Lympho-epithelial Kazal-type Inhibitor Research Articles

MiR-375-3p alleviates the severity of inflammation through targeting YAP1/LEKTI pathway in HaCaT cells.

Atopic dermatitis (AD) is a relapsing inflammatory skin disease with a complicated pathogenesis. This study aimed to investigate whether miR-375-3p could regulate AD through the Yes-associated protein 1 (YAP1) pathway. In this study, inflammatory response was induced by TNF-α and IFN-γ administration in HaCaT cells. We found that viability and inflammatory factor release, including interleukin-1β (IL-1β) and IL-6, were negatively related to miR-375-3p expression in HaCaT cells. We also found that YAP1 overexpression down-regulated lympho-epithelial Kazal type inhibitor (LEKTI) levels and aggravated viability and inflammation in TNF-α and IFN-γ-treated HaCaT cells. Dual-luciferase reporter assay proved the targeted binding of miR-375-3p and YAP1 3'-UTR. Additionally, the protective effect of miR-375-3p on inflammatory response in TNF-α and IFN-γ-treated HaCaT cells could be impeded by YAP1 overexpression. Collectively, our results suggested that miR-375-3p could modulate HaCaT cell viability and inflammation through the YAP1/LEKTI pathway.

256 Treatment of Netherton syndrome with dupilumab

Netherton syndrome (NS) is a rare autosomal-recessive disease that is caused by loss-of function mutations in the SPINK 5 gene encoding for the serine protease inhibitor lympho-epithelial Kazal type inhibitor (LEKTI). LEKTI opposes the function of several epidermal serine proteases including kallikrein 5 (KLK5), kallikrein 7 (KLK7) and kallikrein 14 (KLK14). This results in an activation of the PAR2-TSLP axis and an increase of other Th2 polarizing mediators including CCL-17 and CCL-22. This presumably spurs a Th-2 response consequently leading to increased IgE levels. In NS effective treatment options are missing. In contrast, in atopic dermatitis treatment with dupilumab an antibody directed against the alpha unit of the IL-4 receptor leads to a remarkable success in controlling disease activity which is reflected in decreased IgE levels. Thus, dupilumab represents a worthwhile treatment strategy in NS. Three adult patients with genetically confirmed NS were individually treated with dupilumab 300mg injections every other week for 32 weeks. EASI scores at baseline were 30.0, 29.0 and 18.2. Clinical improvement was observed as early as at week 8 leading to continuously improved EASI scores (w32: -55.82%+/-16.69). In line, IGA scores enhanced in all three patients. DLQI clearly improved with ongoing dupilumab treatment. Serum IgE levels declined steadily (w32: -60.93%+/- 8.57). In contrast, LDH serum levels and blood eosinophil count were unchanged. Further analyses of serum cytokines are underway. Decrease of serum IgE levels clearly correlated with both EASI and DLQI reduction intra- and interindividually. Similarly, EASI improvement correlated with DLQI reduction. In summary, these data for the first time shows that dupilumab is effective in NS patients.

TNF-α and IL-17A induce the expression of lympho-epithelial Kazal-type inhibitor in epidermal keratinocytes

BackgroundSerine proteases have important roles in skin barrier function and desquamation, and the aberrant expression or the dysfunction of serine proteases is associated with the pathogenesis of skin diseases. Serine protease activities are tightly regulated by serine proteases such as kallikrein-related peptidases (KLKs) and serine protease inhibitors such as lympho-epithelial Kazal-type related inhibitor (LEKTI). For a better understating of diseases’ pathogenesis, the regulation mechanism of serine proteases and the inhibitors’ expression in epidermal keratinocytes must be clarified. ObjectivesTo investigate the effects of the cytokines on the expression of LEKTI in epidermal keratinocytes. MethodsNormal human epidermal keratinocytes (NHEKs) were stimulated with panels of inflammatory cytokines. The expression of serine protease inhibitors was analyzed using quantitative real-time PCR and ELISA. LEKTI expression in normal human skin and lesions from psoriasis or atopic dermatitis (AD) were analyzed by immunohistochemically and tape-stripping. Trypsin- and chymotrypsin-like serine protease activities in culture supernatants were measured by using specific substrates. ResultsTNF-α and IL-17A significantly induced the expression of LEKTI in NHEKs. The immunohistochemical and tape-stripping analysis revealed that psoriatic skin lesions had higher LEKTI expression compared to normal skin and AD lesions. Trypsin- and chymotrypsin-like protease activities in the culture media were upregulated 3–5 days later but attenuated 6–7 days later period by these cytokines. ConclusionsIn epidermal keratinocytes, the Th1&Th17 cytokines TNF-α and IL-17A induce the expression of serine protease inhibitor LEKTI, and it might occur to suppress the increase in the serine protease activities under inflammation.

