Lactobacillus plantarum induces genomic DNA-dependent and TLR9-mediated elafin secretion from Caco-2 cells.

BACKGROUND: Lactobacilli show anti-inflammatory effects in the human intestine, and their genomic DNA was identified as one of the anti-inflammatory components. Increased levels of the natural protease inhibitor elafin in the intestine plays an important role in protection against intestinal inflammation. However, there have been no previous reports regarding whether lactobacilli increase elafin levels. OBJECTIVE: This study was performed to investigate whether Lactobacillus plantarum induces elafin secretion from the human epithelial colorectal adenocarcinoma cell line, Caco-2. Moreover, we examined the roles of bacterial genomic DNA and toll-like receptor 9 (TLR9), a specific receptor of bacterial DNA, in this effect. METHODS: Elafin secretion from Caco-2 cells by live and heat-killed L. plantarum was measured. The analysis was also performed using DNase-treated L. plantarum and genomic DNA extracted from L. plantarum. We examined the role of TLR9 in elafin secretion by L. plantarum and its genomic DNA by suppressing TLR9 expression using RNAi in Caco-2 cells. RESULTS: Heat-killed L. plantarum time- and dose-dependently increased elafin secretion, whereas live L. plantarum had no such effect. The elafin secretion by heat-killed L. plantarum was partially abolished by DNase treatment of the bacterium. In addition, L. plantarum genomic DNA also increased elafin secretion. Furthermore, suppression of TLR9 expression partially or completely abolished elafin secretion by heat-killed L. plantarum and its genomic DNA. CONCLUSION: Our results indicated that heat-killed L. plantarum induced genomic DNA-dependent and TLR9-mediated elafin secretion. The anti-inflammatory effects of lactobacilli may be mediated by increases in the levels of elafin in the intestine.

Expression of Elafin in Dermatitis Herpetiformis.

BACKGROUND: Elafin is a serine protease inhibitor that has various epithelial cell regulatory and immunomodulatory effects including inactivation of neutrophil elastases. This later role originated the interest of elafin in certain neutrophil-rich dermatoses. Interestingly, it has been speculated that elafin has a protective role by slowing the deamidation process of gliadin in celiac disease (CD), despite the typical absence of neutrophils in intestinal histologic samplings. Dermatitis herpetiformis (DH) is a chronic recurrent vesicular dermatitis associated with gluten hypersensitivity and also characterized by a neutrophilic infiltrate and granular immunoglobulin A deposits in papillary dermis. MATERIALS AND METHODS: We selected 31 formalin-fixed paraffin-embedded skin specimens of DH that demonstrated typical immunopathologic findings and probed them with rabbit polyclonal immunoglobulinG antielafin antibodies through standard immunohistochemistry analysis. Negative controls consisted of normal skin from elbow and knee surgical re-excisions specimen lacking residual tumor. Positive controls included skin biopsies of active plaque psoriasis, Sweet syndrome, and pyoderma gangrenosum. RESULTS: Similar to what has been previously reported in intestinal sampling of patients with active CD, abnormal expression of elafin was noted in virtually all probed skin biopsies of DH patients with active cutaneous disease. CONCLUSION: Under normal circumstances, keratinocytes overexpress elafin to downregulate a neutrophil mediated inflammatory response. The deficient expression of elafin in the aforementioned probed DH specimens correlates with previous similar elafin underexpression in intestinal samples of active CD. These histological findings suggest that these 2 gluten mediated disorders carry an abnormal elafin underexpression during disease activity.

Cervical Expression of Elafin and SLPI in Pregnancy and Their Association With Preterm Labor.

