Chih-Jung Hsu

Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy

The photoaging process of facial skin is investigated by use of multiphoton fluorescence and second-harmonic generation (SHG) microscopy. We obtain the autofluorescence (AF) and SHG images of the superficial dermis from the facial skin of three patients aged 20, 40, and 70 years. The results show that areas of AF increase with age, whereas areas of SHG decrease with age. The results are consistent with the histological findings in which collagen is progressively replaced by elastic fibers. The AF and SHG changes in photoaging are quantified by a SHG to autofluorescence aging index of dermis (SAAID). Our results suggest that SAAID can be a good indicator of the severity of photoaging.

Self-assembly of dermal papilla cells into inductive spheroidal microtissues on poly(ethylene-co-vinyl alcohol) membranes for hair follicle regeneration

Self-aggregation is key to hair follicle (HF) induction ability of dermal papilla (DP) cells and neogenesis of HF can be achieved by transplanting DP microtissues. However, there is currently lack of a suitable system that allows efficient production of DP microtissues and analysis of DP self-aggregation in vitro. We demonstrate that, at a higher seeding cell density, poly(ethylene-co-vinyl alcohol) (EVAL) membranes facilitate DP self-assembly into many compact spheroidal microtissues that are able to induce new HFs. This self-assembling process is associated with an enhanced cell movement and a declined cell-substrate adhesivity on EVAL. A compromised cell growth is also revealed on EVAL. On the contrary, a more adherent surface allows faster cell expansion but maintains DP cells in a flat morphology. Dynamically, cell migration, intercellular collision and intercellular adhesion contribute to DP microtissue formation on EVAL. Our results suggest that, for large-scale production of DP microtissues for HF regeneration, an adhesive surface is needed for quick cell expansion and a biomaterial with a lower adhesivity is required for self-aggregation. In addition, this system can be a model for investigation of DP self-aggregation in vitro.

Prediction of heat-induced collagen shrinkage by use of second harmonic generation microscopy

Collagen shrinkage associated with denaturation from thermal treatment has a number of important clinical applications. However, individualized treatment is hindered by the lack of reliable noninvasive methods to monitor the process of collagen denaturation. We investigate the serial changes of collagen denaturation from thermal treatment of rat tail tendons at 58 degrees C by use of second harmonic generation (SHG) microscopy. We find that rat tail tendon shrinks progressively from 0 to 9 min of thermal treatment, and remains unchanged in length upon further thermal treatment. The SHG intensity also decreases from 0 to 9 min of thermal treatment and becomes barely detectable from further thermal treatment. Collagen shrinkage and the SHG intensity are well correlated in a linear model. In addition, SHG imaging reveals a tiger-tail-like pattern of collagen denaturation. The bands of denatured collagen progressively widen from increased thermal treatment and completely replace the adjacent bands of normal collagen after 9 min of thermal treatment. Our results show that collagen denaturation in rat tail tendon from thermal treatment is inhomogeneous, and that SHG intensity can be used to predict the degree of thermally induced collagen shrinkage. With additional development, this approach has the potential to be used in biomedical applications.

Epicutaneous sensitization with a protein antigen induces Th17 cells

BACKGROUND Th17 is a newly identified effector T cell lineage which plays a central role in many human inflammatory diseases and experimental animal models. Epicutaneous sensitization with a protein antigen has been proven to induce a Th2-predominant immune response and lead to development of atopic diseases in a murine protein-patch model. OBJECTIVE We sought to assess the generation of Th17 cells in epicutaneous sensitization with a protein antigen and its regulation by environmental elements and genetic background. METHODS BALB/c, C57BL/6, and DO11.10 mice were epicutaneously immunized by patch application of the following: ovalbumin alone, or co-administration of one of TLR ligands, irritant, hapten or superantigens. IL-17 and IL-22 contents in supernatants of in vitro reactivation culture of lymph nodes cells were determined by ELISA. Frequency of IL-17-secreting CD4 T cells was measured by ELISPOT. RESULTS Small but significant amounts of IL-17 and IL-22 could be detected in supernatants of in vitro reactivation culture of lymph nodes cells of mice receiving patch application of ovalbumin. ELISPOT assay for IL-17 also revealed low frequency of IL-17-secreting CD4 T cells in lymph nodes cells in ovalbumin group. All TLR ligands tested including agonists for TLR2, TLR3, TLR4, TLR5, TLR7 and TLR9 as well as many environmental elements including irritant, hapten and superantigen could further promote the generation of Th17 cells. In addition, C57BL/6 mice generate less Th17 cells than BALB/c mice in epicutaneous sensitization. CONCLUSION This study demonstrates Th17 generation and its regulation by environmental elements and genetic background to a protein antigen by epicutaneous route.