286 SerpinB12 is an intrinsic mesotrypsin inhibitor regulating corneocyte desquamation and denucleation

It is suggested that kallikrein-related peptidases (KLKs) are key players in corneocyte desquamation and this process is regulated by lympho-epithelial Kazal-type inhibitor (LEKTI). However, it is unclear how these proteases are activated and how activated KLKs are released from LEKTI in the upper cornified layer. Previously we reported cloning of a new PRSS3 gene product, keratinocyte-specific mesotrypsin from the cDNA library. It has been reported that mesotrypsin is insusceptible to intrinsic inhibitors. Moreover, we found that mesotrypsin effectively activated pro-KLK5 and pro-KLK7. Although LEKTI strongly inhibited KLKs, mesotrypsin was not suppressed by LEKTI but rather degraded them. Surprisingly, we isolated a novel inhibitor and identified it as serpinB12 (Another name is YUKOPIN). Thin skin, such as the eyelid, showed strong mesotrypsin staining, while serpinB12 staining was barely detectable. In contrast, footpad skin, which has a thick cornified layer, showed strong serpinB12 staining at the granular layer. We constructed skin equivalent models, in which serpinB12 was over-expressed or knocked-down. Cornified layers were increased and the epidermis became thicker in serpinB12 over-expression. When serpinB12 was down-regulated, the epidermal structure became very thin. Furthermore, we found that excessive over-expression of serpinB12 induced parakeratosis. Immunohistochemical study showed that serpinB12 was expressed in parakeratic area of diseased skin, such as psoriasis and atopic dermatitis. Our findings provided a new insight into the desquamation mechanisms, in which mesotrypsin and serpinB12 play critical roles. Moreover, our results suggest that serpinB12 would be involved in controlling the thickness of stratum corneum through mesotrypsin regulation, and excessive serpinB12 expression might be the cause of parakeratotic disease condition.

Toll-like receptor signaling induces the expression of lympho-epithelial Kazal-type inhibitor in epidermal keratinocytes

BackgroundLympho-epithelial Kazal-type inhibitor (LEKTI) tightly controls the activities of serine proteases such as kallikrein-related peptidase (KLK) 5 and KLK7 in the epidermis. LEKTI is known to be an essential molecule for the epidermal skin barrier, as demonstrated by SPINK5 nonsense mutation, which results in Netherton syndrome. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns or damage-associated molecular patterns and produce inflammatory cytokines, chemokines, and antimicrobial peptides. However, the effect of TLR signaling on the expression of LEKTI is not clear. ObjectiveTo investigate whether TLR signaling can affect expression of LEKTI in epidermal keratinocytes. MethodsWe stimulated a panel of TLR ligands and investigated the expression of LEKTI in normal human epidermal keratinocytes (NHEKs). We further measured trypsin or chymotrypsin-like serine protease activity in NHEK cultured media under stimulation with TLR3 ligand, poly (I:C). Immunostaining for LEKTI was performed using skin samples from skin infectious diseases. ResultsTLR1/2, 3, 5, and 2/6 ligands induced the expression of LEKTI in NHEKs. The trypsin or chymotrypsin-like serine protease activity in NHEKs was up-regulated with the stimulation of poly (I:C). The gene expressions of KLK6, KLK10, KLK11, and KLK13 were also increased by poly (I:C). An immunohistochemical analysis demonstrated that the expression of LEKTI was up-regulated in the lesions of varicella, pyoderma, and rosacea. ConclusionsTLR signaling induces the expression of LEKTI in epidermal keratinocytes, which might contribute to the control of aberrant serine protease activities in inflammatory skin diseases.