PROBLEM: Elafin and secretory leukocyte peptidase inhibitor (SLPI) are unique among antimicrobial peptides (AMPs). This study aimed to determine the expression levels of these AMPs at the cervix during pregnancy and to investigate their association with preterm labor. METHOD OF STUDY: Cervical epithelial cells were swabbed from normal pregnant women to evaluate the physiological expression of elafin and SLPI. Cross-sectional analysis was conducted to compare cervical expression levels for SLPI and elafin among three women's groups, controls (n = 26), women with threatened preterm labor who delivered at term (t-TPL, n = 23) and TPL who ended in preterm labor (p-TPL, n = 19). RESULTS: Elafin and SLPI proteins were detected in the squamous and glandular cells of the cervix. Cervical SLPI expression levels increased over the course of pregnancy, whereas elafin levels remained unchanged. Cervical mRNA expression levels of elafin and SLPI were significantly higher in p-TPL compared with t-TPL and control groups. CONCLUSION: Constitutive expression of elafin and SLPI in cervical cells during pregnancy suggests their essential roles in local tissue homeostasis and immune defense. The elevations in cervical elafin and SLPI expression in the women with preterm delivery might reflect the local response to the pathogen invasion into the cervix preceding preterm labor.

Modification of sialylation is associated with multidrug resistance in human acute myeloid leukemia.

Aberrant cell surface sialylation patterns have been shown to correlate with tumor progression and metastasis. However, the role of sialylation regulation of cancer multidrug resistance (MDR) remains poorly understood. This study investigated sialylation in modification on MDR in acute myeloid leukemia (AML). Using mass spectrometry (MS) analysis, the composition profiling of sialylated N-glycans differed in three pairs of AML cell lines. Real-time PCR showed the differential expressional profiles of 20 sialyltransferase (ST) genes in the both AML cell lines and bone marrow mononuclear cells (BMMCs) of AML patients. The expression levels of ST3GAL5 and ST8SIA4 were detected, which were overexpressed in HL60 and HL60/adriamycin-resistant (ADR) cells. The altered levels of ST3GAL5 and ST8SIA4 were found in close association with the MDR phenotype changing of HL60 and HL60/ADR cells both in vitro and in vivo. Further data demonstrated that manipulation of these two genes' expression modulated the activity of phosphoinositide-3 kinase (PI3K)/Akt signaling pathway and its downstream target thus regulated the proportionally mutative expression of P-glycoprotein (P-gp) and MDR-related protein 1 (MRP1), both of which are known to be involved in MDR. Blocking the PI3K/Akt pathway by its specific inhibitor LY294002 or by Akt small interfering RNA resulted in the reduced chemosensitivity of HL60/ADR cells. Therefore, this study indicated that sialylation involved in the development of MDR of AML cells probably through ST3GAL5 or ST8SIA4 regulating the activity of PI3K/Akt signaling and the expression of P-gp and MRP1.

Sheep lung segmental delivery strategy demonstrates adenovirus priming of local lung responses to bacterial LPS and the role of elafin as a response modulator.

Viral lung infections increase susceptibility to subsequent bacterial infection. We questioned whether local lung administration of recombinant adenoviral vectors in the sheep would alter the susceptibility of the lung to subsequent challenge with bacterial lipopolysaccharide (LPS). We further questioned whether local lung expression of elafin, a locally produced alarm anti-LPS/anti-bacterial molecule, would modulate the challenge response. We established that adenoviral vector treatment primed the lung for an enhanced response to bacterial LPS. Whereas this local effect appeared to be independent of the transgene used (Ad-o-elafin or Ad-GFP), Ad-o-elafin treated sheep demonstrated a more profound lymphopenia in response to local lung administration of LPS. The local influence of elafin in modulating the response to LPS was restricted to maintaining neutrophil myeloperoxidase activity, and levels of alveolar macrophage and neutrophil phagocytosis at higher levels post-LPS. Adenoviral vector-bacterial synergism exists in the ovine lung and elafin expression modulates such synergism both locally and systemically.

Clinical significance of p95HER2 overexpression, PTEN loss and PI3K expression in p185HER2-positive metastatic breast cancer patients treated with trastuzumab-based therapies.