Erythema ab igne caused by frequent hot bathing.

An 18-year-old woman, a high school student, had had asymptomatic hyperpigmented lesions on her feet, lower legs, upper thighs and buttocks for 3 months. She was otherwise healthy and was not on medication. She did not apply lotions or medicaments to her skin. She also denied applying hot bottles, heating pads or heating blankets to her skin or using other external heating devices. Physical examination revealed symmetrically distributed reticular hyperpigmentation on a faint erythematous background spanning from the buttocks down to the mid-thighs and from the mid-lower legs down to the feet with sparing of the knees and the surrounding skin (Fig. 1). On further questioning, she reported having started to take a hot bath for 60 to 90 min in a bathtub nightly 6 months previously. The bathtub was not deep, so her knees and chest remained above the water when she was bathing, and distribution of the skin lesions corresponded to the areas that were immersed in the water. Skin biopsy specimens were taken from a lesion on her left buttock and left ankle. The microscopic ® ndings of the two specimens were similar, showing hyperkeratosis, epidermal thinning with loss of rete ridges, basal hyperpigmentation and dermal oedema. Slight pigment incontinence and a mild super® cial perivascular lymphocytic in® ltrate were also revealed. Stain for elastin showed an increased amount of elastic ® bres in the upper and middle dermis. Based on the clinical manifestations and microscopic ® ndings, a diagnosis of erythema ab igne was made. The patient was instructed not to take hot baths. Her skin lesions gradually faded during the following months.

Antigen-driven bystander effect accelerates epicutaneous sensitization with a new protein allergen

Exposure to protein allergen epicutaneously, inducing a Th2-dominant immune response, sensitizes the host to the development of atopic disease. Antigen-driven bystander effect demonstrates that polarized T cells could instruct naïve T cells to differentiate into T cells with similar phenotype. In this study, we aimed to determine the contribution of antigen-driven bystander effect on epicutaneous sensitization with a newly introduced protein allergen. BALB/c mice were immunized intraperitoneally with BSA emulsified in alum, known to induce a Th2 response, three weeks before given BSA and OVA epicutaneously. Lymph node cells from these mice restimulated with OVA secreted higher levels IL-4, IL-5 and IL-13 as compared with cells from mice without BSA immunization. In addition, BALB/c mice immunized subcutaneously with BSA emulsified in complete Freunds adjuvant, known to induce a Th1-predominant response, also induced higher Th1 as well as Th2 cytokine response when restimulated with OVA as compared with mice without immunization. We demonstrated that subcutaneous immunization with BSA in CFA induced Th2 as well as Th1 response. The threshold of epicutaneous sensitization to OVA was also reduced, possibly due to increased expressions of IL-4 and IL-10 in the draining lymph nodes during the early phase of sensitization. In conclusion, antigen-driven bystander effect, whether it is of Th1- or Th2-predominant nature, can accelerate epicutaneous sensitization by a newly introduced protein allergen. These results provide a possible explanation for mono- to poly-sensitization spread commonly observed in atopic children.

Cross‐priming with an epicutaneously introduced soluble protein antigen generates Tc1 cells

Epicutaneous sensitization with a protein antigen was demonstrated to induce a predominant type 2 CD4 T cell response with high IgE production in mice. On the other hand, its CD8 T cell responses have not been addressed probably partly because of the generally accepted concept that cross‐priming of soluble protein is an inefficient process. Here, we used an established patch‐applied murine model to demonstrate that cross‐priming with an epicutaneously introduced soluble protein antigen, though inefficient, generated mainly Tc1 cells, but not Tc2 cells. In the presence of an irritant or hapten, the efficiency of this cross‐priming process could be enhanced and more Tc1 cells were generated. CpG oligonucleotides also promote the generation of Tc1 cells. In contrast, lipopolysaccharide and poly (inosinic‐cytidylic) acid [poly (I:C)] have no effect. Together, these results provide supportive evidence of the epicutaneous sensitization of human cutaneous lymphocyte‐associated antigen‐positive CD8 T cells found in the peripheral blood or tissues of patients. The surprising observation of the type 1 character of the generated CD8 T cells will also help us to better understand the complicated pathogenesis of atopic and cutaneous inflammatory diseases.