Homologous Lympho-Epithelial Kazal-type Inhibitor Domains Delay Blood Coagulation by Inhibiting Factor X and XI with Differential Specificity

Despite being initially identified in the blood filtrate, LEKTI is a 15-domain Kazal-type inhibitor mostly known in the regulation of skin desquamation. In the current study, screening of serine proteases in blood coagulation cascade showed that LEKTI domain 4 has inhibitory activity toward only FXIa, whereas LEKTI domain 6 inhibits both FXIa and FXaB (bovine FXa). Nuclear magnetic resonance structural and dynamic experiments plus molecular dynamics simulation revealed that LEKTI domain 4 has enhanced backbone flexibility at the reactive-site loop. A model of the LEKTI-protease complex revealed that FXaB has a narrower S4 pocket compared with FXIa and hence prefers only small side-chain residues at the P4 position, such as Ala in LEKTI domain 6. Mutational studies combined with a molecular complex model suggest that both a more flexible reactive-site loop and a bulky residue at the P4 position make LEKTI domain 4 a weaker but highly selective inhibitor of FXIa.

Expression of LEKTI Correlates With PNI and LVI In SCC of the Oral Tongue and Mechanism of LEKTI Loss in HNSCC

Expression of Lympho-Epithelial Kazal-Type-Inhibitor (LEKTI), a broad spectrum protease inhibitor, is dysregulated in head and neck squamous cell carcinomas (HNSCC) and HNSCC cell lines. Here, we investigated expression of LEKTI in primary tumor specimens of 81 patients with SCC of the oral tongue in correlation with pathologic findings and clinical outcomes. IHC analyses have shown that LEKTI expression is negative in 31, intermediate in 44, and strong in 6 patients. Correlative analyses between LEKTI expression and perineural Invasion (PNI) and lymphovascular invasion (LVI) demonstrated that the relative risk of PNI and LVI were 3.2 (95% CI, 1.2 to 8.9, p = 0.007 by Chi Square Test) and 6.0 (95% CI 1.2 to 40.8, p = 0.01 by Fisher’s Exact Test) respectively in patients with LEKTI-negative tumors compared to those with LEKTI-positive tumors. Kaplan-Meier estimates showed that patients with LEKTI-negative expression had a 20% increased risk of disease recurrence (HR 1.19 and 95% CI 0.61 to 2.33, p = 0.23 by Log rank test) and an 80% increased risk of death from all causes (HR 1.78 and 95% CI 0.34 to 9.41, p = 0.48 by Log rank test). Analysis of the covariates for disease recurrence and death in tongue cohort found no significant differences in age, gender, T-stage, grade, N-stage, and postoperative treatment between patients with LEKTI-negative and LEKTI-positive tumors. Further, we present evidence that transcriptional regulation is a very likely mechanism accounting for loss of LEKTI mRNA and protein from HNSCC. These data confirm our previous in vitro and orthotopic model of tongue cancer findings and shed new light on mechanisms of PNI and LVI in HNSCC.

Molecular and Biochemical Properties of Lympho-Epithelial Kazal-Type-Inhibitor (LEKTI)

Here, we review the literature on the molecular and biochemical properties of Lympho-Epithelial Kazal-Type-Inhibitor (LEKTI). Serine Protease Inhibitor Kazal- type 5 (SPINK5) gene encodes three different LEKTI isoforms. These isoforms are organized into a typical 15, longer than 15 and shorter than 15 inhibitory domains consisting of 1064, 1094 and 916 residues respectively. LEKTI isoforms synthesized as pro-LEKTI proteins are processed intracellular and secreted as bioactive LEKTI fragments into blood. LEKTI potently inhibits activity of plasmin, subtilisin A, cathepsin G, elastase, trypsin, kallikrein (KLK) 5, KLK6, KLK7, KLK13, and KLK14 to varied extents. Mutations in SPINK5 gene resulting in decreased LEKTI function and increased KLK activity causes Netherton syndrome (NS). Low SPINK5 expression/LEKTI function is also associated with Head and Neck Squamous Cell carcinoma (HNSCC), chronic rhinosinusitis and asthma. Thus restoring SPINK5 expression/LEKTI function in NS, HNSCC and asthma holds therapeutic promise.

