluated as potential therapeutic targets for the treatment of multiple types of cancers. In the last few years, extensive evidence has supported the immunoregulatory effects of traditional Chinese medicines (TCMs). The main advantage of TCMs and natural medicine is that they usually contain multiple active components, which can act on multiple targets at the same time, resulting in additive or synergistic effects. The strong immune regulation function of traditional Chinese medicine on immune checkpoints has also been of great interest. For example, Astragalus membranaceus polysaccharides can induce anti-PD-1 antibody responses in animals, and these antibodies can overcome the exhaustion of immune cells under tumor immune evasion. Furthermore, many other TCM molecules could also be novel and effective drug candidates for the treatment of cancers. Therefore, it is essential to assess the application of immune checkpoints in the development of new drugs and TCMs. In this review, we focus on research progress in the field of immune checkpoints based on three topics: (1) immune checkpoint targets and pathways, (2) development of novel immune checkpoint-based drugs, and (3) application of immune checkpoints in the development of TCMs., Graphical abstract In this review, we provide a discussion of the applications of immune checkpoints in the development of novel drugs and TCMs.Image 1

The design of artificial tissues and implantable devices must incorporate the knowledge of how cells and tissues respond to changes in mechanical forces or properties introduced into the body. Mechanobiology is a field that investigates these fundamentals, including not only the biological responses to mechanical signals but also how cells convert external mechanical stimuli into internal chemical signals, and how they communicate mechanically with their environment and with each other. Mechanobiology is thus crucial to the understanding of many clinically relevant biological phenomena, such as morphogenesis, tissue regeneration, and cancer metastasis, and to the development of medical applications such as implants, wound healing, and tissue engineering.

Substrate rigidity has been recognized as an important property that affects cellular physiology and functions. While the phenomenon has been well recognized, understanding the underlying mechanism may be greatly facilitated by creating a microenvironment with designed rigidity patterns. This chapter describes in detail an optimized method for preparing substrates with micropatterned rigidity, taking advantage of the ability to dehydrate polyacrylamide gels for micropatterning with photolithography, and subsequently rehydrate the gel to regain the original elastic state. While a wide range of micropatterns may be prepared, typical composite substrates consist of micron-sized islands of rigid photoresist grafted on the surface of polyacrylamide hydrogels of defined rigidity. These islands are displaced by cellular traction forces, for a distance determined by the size of the island, the rigidity of the underlying hydrogel, and the magnitude of traction forces. Domains of rigidity may be created using this composite material to allow systematic investigations of rigidity sensing and durotaxis.

Publisher Summary This chapter introduces to the basic concept of several computational image restoration techniques that are particularly useful for processing fluorescence images. An optimal computational noise filter should aggressively remove random fluctuations in the image while preserving as much as possible the non random features. Filters that apply a uniform algorithm across the image, such as low-pass convolution and median filters, usually lead to the degradation of image resolution or amplification of artifacts. Better performance may be obtained using filters that adapt to the local characteristics of the image. In anisotropic diffusion, local intensity fluctuations are reduced through a simulated division process by diffusing from pixels of high intensity toward neighboring pixels of low intensity. Mathematical reversal of systemic defects of the microscope is known as image “Deconvolution.” This is because the degradation effects of the microscope can be mathematically described as the convolution of input signals by the pointspread function of the optical system. In many cases, the fluorescence image contains specific characteristics, which may be used as powerful preconditions for image restoration. As optical microscopy continues to push for maximal speed and minimal irradiation, overcoming the limitation in signal-to-noise ratio will likely remain a major challenge for both the retrieval of information from unprocessed images and the application of various restoration algorithms. In addition, as many biological structures contain characteristic features, the application of feature recognition and extraction will play an increasingly important role in both light and electron microscopy.

In conjunction with surface chemistry, the mechanical properties of cell culture substrates provide important biological cues that affect cell behavior including growth, differentiation, spreading, and migration. The phenomenon has led to the increased use of biological and synthetic polymer-based flexible substrates in cell culture studies. However, widely used methods for measuring the Young's modulus have proven difficult in the characterization of these materials, as they tend to be relatively thin, soft, hydrated, and tethered to glass substrates. Here we describe three methods that have been applied successfully to probe the flexibility of soft culture substrates.

