Capsaicin‐induced neurogenic inflammation in the skin in patients with symptoms induced by odorous chemicals

Background: Intradermal injection of capsaicin induces the axonal release of neuropeptides, vasodilatation and flare, e.g. neurogenic inflammation. The spatial profile of neurogenic inflammation in the skin has been studied in various experimental models. Polarization spectroscopy imaging introduced recently may be used for the quantitative assessment of the temporal profile of neurogenic inflammation expressed as erythema intensity. Purpose: In the present study, we aimed to compare capsaicin‐induced erythema intensity with the flare area in patients with symptoms induced by odorous chemicals, thereby comparing the temporal and spatial profiles of neurogenic inflammation. Methods: Sixteen patients fulfilling Cullen's criteria for multiple chemical sensitivity (MCS) and 15 eczema (EC) patients with airway symptoms elicited by odorous chemicals were compared with 29 age‐matched, healthy controls. Participants were administered two intradermal injections of capsaicin 3.3 and 33 μM. Erythema intensity was measured by polarization spectroscopy imaging and flare response was quantified by visual inspection. Results: Erythema intensity and flare area did not differ between patients and controls, and they were not correlated. Erythema intensity and flare area showed a dose‐dependent increase (P<0.05). Erythema intensity increased with age at 3.3 μM but not at 33 μM capsaicin, whereas the flare area increased with age at both concentrations (P<0.05). Conclusion: Capsaicin‐induced erythema intensity and visual flare were normal in patients with MCS and EC patients with airway symptoms from odorous chemicals. Polarized light spectroscopy was a useful method for the measurement of the rapid temporal changes in erythema of experimental reactions.     

Combined Effects of Multiple Endoplasmic Reticulum Stresses on Cytokine Secretion in Macrophage

Cells show various stress signs when they are challenged with severe physiological problems. Majority of such cellular stresses are conveyed to endoplasmic reticulum (ER) and unfolded protein response (UPR) serves as typical defense mechanism against ER stress. This study investigated an interaction between ER stress agents using macropage cell line Raw 264.7. When activated by lipopolysaccharide (LPS), the cell lines showed typical indicators of ER stress. Along with molecular chaperones, the activation process leads to the production of additional infl ammatory mediators. Following activation, the macrophage cell line was further treated with TUN and characterized in terms of chaperone expression and cytokine secretion. When treated with TUN, the activated macrophage cell leads to increased secretion of IL-6 although expression of ER stress markers, GRP94 and GRP78 increased. The secretion of cytokines continued until the addition of BFA which inhibits protein targeting from ER to Golgi. However, secretion of cytokines was ceased upon dual treatments with BFA and TG. This result strongly implies that cells may differently deal with various polypeptides depending on the urgency in cellular function under ER stress. Considering IL-6 is one of the most important signal molecules in macrophage, the molecule might be able to circumvent ER stress and UPR to reach its targeting site.     

Scintillation proximity assay in lead discovery

Background: Scintillation proximity assay (SPA) is a homogeneous scintillant bead-based platform for the measurement of biological processes and plays an important role in the identification of active chemical entities in drug discovery. Objective: The design and development of solid-phase SPA approaches are examined and compared with alternative non-radiometric fluorescence-based technologies. Methods: This review provides background on the principle of SPA and its application to biomolecular interactions from a variety of biological sources. Conclusion: The SPA approach is well suited to the demands of commercial high volume automation and assay miniaturization for target-based high-throughput screening campaigns on synthetic and natural product libraries as well as for benchtop characterization and confirmation studies. In the near future, innovations in the way SPA and fluorescence-based screening strategies are multiplexed will improve our comprehensive understanding of cellular system biology and dramatically advance the lead discovery process for the treatment of complex target-related disorders.     

Diffractive microscopy in dermatopathology

Diffraction is the process by which a beam of light is spread out as a result of passing through a narrow aperture or across an edge. Light diffraction can be produced by closing the aperture diaphragm beyond the recommended setting, by flipping the condenser cone down, or by using an opaque object such as the microscopist's hand to block the column of light and force it to bend around the edge. Any of these techniques results in greater refractility of objects in the path of the light. We studied 77 biopsy specimens from a variety of conditions selected to compare the value of diffractive microscopy, and found that it worked best in the evaluation of alopecia, tumor stroma, hemosiderin, argyria and imipramine pigmentation. In amyloidosis stained with Congo red and silica granuloma, polarized microscopy was superior to diffraction microscopy, and neither diffractive microscopy nor polarized microscopy was superior to routine light microscopy in the evaluation of melanin, chrysiasis or ochronosis.     