LEKTI, A Physiological Inhibitor of Multiple Serine Proteinases, Suppresses Perineural and Lymphovascular Invasion of Human Head and Neck Cancer Cells in A Mouse Orthotopic Model

Serine Protease Inhibitor Kazal-type 5 (SPINK5) gene encodes 3 different LymphoEpithelial Kazal-Type-Inhibitor (LEKTI) isoforms. We identified and cloned LEKTI by its constitutive expression in normal oral mucosa and its loss of expression in matched tumor specimens of patients with head and neck squamous cell carcinoma (HNSCC). Stable re-expression of LEKTI in HNSCC OSC19 cells resulted in reduced migration and invasionand enhanced adhesion on variety of ECM substrates in vitrowith a concomitant reduction inexpression of endogenous MMP-14, MMP-8, KLK5, and ADAM8. Here, we sought to determine the consequences of LEKTI re-expression on the in vivo changes in the tumor growth and invasion using an orthotopic model of tongue cancer. In the tongue tumors of mice, lymphovascular invasion or perineural spread was found in 100% of tumors derived from parental cell lines but was almost totally absent in all tumors derived from LEKTI-expressing clones. Our work suggests that loss of LEKTI expression in primary tumors might correlate with aggressive biologic behavior and restoration of LEKTI expression by pharmacologic means might be beneficial for patients with HNSCC.

Netherton syndrome: defective kallikrein inhibition in the skin leads to skin inflammation and allergy.

Netherton syndrome (NS) is an orphan genetic skin disease with a profound skin barrier defect and severe allergic manifestations. NS is caused by loss of function mutations in SPINK5 encoding lympho-epithelial Kazal-type inhibitor (LEKTI), a secreted multi-domain serine protease inhibitor expressed in stratified epithelia. Studies in mouse models and in NS patients have established that unopposed kallikrein 5 activity triggers stratum corneum detachment and activates PAR-2 signaling, leading to the autonomous production of pro-allergic and pro-inflammatory mediators. This emerging knowledge on NS pathogenesis has highlighted a central role for protease regulation in skin homeostasis but also in the complexity of the disease, and holds the promise of new specific treatments.

LEKTI, A Physiological Inhibitor of Multiple Serine Proteinases, Blocks Migration and Invasion of Head and Neck Squamous Cell Carcinoma (HNSCC) Cells

Serine Protease Inhibitor Kazal-type 5 (SPINK5) gene encodes 3 different Lympho- Epithelial Kazal-Type-Inhibitor (LEKTI) isoforms which are organized into longer than 15, 15, and 13 inhibitory domains. We identified LEKTI by its constitutive expression in normal oral mucosa and lost or down regulated expression in matched tumor specimens of patients with head and neck squamous cell carcinoma (HNSCC). Previously, we showed that recombinant full-length LEKTI and rLEKTI fragments inhibit the activity of plasmin, subtilisin A, cathepsin G, neutrophil elastase, trypsin, caspase 14, and kallikreins (KLK) 5, 6, 7, 13, and 14 to varied extents. Here, we show that LEKTI protein is absent in HNSCC OSC19, Tu138, Tu177, and UMSCC1 lines. We then determined the consequences of LEKTI re-expression on migration and invasion, adhesion and gene expression profile of HNSCCOSC19 and UMSCC1 lines. We demonstrate that LEKTI expressing OSC19 and UMSCC1 clones show markedly reduced migration and invasion. Moreover, LEKTI expressing OSC19 clones show striking morphological changes and enhanced adhesionon type I, III, IV, and V collagens, fibronectin, and laminin5.In addition, we show that exogenous r-LEKTI blocks migration of OSC19-parental cells in a dose and time dependent manner. Microarray analysis identified 186 genes which are differentially regulated in both OSC19 LEKTI clones. MMP-14, KLK5, and ADAM8 are down regulated while MMP-3, LEKTI, DSC2 and DSC3are up-regulated in OSC19 LEKTI clones. RT-PCR and Western blot results confirmed microarray results for MMP-14 and MMP-3in OSC19 LEKTI clones. In addition we discover that MMP-9 protein expression and pro-MMP-9 activity are severely reduced in LEKTI expressing clones as shown by WB and zymogram. Together, this work provides mechanistic insights into how loss of LEKTI protein expression promotes an invasive phenotype in HNSCC tumors.