Publisher Summary This chapter introduces fluorescence imaging of live cells, defining its various features. An inverted microscope generally allows more flexibility and workspace for the culture and manipulation of live cells than an upright microscope. All lenses for fluorescence imaging should be checked upon delivery for the quality of point-spread function, using fluorescent beads as the sample. The system should include both a mercury arc lamp and a 100-W quartz-halogen lamp for epi-illumination, coupled through a selection mirror to the microscope. Contrary to common practice, the most suitable lamp for fluorescence imaging of live cells is often a 100-W quartz-halogen lamp. The mercury arc lamp should consist of a well shielded housing, power supply, and power supply cable to minimize the potentially damaging electromagnetic wave during ignition. Cooled CCD cameras are used in most fluorescent imaging applications. In choosing a camera, important parameters include quantum efficiency, noise level, pixel size and full-well capacity, and scanning frequency.

The optical flow algorithm presented here is a robust method that rapidly yields a high-density field of substrate displacement vectors based on two optical images. We found that one of the limiting factors, at least for inexperienced experimentalists, is the consistency of focusing or the drift in microscope focus. However, with properly collected images the standard error of the measurement was estimated to be on the order of +/- 0.10 pixels. Finally, although the discussion has been focused on the displacement of flexible substrata, a similar method should be applicable for detecting movements on other types of images, as long as the movement involves a certain degree of local coordination.

We have described a powerful tool for the study of mechanical interactions between cells and their physical environment. Although the approach has already been used in a variety of ways to measure traction forces and to characterize active and passive responses of cultured cells to mechanical stimulation, it can be extended easily and combined with other microscopic approaches, including fluorescent analog imaging (Beningo et al., 2001), photobleaching, calcium imaging, micromanipulation, and electrophysiology. This method will be particularly useful for studying the functions of various components at focal adhesions, and the effects of mechanical forces on focal adhesion-mediated signal transduction. In addition, the method can be extended to a 3D setting, e.g., by sandwiching cultured cells between two layers of polyacrylamide to create an environment mimicking that in the tissue of a multicellular organism. Whereas chemical interactions between cells and the environment have been investigated extensively, many important questions remain as to the role of physical forces in cellular functions and the interplay between chemical and physical mechanisms of communication. The present approach, as well as other approaches capable of probing physical interactions, should fill in this important gap in the near future.

Publisher Summary The performance of optical microscopes is limited both by the aperture of the lens that causes light from a point source to spread (or diffract) over a finite volume, and by the cross-contamination of light that originates from out of focus planes. To overcome these limitations, approaches have been developed both to improve the microscope design, as exemplified by confocal scanning microscopy, and to reverse mathematically the degrading effects of the conventional microscope. This latter approach is commonly referred to as “deconvolution,” because the degradation effects of the microscope can be described mathematically as the convolution of input signals by the point spread function of the optical system. This chapter introduces the basic rationale of these two methods in languages easily understood by biologists. It also provides details for the implementation of nearest- neighbor deconvolution using readily available hardware and software. A number of deconvolution algorithms have been tested for restoring fluorescence images. The most straightforward approach—three-dimensional (3-D) inverse filtering (Agard et al ., Holmes and Liu) attempts to reverse the effects of image degradation through direct calculations. The two methods currently in wide use are constrained iterative deconvolution and nearest-neighbor deconvolution.

The technique of fluorescence immunolocalization has evolved steadily since its first application in the mid-1960s, incorporating innovations in probe chemistry, microscopy, and image detection. This chapter provides an overview of the current status of indirect immunofluorescence for those starting to use the method. It includes both general considerations from cell culture to image detection and several protocols that should serve as an entry point for this technique.

Publisher Summary This chapter focuses on the needle microinjection and scrape loading of protein molecules into cultured somatic cells. The presence of a fluorescent marker in the injection solution can greatly facilitate microinjection. It allows the examination of flow and easy identification of injected cells. It is critical to obtain an optimal shape of the microneedle by adjusting the temperature and force of the pipette puller. Each needle can be used for more than 50 cells, assuming that the solution for microinjection is reasonably clean. The volume delivered by needle microinjection is usually in the range 1 to 10% cell volume. With some practice, the variability can be maintained to within a factor of 2. A number of factors can affect the volume, including the pressure, tip size, accumulation of proteins at the tip, and presence of any air bubbles in the needle. Compared with needle microinjection, the amount of delivery varies more extensively among scrape-loaded cells. The yield of injected cells and the extent of cell damage also vary among cell types. Cell damage often manifests as the appearance of large vacuoles.