BACKGROUND: Overexpression of p185HER2 is an established poor prognostic factor in breast cancer, portending an aggressive course and potential for early metastasis. On the other hand, monoclonal antibody trastuzumab is widely used in the clinic to target this overexpressed oncogene. Unfortunately, ~30-40% of all patients overexpressing HER2 respond to trastuzumab, warranting further research regarding the structure and additional modulation of the receptor. In this study, we aimed to investigate the response to trastuzumab in terms of the potential roles of several oncogenic pathways (phosphatase and tensin homologue (PTEN) and phosphatidylinositol 3-kinase (PI3K)) and a truncated receptor protein, p95HER2, retrospectively. MATERIALS AND METHODS: Paraffin-embedded primary tumour tissues of 100 HER2-positive metastatic breast cancer patients who received trastuzumab with combination cytotoxic chemotherapy were analysed with immunohistochemical method for p95HER2, p85 (PI3K) and PTEN. Relationship between variables were tested via χ(2), Fischer's exact test and Mann-Whitney U tests, wherever appropriate. Progression-free survival (PFS) and overall survival (OS) periods were calculated with Kaplan-Meier method and survival curves of subgroups were compared with log-rank test. RESULTS: Percentage of patients was found to be 33%, 57% and 42% positive for p95 expression, PTEN and PI3K, respectively. p95-expressing tumours had statistically lower response rates for trastuzumab than tumours not expressing p95 (P=0.001). On the contrary, PTEN-expressing tumours had statistically higher response rates for trastuzumab than tumours not expressing PTEN (P=0.012). PI3K expression had no significant effect on trastuzumab response. Median PFS for p95-expressing and not expressing tumours were 8 months (95% CI, 2.5-13.4 months) and 22 months (95% CI, 9.9-34 months), respectively (P=0.0001). Median PFS for PTEN-expressing and not expressing tumours were 15.3 months (95% CI, 12.6-34 months) and 12.1 months (95% CI, 7.9-16.2 months), respectively (P=0.04). Median OS for p95-expressing and not expressing tumours were 24 months (95% CI, 8.3-40.4 months) and 29.1 months (95% CI, 8.6-43.2 months), respectively (P=0.045). Median OS for PTEN-expressing and not expressing tumours were 25.1 months (95% CI, 7.5-40.1 months) and 26.8 months (95% CI, 8.1-42 months), respectively, which was not statistically significant (P=0.5). Level of PI3K expression had no effect on PFS and OS in our patient population. Presence of visceral metastases HR=2.38 ((95% CI, 1.2-4.5), P=0.009), p95 expression HR=2.1 ((95% CI, 1.1-3.7), P=0.03) and response to trastuzumab HR=2.2 ((95% CI, 1.18-4.47), P=0.014) are identified as factors independently affecting PFS. Response to trastuzumab HR=1.7 ((95% CI, 1.14-3.47), P=0.013) was identified as the single parameter influencing survival by Cox regression analysis. CONCLUSIONS: Presence of p95 predicted a poorer response to trastuzumab treatment, shorter PFS and OS in our HER2-positive metastatic breast cancer cohort. In addition, loss of PTEN predicted a poorer response to trastuzumab treatment and shorter PFS but not OS. We could not find an effect of PI3K expression on the above-mentioned parameters.

Everolimus in combination with letrozole inhibit human breast cancer MCF-7/Aro stem cells via PI3K/mTOR pathway: an experimental study.