Keratinocyte-Specific Mesotrypsin Contributes to the Desquamation Process via Kallikrein Activation and LEKTI Degradation

Kallikrein-related peptidases (KLKs) have critical roles in corneocyte desquamation and are regulated by lymphoepithelial Kazal-type inhibitor (LEKTI). However, it is unclear how these proteases are activated and how activated KLKs are released from LEKTI in the upper cornified layer. Recently, we reported cloning of a PRSS3 gene product, keratinocyte-specific mesotrypsin, from a cDNA library. We hypothesized that mesotrypsin is involved in the desquamation process, and the aim of the present study was to test this idea by examining the effects of mesotrypsin on representative desquamation-related enzymes pro-KLK5 and pro-KLK7. Incubation of mesotrypsin and these zymogens resulted in generation of the active forms. KLK activities were effectively inhibited by recombinant LEKTI domains D2, D2-5, D2-6, D2-7, D5, D6, D6-9, D7, D7-9, and D10-15, whereas mesotrypsin activity was not susceptible to these domains, and in fact degraded them. Immunoelectron microscopy demonstrated that mesotrypsin was localized in the cytoplasm of granular cells and intercellular spaces of the cornified layer. Proximity ligation assay showed close association between mesotrypsin and KLKs in the granular to cornified layers. Age-dependency analysis revealed that mesotrypsin was markedly downregulated in corneocyte extract from donors in their sixties, compared with younger donors. Collectively, our findings suggest that mesotrypsin contributes to the desquamation process by activating KLKs and degrading the intrinsic KLKs' inhibitor LEKTI.

The Th2 cytokine, interleukin‐4, abrogates the cohesion of normal stratum corneum in mice: implications for pathogenesis of atopic dermatitis

There is mounting evidence that Th2 cytokines adversely affect skin barrier functions and contribute to the pathogenesis of atopic dermatitis (AD). AD is also characterized by abnormal cohesion in the stratum corneum (SC). However, the contribution of Th2 cytokines to this abnormality remains unknown. This study examined the effects of IL-4, a prototypic Th2 cytokine, on the cohesion of the SC. Structural and physiological assessments revealed that repeated intradermal injections of IL-4 compromised the cohesion of the SC of normal hairless mice. Two potential mechanisms were explored to account for the altered cohesion. First, IL-4 decreased the amount of corneodesmosomes and down-regulated the expression of desmoglein 1, but not of corneodesmosin (CDSN) or loricrin expression, in murine skin and in cultured human keratinocytes (KC). IL-4 did not affect the skin surface pH, and in situ zymography revealed no net change in total serine protease activity in the IL-4-treated SC. Yet, IL-4 enhanced expression of kallikrein (KLK)7, while simultaneously down-regulating KLK5 and KLK14. Finally, IL-4 did not alter the expression of the lympho-epithelial Kazal-type inhibitor (LEKTI) in KC. This study suggests that IL-4 abrogates the cohesion of SC primarily by reducing epidermal differentiation.

