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Objective:To investigate the relationship between the levels of serum cytokines and chemokines and the prognosis of patients with acute B-ALL after receiving chimeric antigen receptor (CAR)-T cell immunotherapy and acute graft-versus-host disease (aGVHD) in patients after bridging allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods:According to the case-control principle, Forty-two patients with B-ALL who received CD19-CAR-T cell immunotherapy bridged to allo-HSCT at Heibei Yanda Ludaopei Hospital from September 18, 2019 to May 9, 2022 were enrolled. Mann-Whitney U test was used to compare the changes of aGVHD-related cytokines and chemokine levels between CAR-T cell immunotherapy and bridging transplantation in different patients at the same time. Their plasma levels of cytokines and chemokines related to aGVHD were monitored at the day before CAR-T therapy and after CAR-T treatment at day 4, 7,14,21,28. The receiver operating characteristic curve was drawn to evaluate the predictive value of cytokines and chemokines in predicting the occurrence and the death of aGVHD patients. Kaplan-Meier method and Log-rank tests were used for Overall survival (OS) analysis. Results:Twenty-four of total 42 patients had aGVHD, of which 11 patients died and 31 patients survived. There was no significant difference in cytokines and chemokines between the aGVHD group and the non-aGVHD group on the day before CAR-T cell treatment. According to statistical analysis, the serum Elafin levels of aGVHD group was higher than that of non-aGVHD group at the 21st day [4 482 (2 811, 6 061) ng/L vs 2 466 (1 948, 3 375) ng/L, Z=3.145, P=0.001] and the 28st day [4 391 (2 808, 5594) ng/L vs 2 463 (1 658, 2 830) ng/L, Z=2.038, P=0.048] separately. At the 14th day, serum cytokines and chemokines levels between the two group were as follows,MIP-1 α [21.02 (12.36, 30.35) ng/L vs 5.56 (3.64, 10.79) ng/L], sCD25 [422.47 (257.99, 1 233.78) IU/ml vs 216.11 (133.75,457.39) IU/ml], Elafin [4 101 (2 393, 5 006) ng/L vs 2 155 (1 781, 3 033) ng/L], IL-6 [119.08 (23.97, 183.43) ng/L vs 8.39 (2.91, 17.42) ng/L] and IL-8 [13.56 (12.50, 24.52) ng/L vs 2.83 (1.73,6.87) ng/L] were at higher levels ( Z=2.653, P=0.007; Z=2.176, P=0. 030; Z=2.058, P=0.041; Z=3.329, P<0.001; Z=3.162, P=0.001). The KM survival curve showed that the cumulative survival rates of patients with higher serum levels of MIP-1α, sCD25, Elafin, IL-6 and IL-8 were lower than those with low levels at day 14, and the difference was statistically significant (χ 2=12.353, 4.890, 6.551, 10.563, 20.755, P<0.05). Conclusion:The outcomes of patients treated with CAR-T cell therapy bridged to allo-HSCT was correlated with serum MIP-1α, sCD25, Elafin, IL-6 and IL-8 levels after receiving CAR-T therapy. High concentrations of MIP-1α, sCD25, Elafin, IL-6 and IL-8 suggest poor prognosis and can be used as biomarkers to suggest appropriate clinical selection of therapy.
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Objective:To investigate the effect of recombinant Elafin on A549 apoptosis induced by paraquat and the underlying mechanism.Methods:pEGFP-C1-Elafin was transformed into A549 competent cells by electroporation.Transformed A549 was cultured for additional 24 h before Elafin mRNA was detected by RT-PCR and cocultured with or without various concentration of paraquat( PQ ) for different duration.A549 apoptosis percentage and reactive oxygen species ( ROS ) content were tested by flow cytometry .Nuclear factor erythroid like-2(Nrf2) heme oxygenase-1(HO-1) were examed by Western blot .Results:Elafin expressed successfully in A549 after transformation.PQ caused A549 apoptosis in a concentration dependent manner and leaded to ROS content increasing and Nrf2 and HO-1 decreasing.However,elafin could inhibit ROS production and A549 apoptosis,upregulate Nrf2 and HO-1.Conclusion:Elafin could restrict A549 apoptosis to some extent and the possible mechanism lied in its ability to upregulate Nrf2 ex-pression.
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Objective To explore the different effect of elafin incubated by different bacteria on P.aeruginosa(Pa) bioflim. Methods To cultivate the A549 cells in vitro, the pEGFP-N1-elafin eukaryotic expression vectors have been transfected to the cells by LipofectamineTM 2000. Elafin transfected cells incubated by the supernatant of S. epidermidis (S.epidermidis group), Pa (Pa group) and E.coli (E.coli group) respectively for 24 hours,the A549 cells were transfected. Then the levels of elafin were detected by ELISA and Western blot. To establish the Pa biofilm model in vitro,a rapid silver nitrate staining procedure and scanning electronic microscope (SEM)demonstrated bacterial biofilm. After biofilm carriers were put into each group and incubated for 8 hours, we measured the proportion of bacteria biofilm by silver nitrate staining and observed the structure of biofilm by SEM. Results The Pa and E.coli groups(especially Pa) raised the content of elafin in cells and the level of secretion increasing as compared to the normal group, while the S. epidermidis group had no change. Both silver nitrate staining procedure and SEM demonstrated the prestnce of bacterial biofilms. The the proportion of bacteria biofilm and the structure of BF in Pa and E.coli groups were changed, especially in Pa group. Conclusion There was a specificity for bacteria to induce the express of elafin. The inducing effect of Pa was more significant than that of E.coli.
