Construction of a Spink5 conditional knockout mouse model and analysis of its phenotype / 中华皮肤科杂志

Objective:To construct a serine protease inhibitor Kazal type-5 (Spink5) conditional knockout mouse model, and to identify its phenotype.Methods:B cell-specific Spink5 conditional knockout mice of genotype Mb1 cre/+Spink5 floxp/floxp were constructed by using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein 9 (Cas9) technology, and served as the knockout group. Mice of genotype Mb1 +/+Spink5 floxp/floxp served as the control group. The mice of genotype Mb1 cre/+Spink5 floxp/floxp or Mb1 +/+Spink5 floxp/floxp were sacrificed when they were 4 to 6 weeks old, splenic mononuclear cells were isolated, and B lymphocytes and non-B lymphocytes were sorted by flow cytometry and fluorescence-activated cell sorting. Genotype identification was performed by PCR, and protein expression of lymphoepithelial Kazal-type-related inhibitor (LEKTI) was determined by Western blot analysis. Skin tissues were resected from the mice, and subjected to hematoxylin-eosin staining for measuring the epidermal thickness. Immunofluorescence staining was performed to determine fluorescence intensity of LEKTI protein in the mouse skin tissues. Paired t test or two-independent-sample t test was used for comparisons between groups. Results:Genotype identification results demonstrated that the stable B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed. Western blot analysis revealed that the relative protein expression of LEKTI in the B lymphocytes in the knockout group was 0.01 ± 0.02, which was significantly lower than that in the non-B lymphocytes in the knockout group (0.66 ± 0.11, t = 9.99, P < 0.001) , and that in the B lymphocytes in the control group (1.08 ± 0.13, t = 13.78, P < 0.001) . Among 39 mice in the knockout group, 4 presented with dry skin and scattered scaly hypertrophic maculopapules. The epidermal thickness of the lesional skin tissues in the knockout group was 90.42 ± 21.31 μm, significantly higher than that of the non-lesional skin tissues in the knockout group (29.71 ± 3.63 μm, t = 5.05, P = 0.002) and that of normal skin tissues in the control group (12.42 ± 2.21 μm, t = 6.74, P < 0.001) . Immunofluorescence staining showed no significant difference in the fluorescence intensity of LEKTI protein among the lesional skin tissues (46.21 ± 1.21) , non-lesional skin tissues (46.62 ± 2.13) in the knockout group and normal skin tissues in the control group (47.69 ± 1.71, P > 0.05) . Conclusion:The B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed, which provides a basis for further exploring mechanisms underlying skin barrier defects and immune dysfunction in Netherton syndrome.

Pathophysiology of psoriasis.

Psoriasis is a chronic inflammatory papulosquamous disease characterized by multiple remissions and relapses. For long, it was believed to be primarily a disorder of keratinization. However, the successful use of traditional immunosupressants and newer immunomodulatory agents in the treatment of psoriasis led to the belief that psoriasis is primarily a disease of Th1 cell immune dysregulation. Recent developments have brought up several new findings such as the role of Th17 cells and evidence of skin barrier dsysfunction in psoriasis, akin to atopic dermatitis. The present review aims to focus on these new developments and explain the pathogenesis of psoriasis on the basis of currently available information.

Pathophysiology of psoriasis.

Psoriasis is a chronic inflammatory papulosquamous disease characterized by multiple remissions and relapses. For long, it was believed to be primarily a disorder of keratinization. However, the successful use of traditional immunosupressants and newer immunomodulatory agents in the treatment of psoriasis led to the belief that psoriasis is primarily a disease of Th1 cell immune dysregulation. Recent developments have brought up several new findings such as the role of Th17 cells and evidence of skin barrier dsysfunction in psoriasis, akin to atopic dermatitis. The present review aims to focus on these new developments and explain the pathogenesis of psoriasis on the basis of currently available information.

Clinical use of a ceramide-based moisturizer for treating dogs with atopic dermatitis

In humans, skin barrier dysfunction is thought to be responsible for enhanced penetration of allergens. Similar to conditions seen in humans, canine atopic dermatitis (CAD) is characterized by derangement of corneocytes and disorganization of intercellular lipids in the stratum corenum (SC) with decreased ceramide levels. This study was designed to evaluate the effects of a moisturizer containing ceramide on dogs with CAD. Dogs (n = 20, 3~8 years old) with mild to moderate clinical signs were recruited and applied a moisturizer containing ceramide for 4 weeks. Transepidermal water loss (TEWL), skin hydration, pruritus index for canine atopic dermatitis (PICAD) scores, and canine atopic dermatitis extent and severity index (CADESI) scores of all dogs were evaluated. Skin samples from five dogs were also examined with transmission electron microscopy (TEM) using ruthenium tetroxide. TEWL, PICAD, and CADESI values decreased (p < 0.05) and skin hydration increased dramatically over time (p < 0.05). Electron micrographs showed that the skin barrier of all five dogs was partially restored (p < 0.05). In conclusion, these results demonstrated that moisturizer containing ceramide was effective for treating skin barrier dysfunction and CAD symptoms.

Atopic Dermatitis and Skin Barrier

Atopic dermatitis (AD) is an inflammatory skin disorder characterized by itching, chronically relapsing course, typical stribution, and family history of allergies. The pathogenesis of AD is unknown but the development of AD is undoubtedly influenced by immunologic abnormality. But the broad clinical spectrum of this disease cannot be explained simply by allergy or immunological abnormalities. In recent studios revealed skin barrier dysfunctions in AD. We consider immunological abnormality and skin harrier dysfunction as two core defects of AD. The wide range of clinical manifestation of AD can be more easily comprehended by partial overlapping of these two core abnormalities. These two core abnormalities influences each other. This sequence results in a 'vicious cycle', which plays the most important role in pathogenesis of AD.