Circumscribed palmo‐plantar hypokeratosis: a disease of desquamation? Immunohistological study of five cases and literature review

Circumscribed palmoplantar hypokeratosis (CPH) is a recently recognized, rarely reported dermatosis that shows characteristic clinicopathological features; however, its pathogenesis remains unknown. The aim of this study was to get further insight into the pathogenesis of CPH. An immunohistological study was performed on five cases of CPH to investigate the expression of several epidermal proliferation and differentiation proteins, with emphasis on those involved in corneocyte desquamation [including corneodesmosin, kallikrein 5 and lympho-epithelial Kazal type inhibitor (LEKTI)]. In three of five cases, a decreased expression of LEKTI, corneodesmosin and filaggrin was found, along with an increased expression of kallikrein 5 and keratin 6. The expression of several other antigens (including involucrin, Ki67, p63, CD138/syndecan I, EGF-R) did not present a consistently different pattern as compared with the unaffected epidermis. The immunohistopathologic features of CPH suggest that an altered (accelerated) corneocyte desquamation process could be the main pathological mechanism underlying the development of lesions.

Isolation of SPINK6 in Human Skin: SELECTIVE INHIBITOR OF KALLIKREIN-RELATED PEPTIDASES

Kallikrein-related peptidases (KLKs) play a central role in skin desquamation. They are tightly controlled by specific inhibitors, including the lymphoepithelial Kazal-type inhibitor (LEKTI) encoded by SPINK5 and LEKTI-2 encoded by SPINK9. Herein, we identify SPINK6 as a selective inhibitor of KLKs in the skin. Unlike LEKTI but similar to LEKTI-2, SPINK6 possesses only one typical Kazal domain. Its mRNA was detected to be expressed at low levels in several tissues and was induced during keratinocyte differentiation. Natural SPINK6 was purified from human plantar stratum corneum extracts. Immunohistochemical analyses revealed SPINK6 expression in the stratum granulosum of human skin at various anatomical localizations and in the skin appendages, including sebaceous glands and sweat glands. SPINK6 expression was decreased in lesions of atopic dermatitis. Using KLK5, KLK7, KLK8, KLK14, thrombin, trypsin, plasmin, matriptase, prostasin, mast cell chymase, cathepsin G, neutrophil elastase, and chymotrypsin, inhibition with recombinant SPINK6 was detected only for KLK5, KLK7, and KLK14, with apparent K(i) values of 1.33, 1070, and 0.5 nm, respectively. SPINK6 inhibited desquamation of human plantar callus in an ex vivo model. Our findings suggest that SPINK6 plays a role in modulating the activity of KLKs in human skin. A selective inhibition of KLKs by SPINK6 might have therapeutic potential when KLK activity is elevated.

Therapeutic Implications of a Barrier-based Pathogenesis of Atopic Dermatitis

In this review, I first provide relevant background information about normal epidermal barrier structure and function. I then update recent information about how inherited defects in either filaggrin and/or in the serine protease inhibitor, lymphoepithelial Kazal-type inhibitor 1, converge to stimulate the development of atopic dermatitis (AD). Next I explain the multiple mechanisms whereby a primary barrier abnormality in AD can lead to inflammation. Furthermore, I explore how certain acquired stressors, such as a reduced external humidity, high pH soaps/surfactants, psychological stress, as well as secondary Staphylococcus aureus infections initiate or further aggravate AD. Finally, and most importantly, I compare various therapeutic paradigms for AD, highlighting the risks and benefits of glucocorticoids and immunomodulators vs. corrective, lipid replacement therapy.

Identification of Lympho-Epithelial Kazal-Type Inhibitor 2 in Human Skin as a Kallikrein-Related Peptidase 5-Specific Protease Inhibitor

Kallikreins-related peptidases (KLKs) are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI). Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink)5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5.

Atopic dermatitis: Therapeutic concepts evolving from new pathophysiologic insights

Recent insights into the relevance of the epidermal barrier function and its interaction with components of the innate and adaptive immune responses in patients with atopic dermatitis (AD) give rise to a number of novel potential treatment options. In particular, the identification of loss-of-function mutations in the barrier protein filaggrin and of a diminished expression of certain antimicrobial peptides in AD skin stimulates new concepts to think beyond the T(H)1/T(H)2 paradigm. This review will focus on these most recent discoveries and will discuss new and corresponding proof-of-concept trials in patients with AD. It will further speculate on novel ways to restore the homeostasis among the 3 major components in AD skin suspected to be clinically relevant.