This study evaluated the effects of an mTOR inhibitor everolimus alone or in combination with letrozole on MCF-7/Aro (MCF-7 cells stably transfected with CYP19) in vitro and in vivo. In vitro studies, full-length CYP19 (aromatase) was cloned in a plasmid transfer vector pH ß-Aro and then transfected into MCF-7 stem cells which were ESA(+)CD44(+)CD24(-/low) sorted by flow cytometry. MTT assays were used to quantify the inhibitory effect of the drugs on MCF-7/Aro stem cells (SCs) and non-stem cells (NSCs). Apoptosis and the cell cycle distributions of stem cells were examined by flow cytometry. The tumorigenicity of stem cells after treatment was investigated by soft agar colony formation assays. In vivo studies, the BALB/c mice were injected with MCF-7/Aro SCs, and the different treatments were administered. After necropsy, the expression of KI67, CD31, AKT1, phospho-AKT (Thr308), and mTOR was analyzed by immunohistochemistry. In vitro, compared with MCF-7/Aro NSCs, there were greater resistance to the standard treatment doses of letrozole and everolimus in MCF-7/Aro SCs (17- and 15-fold, respectively). Treatment with everolimus or letrozole resulted in growth inhibition of SCs in a dose-dependent manner. Compared with single-agent therapy, the combination of everolimus with letrozole was more effective in the inhibition of cell growth (P < 0.001) and tumorigenicity (P < 0.01). In addition, an increase in G1 cell cycle arrest and increases in early apoptosis were observed in the combination treatment group compared with either single-agent group. In vivo, the xenograft tumor sizes were significantly decreased in everolimus alone group compared to control group, and everolimus plus letrozole therapy was much more effective compared with either single agent alone (P < 0.01). Compared with everolimus alone, the combination of everolimus and letrozole reduced the expression of KI67, mTOR, and phospho-AKT (Thr308; P < 0.01). Everolimus has effective inhibition on aromatase-overexpressing stem cell in vitro and in vivo. The combination everolimus and letrozole could be more effective than either drug alone.

miR-34a regulates cisplatin-induce gastric cancer cell death by modulating PI3K/AKT/survivin pathway.

The purposes of this study were to determine the expression profiles of microRNA-34a (miR-34a) in human gastric cancer cell line (SGC-7901) and cisplatin-resistant cell lines (SGC-7901/DDP), and to establish the correlation between miR-34a expression profile and the sensitivity of human gastric cancer cell to cisplatin-based pattern, thereby providing new methods and strategies for treating gastric cancer. Gastric cancer cell line (SGC-7901) and cisplatin-resistant cell line (SGC-7901/DDP) were cultivated in vitro, respectively. Quantitative real-time PCR (qRT-PCR) and Western blot were utilized to determine the expression profiles of miR-34a and survivin in both gastric cancer cell lines. With miR-34a mimic and miR-34a inhibitor transfected into SGC-7901 and SGC-7901/DDP for 48 h, post-transfection changes of miR-34a expression was determined; the effects of miR-34a ectopic expression on the viability of cisplatin-induce gastric cancer cell were assayed by the MTT method. The effects of miR-34a ectopic expression on apoptosis of cisplatin-induce gastric cancer cell were determined by Annexin V/propidium iodide (PI) double staining method and flow cytometry. The effects of miR-34a ectopic expression on the AKT and p-AKT expression of cisplatin-induce gastric cancer cells were determined by Western blot and flow cytometry with the PI3K pathway inhibitor Wortmannin. As shown by qRT-PCR and Western blot analyses, the expression of miR-34a in cisplatin-resistant cell lines decreased significantly in comparison to that of SGC-7901 cell line (p < 0.05), while significant up-regulation of survivin expression was also observed (p < 0.05). Compared with the control group, the expression of miR-34a increased significantly in SGC-7901 cells transfected with miR-34a mimic for 48 h (p < 0.01). After miR-34a inhibitor transfection, the expression of miR-34a decreased significantly (p < 0.05). The viability of cisplatin-induce gastric cancer cells increased significantly (p < 0.05) with significant decrease of apoptosis after miR-34a expression inhibition, as demonstrated by MTT and flow cytometry with miR-34a over-expression, the viability of cisplatin-induce gastric cancer cells decreased significantly (p < 0.05), with significant apoptosis increase (p < 0.05). As shown by Western blot and flow cytometry, in comparison to the control group, Wortmannin could inhibit miR-34a inhibitor and DDP induced up-regulation of p-AKT significantly (p < 0.05) and stimulated apoptosis. In conclusion, miR-34a expression was down-regulated in cisplatin-resistant cell lines. miR-34a over-expression could improve the sensitivity of gastric cancer cells against cisplatin-based chemotherapies, with PI3K/AKT/survivin signaling pathway possibly involved in the mechanism.

Elafin expression in mucosa of fallopian tubes is altered by hydrosalpinx.