Regulation of immune responses to Strongyloides venezuelensis challenge after primary infection with different larvae doses

Nematode infections are generally followed by high rates of reinfection, leading to elevated prevalence in endemic areas. Therefore, the effective control of nematode infections depends on understanding the induction and regulation of protective mechanisms. However, most experimental models for protective immune response against nematodes use high parasite exposure, not always reflecting what occurs naturally in human populations. In this study, we tested whether infecting mice with different Strongyloides venezuelensis larvae loads would affect protective responses against reinfection. Interestingly, we found that a previous infection with 10–500 larvae conferred high rate of protection against reinfection with S. venezuelensis in mice, by destroying large numbers of migrating larvae. However, low‐dose priming did not abolish adult worm maturation, as detected in high‐dose primed group. Results also indicated that a previous low‐dose infection delayed the development of cellular infiltrate, while a high inoculum rapidly induced these inflammatory features. Cytokine production by splenocyte cultures of challenge infected mice demonstrated that low‐dose priming had increased production of IL‐4 and IFN‐γ, while high‐dose induced IL‐4 production but not IFN‐γ. Our data support the hypothesis that low‐dose nematode infection does not induce a polarized type‐2 immune response, allowing adult worm survival.     

RhoB induces the production of proinflammatory cytokines in TLR-triggered macrophages

Toll-like receptors (TLRs) are the primary sensors detecting conserved molecular patterns on microorganisms, thus acting as important components of innate immunity against invading pathogens. Many positive and negative regulators of TLR-triggered signaling have been identified. The Rho GTPase RhoB plays a key role in cell migration, division and polarity; however, the function and regulatory mechanisms of RhoB in TLR ligand-triggered innate immune responses remain to be investigated. Here, we report that the expression of RhoB is induced by TLR agonists (lipopolysaccharide (LPS), CpG, poly(I:C)) in macrophages. Knockdown of RhoB expression markedly decreased TLR ligand-induced activation of mitogen activated protein kinases and nuclear factor-κB (NF-κB), and the production of tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1β in macrophages stimulated with TLR ligands. Furthermore, we demonstrated that RhoB interacts with major histocompatibility complex class II (MHCII) α chain, but not β chain, in endosomes of macrophages. Knockdown of MHCII expression greatly reduced the interaction of RhoB with Btk, and attenuated the induction of NF-κB and interferon β activity by RhoB upon LPS stimulation. These findings suggest that RhoB is a positive physiological regulator of TLRs signaling via binding to MHCII in macrophages, and therefore RhoB may be a potential therapeutic target in inflammatory diseases.     

Intratumoral heterogeneity of macrophages and fibroblasts in breast cancer is associated with the morphological diversity of tumor cells and contributes to lymph node metastasis

Recent studies have highlighted the heterogeneity of the tumor microenvironment (ME) and the importance of its analysis to the understanding of its impact on clinical outcomes. In this study, we aimed to analyze the intratumoral distribution of macrophages and fibroblasts in breast cancer (BC) based on the morphological diversity of tumor cells (tubular, alveolar, solid, trabecular and discrete structures) and the clinicopathological parameters of the disease. Thirty-six patients with invasive breast carcinoma of no special type were included in the study. The distribution of macrophages and fibroblasts in the MEs of different morphological structures was assessed using laser microdissection-assisted quantitative RT-PCR analysis of marker genes and double immunofluorescence staining for the CD68, RS1, aSMA, and FAP proteins. Gene expression microarrays were used to determine the expression of genes involved in the regulation of macrophage and fibroblast phenotypes in different morphological structures. We found that different macrophage and fibroblast subpopulations were simultaneously observed in the MEs of morphologically distinct structures but that the frequency of their detection and number of cells detected varied significantly among these structures. In particular, macrophages and fibroblasts were more frequently detected in the ME of solid structures and were rarely observed in tubular structures. A high number of CD68⁺RS1⁺ macrophages in the ME of solid structures was found to be associated with an increased frequency of lymph node metastasis in luminal B HER2⁻ BC. In contrast, in luminal B HER2⁺ BC, lymph node involvement was related to the high representation of aSMA⁺FAP⁺ fibroblasts around trabecular structures. Morphologically distinct structures differed in the mechanisms regulating the macrophage and fibroblast phenotypes. The highest number of overexpressed genes controlling macrophage and fibroblast functions was observed in discrete groups of tumor cells, and the lowest number was observed in alveolar and solid structures. Taken together, our findings indicate the heterogeneous distribution of macrophages and fibroblasts in breast tumors and its close relation to the intratumoral morphological diversity of BC and contribution to lymph node metastasis.