Expression of LEKTI correlates with perineural invasion in oral cavity carcinoma

6036 Background: Invasion may result from imbalance of proteinase enzymes and their inhibitors that favors degradation of extracellular matrix (ECM). Lymphoepithelial kazal-type inhibitor (LEKTI) is a serine proteinases inhibitor we identified by constitutive expression in normal oral mucosa and lost or downregulated expression in patients with head and neck squamous cell carcinoma (HNSCC). Previously, we found that expression of LEKTI in HNSCC cell lines regulates the expression and secretion of matrix metalloproteinases critical to ECM degradation. Furthermore, using an orthotopic mouse model, we found increased perineural invasion (PNI) in tongue tumors derived from OSC- 19 cells compared to those derived from clones expressing LEKTI. We hypothesized that loss of LEKTI expression in primary tumors correlates with aggressive biologic behavior in patients with HNSCC. Methods: We assembled a retrospective, inception cohort of 81 consecutive, previously untreated patients who underwent surgery as initial treatment for SCC of the oral tongue with at least 18 months of follow-up. Primary tumor specimens were recut and stained with a purified mouse anti-LEKTI monoclonal antibody. Slides were reviewed and scored by two independent investigators. Surgical pathology reports were reviewed for covariate and clinical follow-up data. Time-to-event analysis was performed by Kaplan-Meier method for patients stratified by LEKTI-staining pattern. Results: LEKTI expression was negative in 31, intermediate in 44, and strong in 6 patients. PNI was present in 12 of 31 patients with LEKTI-negative tumors, in contrast to 6 of 50 patients with LEKTI-positive tumors. Therefore, the relative risk of PNI was 3.2 (95% CI from 1.2 to 8.9) in patients with LEKTI-negative tumors compared to those LEKTI-positive tumors (p = 0.007 by Chi Square Test). Patients with LEKTI-negative expression had a 20% increased risk of disease recurrence (HR 1.2 and 95% CI 0.61 to 2.33, p = 0.23 by Logrank test) and an 80% increased risk of death from all causes (HR 1.8 and 95% CI 0.34 to 9.41, p = 0.48). Conclusions: Loss of LEKTI expression in HNSCC results in a cellular phenotype with locally aggressive behavior. Our findings shed new light on mechanisms of PNI, offer prognostic value, and may aid in the selection of therapeutic approaches. No significant financial relationships to disclose.

Epithelial Genes in Chronic Rhinosinusitis with and without Nasal Polyps

Genetic studies on chronic inflammatory diseases have resulted in an emphasis on the epithelial interface with the environment and the genes that influence this interaction. This study examines the expression of key epithelial genes implicated in the pathogenesis of other inflammatory disorders for their role in chronic rhinosinusitis (CRS). Epithelial cells were collected from the inferior turbinate, middle turbinate, and/or uncinate from 62 subjects undergoing sinonasal surgery. Patient groups included 21 CRS patients with nasal polyposis, 23 CRS patients without nasal polyposis, and 18 controls. Samples were analyzed for S100A7, S100A8, S100A9, SLC9A3R1, G-protein-coupled receptor for asthma, and serine protease inhibitor kazal type 5 (SPINK5) by quantitative real-time polymerase chain reaction. Immunohistochemistry (IHC) was performed to analyze expression of SPINK5 lympho epithelial kazal-type inhibitor (LEKTI) in sinonasal samples. Expression of S100A7 and S100A8 was significantly decreased in CRS with and without nasal polyps when compared with controls. S100A9 expression was significantly decreased in CRS without nasal polyps, and SPINK5 expression was significantly decreased in CRS with nasal polyps. SPINK5 (LEKTI) protein was detected in sinonasal tissue and was significantly decreased in polyp samples using IHC. This study shows marked reductions in the level of expression of several genes involved in epithelial barrier maintenance and repair in the inflammatory state of CRS.