Elafin is a natural antimicrobial molecule and a member of the antileukoproteinase (Trappin) family. It is normally expressed in the mucosae of fallopian tubes. Hydrosalpinx is a chronic inflammatory process of the fallopian tubes. The objective of this study is to compare the localization of elafin protein and levels of elafin messenger RNA (mRNA) in the mucosa of oviducts with and without hydrosalpinx. Immunohistochemical analysis was performed on tissue sections of hydrosalpinx (n = 10) and normal tubes (n = 22) from paraffin-embedded blocks, obtained from patients who underwent salpingectomy for benign conditions. The main outcome measure was the intensity of staining with 3,3'-diaminobenzidine calculated by ImageJ software and mRNA expression by real-time polymerase chain reaction. The mean intensity of elafin (mean ± standard deviation) in mucosae of the fallopian tubes was 69.68 ± 24.55 in controls and 32.03±18.16 in patients with hydrosalpinx (P < .0001). Elafin mRNA levels were reduced in hydrosalpinx, although not significantly (P = .05, n = 9 from each group). Therefore, tubal epithelium of women with hydrosalpinx seems to have a lower expression of elafin, an elastase inhibitor and a natural antimicrobial molecule, compared to normal tubes.

Loss of fatty acid synthase inhibits the "HER2-PI3K/Akt axis" activity and malignant phenotype of Caco-2 cells.

BACKGROUND: Fatty acid synthase (FASN) is frequently activated and overexpressed in human cancers, and plays a crucial role in the carcinogenesis of various cancers. In this study, our aims were to explore the role of FASN in regulating the "HER2-PI3K/Akt axis" activity and malignant phenotype of colorectal cancer. METHODS: Caco-2 cells with a high expression of both HER2 and FASN were selected for functional characterization. Caco-2 cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid, followed by the RT-qPCR and western blot to examine the expression of FASN, HER2, PI3K and Akt. The MTT and colony formation assays were used to assess the proliferation potential. The migration was investigated by the transwell, and the apoptosis and cell cycle were assayed by the flow cytometry. RESULTS: Notably, the expression of FASN, HER2, PI3K and Akt were downregulated upon a silence of FASN. The proliferation was decreased after a downregulation of FASN, which was consistent with an increased apoptosis rate. The migration was also impaired in FASN-silenced cells. CONCLUSION: A downregulation of FASN effectively inhibits the activity of "HER2-PI3K/Akt axis" and alters the malignant phenotype in colorectal cancer cells.

Elafin, an inhibitor of elastase, is a prognostic indicator in breast cancer.

INTRODUCTION: Elafin is an elastase-specific inhibitor with increased transcription in normal mammary epithelial cells compared to mammary carcinoma cells. In this report, we test the hypothesis that inhibition of elastase, through induction of elafin, leads to inhibition of human breast cancer cell viability and, therefore, predicts survival in breast cancer patients. METHODS: Panels of normal and immortalized breast epithelial cells, along with breast carcinoma cells, were used to examine the impact of adenoviral-mediated elafin expression or shRNA-mediated inhibition of elastase on the growth of cells and xenografts in nude mice. To determine the prognostic significance of decreased elafin in patients with invasive breast cancer, previously published gene array datasets were interrogated. RESULTS: Elafin expression had no effect on non-tumorigenic cells but resulted in marked inhibition of cell growth in breast cancer cell lines. Control-treated xenografts generated a tumor burden that necessitated sacrifice within one month of initial treatment, whereas xenograft-bearing mice treated with Ad-Elafin were alive at eight months with marked reduction in tumor growth. Elastase inhibition mimicked these results, showing decreased tumor cell growth in vitro and in vivo. Low expression of elafin gene correlated with significantly reduced time to relapse, and when combined with high expression of elastase gene was associated with decreased survival in breast cancer patients. CONCLUSION: Our data suggest that elafin plays a direct role in the suppression of tumors through inhibition of elastase and thus serves as a prognostic indicator for breast cancer patients.

Elafin selectively regulates the sensitivity of ovarian cancer cells to genotoxic drug-induced apoptosis.