Publisher Summary This chapter describes a method for the fluorescent labeling of the myosin sulfhydryl groups with tetramethylrhodamine, a probe with optimal fluorescence properties for applications in living cells. The method of labeling applies to both skeletal and smooth muscle myosins, although the discussion will focus on the smooth muscle myosin. The method uses tetramethylrhodamine iodoacetamide (TRIA) as the reagent for attaching the tetramethylrhodamine fluorlophore to the sulfhydryl groups of the myosin molecule. The procedure is similar to that for other iodoacetamides as summarized by Reisler. However, special steps are taken to overcome the problems of the relatively low solubility of TRIA and the difficulty in removing unreacted TRIA from proteins. All the buffers should be prepared at 4° and all steps are performed at 4°. The labeled myosin should contain no detectable free dye molecules. The extent of labeling is measured by standard methods: the protein concentration is determined by the Lowry assay, the concentration of tetramethylrhodamine is determined by measuring the absorbance at 555 nm. The molar concentrations of the protein and the fluorophore are then calculated based on the molecular weight of myosin (470,000) and the extinction coefficient of bound tetramethylrhodamine 5 (47,000 M -1 cm -1 ). The labeling protocol yields typically a conjugate with four fluorophores per smooth muscle myosin molecule.

Publisher Summary This chapter describes the use of fluorescent analog cytochemistry for tracing functional protein components in living cells. Fluorescent analog cytochemistry involves preparation of fluorescently labeled cellular components, followed by the introduction of the analogs into living cells. It is found that when properly prepared, the conjugates can maintain all or most of the original properties and functions. In addition, after entering the cell, many conjugates have been observed to associate with normal physiological structures. The first step of fluorescent analog cytochemistry involves purification and fluorescent labeling of cellular components. Requirements for the methods involved, such as gel electrophoresis and column chromatography, are similar to those for a typical biochemistry laboratory. It is found that following purification of the protein under study, chemical reactions are used to couple a fluorescent probe to the protein. The optimal probe to use and the labeling condition vary with each protein, and must be determined empirically. It is suggested that fluorophores with relatively long wavelengths of excitation and emission and high quantum yields are preferable.

Publisher Summary This chapter describes the fluorescent analog cytochemistry of contractile proteins. Fluorescent analog cytochemistry is specifically designed to follow the distribution and interaction of molecular components in single living cells. The technique involves four major steps which include purification and fluorescent labeling of cellular components, biochemical and spectroscopic characterization in vitro , and incorporation into or onto living cells. The fluorescent-labeling reagents should be chosen based on several criteria. The fluorophore should absorb in the visible range, optimally 450–650 nm in wavelength, so that radiation damage to living cells and interference from autofluorescence can be minimized. The average number of fluorophores per protein molecule must be determined to allow proper evaluation of biochemical assays. The concentration of fluorophore can be determined most conveniently by light adsorption. Spectroscopic characterization is very important for the interpretation of fluorescence signals from injected cells. The fluorescence image of injected cells can be recorded using photography, and high-quality records can be obtained from nonmotile processes.

Publisher Summary This chapter discusses the microscope culture system and cell culture chambers. Keeping live, healthy cells on the microscope is crucial for many types of experiments, in particular, fluorescence microscopy of dynamic processes. It is found that although cultured cells may be observed for a short period of time without any special device, a culture system is usually necessary for observations which last for more than 10 min. The major requirements for maintaining the cell are temperature, pH, osmolarity, and nutrients. Cultured mammalian cells should generally be maintained at a temperature close to 37°C. It is observed that with the exception of temperature-sensitive mutants, most cells can tolerate a slightly lower temperature, as well as slight variations in the temperature. The required performance of a microscope culture system depends on the period of observation, the nature of the experiment, and the property of the cell. On the one hand, for short-term observations, the best design may be simply a sealed cover slip. The most sophisticated chambers incorporate mechanisms for the control of various parameters, without the need of a separate microscope incubator. It is found that for open chambers, even with an oil overlay, the pH of the bicarbonate buffer must be maintained by a continuous supply of CO 2 .