Raman Microscopy: A Noninvasive Method to Visualize the Localizations of Biomolecules in the Cornea.

PURPOSE: In vivo and in situ visualization of biomolecules without pretreatment will be important for diagnosis and treatment of ocular disorders in the future. Recently, multiphoton microscopy, based on the nonlinear interactions between molecules and photons, has been applied to reveal the localizations of various molecules in tissues. We aimed to use multimodal multiphoton microscopy to visualize the localizations of specific biomolecules in rat corneas. METHODS: Multiphoton images of the corneas were obtained from nonlinear signals of coherent anti-Stokes Raman scattering, third-order sum frequency generation, and second-harmonic generation. RESULTS: The localizations of the adhesion complex-containing basement membrane and Bowman layer were clearly visible in the third-order sum frequency generation images. The fine structure of type I collagen was observed in the corneal stroma in the second-harmonic generation images. The localizations of lipids, proteins, and nucleic acids (DNA/RNA) was obtained in the coherent anti-Stokes Raman scattering images. CONCLUSIONS: Imaging technologies have progressed significantly and been applied in medical fields. Optical coherence tomography and confocal microscopy are widely used but do not provide information on the molecular structure of the cornea. By contrast, multiphoton microscopy provides information on the molecular structure of living tissues. Using this technique, we successfully visualized the localizations of various biomolecules including lipids, proteins, and nucleic acids in the cornea. We speculate that multiphoton microscopy will provide essential information on the physiological and pathological conditions of the cornea, as well as molecular localizations in tissues without pretreatment.

SHG-specificity of cellular Rootletin filaments enables naïve imaging with universal conservation.

Despite growing demand for truly naïve imaging, label-free observation of cilium-related structure remains challenging, and validation of the pertinent molecules is correspondingly difficult. In this study, in retinas and cultured cells, we distinctively visualized Rootletin filaments in rootlets in the second harmonic generation (SHG) channel, integrated in custom coherent nonlinear optical microscopy (CNOM) with a simple, compact, and ultra-broadband supercontinuum light source. This SHG signal was primarily detected on rootlets of connecting cilia in the retinal photoreceptor and was validated by colocalization with anti-Rootletin staining. Transfection of cells with Rootletin fragments revealed that the SHG signal can be ascribed to filaments assembled from the R234 domain, but not to cross-striations assembled from the R123 domain. Consistent with this, Rootletin-depleted cells lacked SHG signal expected as centrosome linker. As a proof of concept, we confirmed that similar fibrous SHG was observed even in unicellular ciliates. These findings have potential for broad applications in clinical diagnosis and biophysical experiments with various organisms.

The human cathelicidin LL-37 host defense peptide upregulates tight junction-related proteins and increases human epidermal keratinocyte barrier function.

Both psoriasis and atopic dermatitis (AD) are not only associated with an impaired stratum corneum barrier, but also with abnormal expression of the tight junction (TJ) proteins. Because host defense peptides, including LL-37, are overexpressed in lesional psoriatic skin but are downregulated in lesional AD skin, we hypothesized that LL-37 might regulate the TJ function in keratinocytes. We demonstrated that LL-37 selectively increased the expression of several claudins and occludin, and enhanced their membrane distribution. Furthermore, LL-37 elevated the transepithelial electrical resistance while reducing the paracellular permeability of keratinocyte layers, and this activity was weakened by the claudin inhibitor ochratoxin A. A characterization of the molecular mechanism underlying the regulation of the TJ barrier by LL-37 revealed that LL-37 induced the activation of the Rac1, atypical PKC, glycogen synthase kinase-3 and PI3K pathways, and the specific inhibition of these pathways reversed the LL-37-mediated regulation of TJ function. In addition, LL-37 enhanced the expression of differentiation markers under the control of ochratoxin A, suggesting an association between LL-37-induced TJ function and keratinocyte differentiation. These data provide novel evidence that, in addition to its antimicrobial and other immunoregulatory functions, LL-37 contributes to cutaneous immunity by strengthening the skin's barrier function.

Host defense (Antimicrobial) peptide, human ß-defensin-3, improves the function of the epithelial tight-junction barrier in human keratinocytes.

Human ß-defensins (hBDs) are host defense peptides that not only exhibit microbicidal properties but also stimulate various cellular activities, including keratinocyte proliferation, migration, and wound healing. hBDs are overexpressed in the skin in cases of psoriasis but are downregulated in atopic dermatitis skin, although both diseases are associated with stratum corneum barrier defects. Because the tight-junction (TJ) barrier is also dysfunctional in both atopic dermatitis and psoriasis patients, we hypothesized that hBDs may regulate the TJ barrier function in keratinocytes. We observed that, among the hBDs tested, only hBD-3 increased the expression of several claudins and their localization along the cell-cell borders. In addition, hBD-3 elevated the transepithelial electrical resistance and reduced the paracellular permeability of keratinocyte layers, and this effect was reversed by the claudin inhibitor ochratoxin A, CCR6 antibody, and CCR6 small interfering RNA. Moreover, hBD-3 enhanced the activation of Rac1, atypical protein kinase C, glycogen synthase kinase-3, and phosphatidylinositol 3 kinase, which are required for the hBD-3-mediated regulation of the TJ barrier function, as evidenced by the effects of their respective inhibitors. Collectively, our findings provide evidence regarding the contribution of host defense peptides to the innate immunity of skin by regulating TJ barrier function, in addition to their antimicrobial and other immunomodulatory activities.

[Acquired hemophilia A with rectus abdominis muscle and intrapelvic hematomas after aortic arch replacement].

A 60-year-old man demonstrated a bleeding tendency at enterostomy about 7 months after aortic arch replacement. Abdominal muscle hematoma and pelvic hematoma were also detected. He was diagnosed as having acquired hemophilia A based on prolonged APTT (65.9 sec), reduced FVIII activity (>1%), and the presence of FVIII inhibitor (19.5 BU/ml). Severe anemia was recognized. Recombinant activated factor FVIII (9.6 mg) was administered 25 times, combined with steroid pulse therapy. Usually postoperative acquired hemophilia is recognized in the immediate postoperative period or about 3 months later, while in this case, acquired hemophilia appeared 7 months postoperatively.