OBJECTIVE: Elafin has been reported to be abundantly expressed in human epithelial ovarian carcinoma (EOC), however, its functions are poorly understood. Here, we evaluated the role of elafin in modulating the sensitivity of human EOC cells to chemotherapeutic drugs. METHODS: Elafin expression was determined by ELISA in 9 established human EOC cell lines. A lentivirus encoding elafin-specific shRNA was used to down-regulate elafin expression in OVCAR3 and OV433 cells, and a plasmid encoding elafin was used to ectopically express elafin in elafin-negative SKOV3 cells. Sensitivity to cisplatin and other genotoxic agents and to paclitaxel, an inhibitor of microtubule depolymerization, was examined in OVCAR3, OV433 and SKOV3 sublines. Cell viability was determined by the MTT assay, apoptosis by annexin V/7-AAD staining and caspase activation by fluorimetric assay. RESULTS: Knockdown of the elafin gene decreases cisplatin IC50 by at least 2-folds in OVCAR3 and OVCAR433 cells (p<0.01) but does not affect paclitaxel IC50. The sensitivity to other genotoxic agents such as carboplatin, cyclophosphamide and 5-fluorouracil was also increased by silencing the expression of elafin. Apoptosis and caspase-3 activation were significantly augmented in cisplatin-treated OVCAR3 cells with silenced elafin. Overexpression of elafin in SKOV3 cells made them more resistant to cisplatin and decreased cisplatin-induced apoptosis and caspase activation (p<0.01). CONCLUSIONS: Expression of elafin decreases the sensitivity of human EOC cells to several genotoxic agents, which may have an important implication in predicting the response of patients with EOC to chemotherapy in the clinic.

The impact of salt water soaks on biophysical and molecular parameters in psoriatic epidermis equivalents.

BACKGROUND/AIMS: The exact mechanisms of action of balneophototherapy are incompletely understood. We aimed to investigate the effect of salt water soaks on ultraviolet (UV) transmission and the expression of molecular parameters of psoriasis. METHODS: We studied UV transmission and the expression of antimicrobial peptides and skin-derived antileukoproteinase (SKALP/elafin) in psoriatic epidermis equivalents which were pretreated with tap water and differently concentrated salt water solutions. Moreover, we performed in vivo phototoxicity tests in healthy subjects. RESULTS: Highly concentrated salt water soaks significantly increase UV transmission through psoriatic epidermis equivalents, in particular within the wavelength range of 305- 360 nm. In vivo tests revealed increased photosensitivity following highly concentrated salt water baths. A significant decrease in human ß-defensin-2 (hBD-2) and SKALP/elafin is observed after highly concentrated NaCl soaks. CONCLUSION: An increase in UV transmission following highly concentrated salt water soaks likely causes enhanced UV gain within the viable epidermis. Moreover, our data indicate that salt water soaks seem to influence the protein profiles of hBD-2 and SKALP/elafin.

The neutrophil elastase inhibitor elafin triggers rb-mediated growth arrest and caspase-dependent apoptosis in breast cancer.

Elafin, an endogenous inhibitor of neutrophil elastase, is expressed in human mammary epithelial cells but is transcriptionally downregulated in breast cancer cells. We hypothesized that elafin may exert a tumor-suppressive activity in the context of breast cancer. In this study, we show that the retinoblastoma (Rb) pathway governs the antitumor properties of elafin. In breast cancer cells with functional Rb, the expression of elafin triggered Rb-dependent cell cycle arrest. Elafin also exhibited suppressive activity in breast cancer cell lines lacking Rb, but this was associated with an induction of caspase-3-dependent, p53-independent apoptotic cell death. Normal mammary epithelial cells were not affected by elafin. Collectively, these results argue that elafin mediates tumor-suppressive effects that are cytostatic or cytotoxic depending on the Rb status. Our findings suggest that elafin could be engineered as a therapeutic modality to treat breast cancer without toxicity to normal proliferating cells.

Elafin is a biomarker of graft-versus-host disease of the skin.