Low-Energy Visible Light Irradiation Modulates Immune Responses Induced by Epicutaneous Sensitization with Protein Antigen

Epicutaneous sensitization has been an important route for protein allergen sensitization in atopic disease. Although the skin is irradiated by sunlight daily, the influence of visible light on epicutaneous sensitization has not been explored. In this study, by using a well-established murine protein-patch model, we show that low-energy visible light (LEVL) irradiation could differentially modulate the predominant Th2 immune response induced by epicutaneous sensitization with protein antigen. When the induced Th2 response was strong, as usually observed in BALB/c mice, LEVL irradiation suppressed the response. In contrast, LEVL irradiation enhanced the weaker Th2 response in C57BL/6 mice. Increased IL-18 and decreased TGF-beta expression in draining lymph nodes after LEVL irradiation was observed in BALB/c mice, but not in C57BL/6 mice. LEVL irradiation also enhanced IL-18 expression in skin and reduced the downregulation of CD24 expression on epidermal Langerhans cells in draining lymph nodes of BALB/c mice. Collectively, these results provide evidence for immunomodulatory effects of LEVL irradiation and will help us develop a useful strategy for prevention of allergen sensitization.

Monitoring photoaging by use of multiphoton fluorescence and second harmonic generation microscopy

It is a field of great interest to develop therapies to rejuvenate photoaged skin. However, the treatment response can not be ideally determined due to lack of a reliable non-invasive method to quantify photoaging. In this study, the photoaging process of skin is investigated by use of a multiphoton fluorescence and second harmonic generation microscopy. We obtain the autofluorescence and second harmonic generation images of superficial dermis from facial skin of individuals of different ages. The results show that autofluorescence signals increase with age while second harmonic generation signals decrease with age. The results are consistent with the histological findings in which collagen is progressively replaced by elastic fibers. In the case of severe photoaging, solar elastosis can be clearly demonstrated by the presence of thick curvy autofluorescent materials in the superficial dermis. We propose a second harmonic generation to autofluorescence aging index of dermis to quantify the photoaging changes. This index is shown to be a good indicator of photoaging. Our results suggest that multiphoton fluorescence and second harmonic generation microscopy can be developed into a non-invasive imaging modelity for the clinical evaluation of photoaging.

Efficacy and safety of secukinumab in Taiwanese patients with moderate to severe plaque psoriasis: Subanalysis from ERASURE phase III study.

The efficacy and safety of secukinumab, a fully human anti‐interleukin‐17A monoclonal antibody, has been evaluated for moderate to severe plaque psoriasis in global trials which have included a low proportion of Asian subjects. We analyzed the efficacy and safety of secukinumab in Taiwanese patients in a phase III global clinical trial (ERASURE). Fifty‐one Taiwanese patients were randomized into s.c. placebo, 150 and 300 mg secukinumab treatment groups. The proportions of patients who achieved 75% or more improvement in Psoriasis Area and Severity Index (PASI‐75) at week 12 were 87.5% with 300 mg secukinumab, 70% with 150 mg secukinumab, 0% with placebo. Of the patients receiving 300 mg secukinumab, 68.8% achieved PASI‐90 at week 12. Analysis of overall patients receiving 300 mg secukinumab for 12 weeks showed that the proportion of PASI‐75 responders was less in patients with body mass index of 25 or more than less than 25. During the entire 52 weeks, the incidence of adverse events (AE) was consistent with the overall population in ERASURE. The most common AE (cases/per 100 patient‐year) during the entire treatment period were upper respiratory tract infection and pruritus. The duration of upper respiratory tract infection per 100 patient‐year was approximately 399 days in placebo, 1261 days in 150 mg secukinumab and 1805 days in 300 mg secukinumab. The safety and efficacy of secukinumab in Taiwanese patients was compatible with the global phase III study in the treatment of moderate to severe plaque psoriasis.