Graft-versus-host disease (GVHD), the major complication of allogeneic bone marrow transplantation, affects the skin, liver, and gastrointestinal tract. There are no plasma biomarkers specific for any acute GVHD target organ. We used a large-scale quantitative proteomic discovery procedure to identify biomarker candidates of skin GVHD and validated the lead candidate, elafin, with enzyme-linked immunosorbent assay in samples from 492 patients. Elafin was overexpressed in GVHD skin biopsies. Plasma concentrations of elafin were significantly higher at the onset of skin GVHD, correlated with the eventual maximum grade of GVHD, and were associated with a greater risk of death relative to other known risk factors (hazard ratio, 1.78). We conclude that elafin has significant diagnostic and prognostic value as a biomarker of skin GVHD.

Overexpression of elafin in ovarian carcinoma is driven by genomic gains and activation of the nuclear factor kappaB pathway and is associated with poor overall survival.

Ovarian cancer is a leading cause of cancer mortality in women. The aim of this study was to elucidate whether whey acidic protein (WAP) genes on chromosome 20q13.12, a region frequently amplified in this cancer, are expressed in serous carcinoma, the most common form of the disease. Herein, we report that a trio of WAP genes (HE4, SLPI, and Elafin) is overexpressed and secreted by serous ovarian carcinomas. To our knowledge, this is the first report linking Elafin to ovarian cancer. Fluorescence in situ hybridization analysis of primary tumors demonstrates genomic gains of the Elafin locus in a majority of cases. In addition, a combination of peptidomimetics, RNA interference, and chromatin immunoprecipitation experiments shows that Elafin expression can be transcriptionally upregulated by inflammatory cytokines through activation of the nuclear factor kappaB pathway. Importantly, using a clinically annotated tissue microarray composed of late-stage, high-grade serous ovarian carcinomas, we show that Elafin expression correlates with poor overall survival. These results, combined with our observation that Elafin is secreted by ovarian tumors and is minimally expressed in normal tissues, suggest that Elafin may serve as a determinant of poor survival in this disease.

Trappin-2/Elafin: a novel innate anti-human immunodeficiency virus-1 molecule of the human female reproductive tract.

Trappin-2/Elafin is a serine protease inhibitor that plays a major role as an anti-inflammatory mediator at mucosal surfaces. In addition, Trappin-2/Elafin has antibacterial activity against Gram-positive and Gram-negative bacterial and fungal pathogens. In this study we examined the production of Trappin-2/Elafin by epithelial cells from the human upper and lower female reproductive tract as well as its activity as an anti-human immunodeficiency virus (HIV)-1 molecule. We found that primary uterine, Fallopian tube, cervical and ectocervical epithelial cells produce Trappin-2/Elafin constitutively and that production of Trappin-2/Elafin is enhanced following stimulation with Poly(I:C), especially by the uterine cells. Given the presence of Trappin-2/Elafin in the reproductive tract, we tested the ability of recombinant Trappin-2/Elafin to inhibit HIV-1, an important sexually transmitted pathogen. We found that recombinant Trappin-2/Elafin was able to inhibit both T-cell-tropic X4/IIIB and macrophage-tropic R5/BaL HIV-1 in a dose-dependent manner. The inhibitory activity was observed when virus was incubated with Trappin-2/Elafin but not when Trappin-2/Elafin was added to cells either before infection or after infection. This suggests that the mechanism of inhibition is likely to be a direct interaction between HIV-1 and Trappin-2/Elafin. Additionally, we measured the levels of secreted Trappin-2/Elafin in cervico-vaginal lavages (CVL) from both HIV-positive and HIV-negative women and found that average levels of secreted Trappin-2/Elafin were higher in the CVL from HIV-negative women, although the values did not reach statistical significance. We also found that women at the secretory phase of the menstrual cycle produced more Trappin-2/Elafin in CVL relative to women at the proliferative phase of the menstrual cycle. Our data suggest that Trappin-2/Elafin might be an important endogenous microbicide of the female reproductive tract that is protective against HIV-1.

Human gammadelta T cells produce the protease inhibitor and antimicrobial peptide elafin.

Human gammadelta T cells rapidly secrete pro-inflammatory cytokines in response to T cell receptor-dependent recognition of pyrophosphates produced by many bacteria and parasites. In further support of an important role of gammadelta T cells in the immune defence against infection, human gammadelta T cells have been shown to produce the antimicrobial peptide LL37/cathelicidin. In this study, we have investigated whether gammadelta T cells can produce additional antimicrobial peptides. To this end, we have screened human gammadelta T cell clones by RT-PCR for mRNA expression of a broad range of antimicrobial peptides. While alpha-defensins were absent and beta-defensins (HBD1) present only in rare gammadelta T cell clones, elafin mRNA was induced by supernatant of Pseudomonas aeruginosa grown under static conditions. Elafin is a protease inhibitor that also displays antimicrobial activity. Constitutive intracellular expression of elafin was demonstrated by flow cytometry and Western blot analysis. Furthermore, trappin-2 (pre-elafin) could be immunoprecipitated in cell lysates but also in the supernatant of gammadelta T cells stimulated by Ps. aeruginosa supernatant. Taken together, our studies reveal a novel effector function of gammadelta T cells which might be important for local immune defence.

Expression of secretory leukocyte protease inhibitor and elafin in human fallopian tube and in an in-vitro model of Chlamydia trachomatis infection.

BACKGROUND: Secretory leukocyte protease inhibitor (SLPI) and elafin are anti-protease and anti-microbial molecules with a role in innate immune defence. They have been demonstrated at multiple mucosal surfaces including those of the female reproductive tract. METHODS AND RESULTS: This study details their expression in human Fallopian tubes (ampullary region) throughout the menstrual cycle (n = 18) and from women with ectopic pregnancy (n = 6), and examined their regulation by infection with Chlamydia trachomatis in an in-vitro model. Quantitative real-time PCR analysis showed that SLPI and elafin were constitutively expressed in the Fallopian tube during the menstrual cycle but were increased in ectopic pregnancy (P < 0.05 versus early-mid luteal phase, P < 0.01 versus all phases, respectively). SLPI and elafin were immunolocalised to the Fallopian tube epithelium in biopsies from non-pregnant women and those with ectopic pregnancy. An in-vitro culture model of C. trachomatis infection of the OE-E6/E7 oviductal epithelial cell line showed that elafin mRNA expression was upregulated in response to chlamydial infection. CONCLUSION: These data suggest that SLPI and elafin have a role in the innate immune defence of the Fallopian tube in infection and ectopic pregnancy. Their role is likely to include regulation of protease activity, wound healing and tissue remodelling.

Altered secretory leukocyte protease inhibitor expression in the uterine decidua of tubal compared with intrauterine pregnancy.

BACKGROUND: The role of the innate immune system in tubal implantation remains undefined. This study compared expression of two key mediators of innate immunity, secretory leukocyte protease inhibitor (SLPI) and elafin, in the uterine decidua of women with intrauterine and tubal pregnancies. METHODS: Uterine decidua was collected from women (18-45 years) undergoing surgical termination of pregnancy (n = 7), surgical management of spontaneous abortion (n = 6) and tubal pregnancy (n = 10). Using quantitative RT-PCR and immunohistochemistry, mRNA and protein expression patterns of SLPI and elafin were compared. RESULTS: Relative SLPI mRNA expression was significantly higher in decidua of women with tubal pregnancy (12.37 +/- 2.66) compared with spontaneous abortion (5.09 +/- 2.22, P < 0.0185). There was no difference demonstrated in elafin mRNA expression. SLPI and elafin protein expression were demonstrated in the decidual leukocyte populations and epithelium. There was no obvious qualitative difference in levels of SLPI and elafin protein expression or their distribution in the uterine decidua of women with termination of pregnancy, spontaneous abortion or tubal pregnancy. CONCLUSIONS: Herein we demonstrate novel differences in gene expression of uterine decidua of tubal pregnancy compared with spontaneous abortion thereby contributing further to current knowledge of mechanisms involved in extrauterine implantation. The altered expression of SLPI may be a consequence of, or predispose to, tubal pregnancy.