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Objective To explore the molecule mechanism of protective effects of transfecting human elafin gene recombinant plasmid to NCI-H292.Methods Constructe eukaryotic expression vector pEGFP-C1-elafin and transfectied it into NCI-H292 cells,then co-incubated with polymorphonuclear granulocyte(PMN) with stimulation by lipopolysaccharide(LPS).Afater 24 hour,Western bolt and MTT technique were respectively performed to detect expression of ZO-1 in cells and adhesion capability of cells with collagen.Results After stimulation with LPS,Western blot showed that the 220 ku onula occludens-1(ZO-1) protein in transfecting pEGFP-C1 NCI-H292 cells extracts were obviously decreased,in transfecting pEGFP-C1-elafin NCI-H292 cells extracts.And MTT technique showed that compared with transfecting pEGFP-C1-elafin cells,adhesion capability of transfecting pEGFP-C1 cells with collagen were obviously decreased.Conclusion Transfection of recombinant human elafin gene into airway epithelial cells could maintain airway epithelium and enhance the capability of airway to inflammatory injury.
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OBJECTIVE To observe whether the natural antibacterial polypeptide elafin after transfected into airway epithelial cells(A549 cells)has resistant effects on Pseudomonas aeruginosa bioflim.METHODS To establish the P.aeruginosa biofilm model in vitro,a rapid silver nitrate staining procedure was used to demonstrate bacterial biofilm.After cultivating the cells in vitro,the constructed pEGF-N1-elafin eukaryotic expression vector had transfected into A549 cells by LipofectamineTM 2000.Elafin transfected cells were incubated by pig Pancreas Elastase(NE group),the supernatants of PAE(PAE group)and Escherichia coli(ECO group),respectively,for 24 hours.Then the content of elafin in cells and the level of elafin secretion were detected by Western blot and ELISA respectively.After biofilm carriers were put into each group and incubated for 8 hours,we observed the ratio of cells shape breakage at 4,8,12 and 24 hour,respectively.RESULTS The NE,PAE and ECO groups induced the content of elafin in cells and the level of secretion increased compared to the no induced group(normal group),while the PAE group and NE group were higher than those of ECO group.The ratio of cells breakage in induced elafin groups was lower than that of in normal group(P
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BACKGROUND: Elafin is a potent anti-elastase in human saliva, and is supposed to play a role in preventing oral ulceration. The expression of elafin was observed in the oral lichen planus (OLP), one of the most common noninfectious oral mucosal diseases, which frequently manifests as extensive ulceration on the involved oral mucosa. METHODS: 50 OLP, 10 oral leuko-plakia, 3 inflammatory oral ulcers, and 3 normal oral mucosa cases were fixed with 10% buffered formalin, and immunohistochemically stained with monoclonal elafin antibody. Representative specimens were fixed with 4% paraformaldehyde, and RNA in situ hybridization, with an elafin RNA probe, was performed. RESULTS: With both the immunohistochemistry and RNA in situ hybridization the expression of elafin was more decreased in the OLP compared to the normal mucosa, while in the hyperplastic epithelium of the leukoplakia and inflammatory ulcers the expressions of elafin was more intense. In the thin epithelia of the reticular and atrophic OLPs the expressions of elafin were reduced compared to the normal mucosa, and became almost negative in the epithelium of the erosive OLP. CONCLUSIONS: These data suggested that the extensive ulceration of the OLP was closely relevant to the reduced expression of elafin in the involved epithelium
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Humans , Elafin , Epithelium , Formaldehyde , Immunohistochemistry , In Situ Hybridization , Leukoplakia , Lichen Planus , Lichen Planus, Oral , Mouth Mucosa , Mucous Membrane , Oral Ulcer , RNA , Saliva , UlcerABSTRACT
Objective To construct the recombinant adenovirus for nature antibiotic elafin protein and observe its expression in primary airway epithelia.Methods The elafin gene was amplified by RT-PCR from lung tissues,then the fragment was inserted into the multiple clone site of pAdTrack-CMV.The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183.The candidate clone was further analyzed by restriction endonuclease digestion,PCR,and sequencing.Then the recombinant adenovirus plasmid was digested with PacⅠ and transfected into 293 cells for packaging and amplification.Infection titer and rate were monitored by green fluorescent protein(GFP)expression.Finally the primary airway epithelium was infected with Ad-elafin,and the GFP expression was observed by fluorescence microscope in epithelial cells 24 h after transfection.The elafin protein was detected by ELISA in the supernatant.Results Restriction endonuclease and PCR confirmed that elafin gene was cloned into the adenovirus vector successfully.Twenty-four hours after transfection,the GFP expression was seen by fluorescence microscopy.The concentration of elafin protein was(2.2?0.4)ng/ml in supernatant of transfected group,while that of control group was(1.9?0.3)ng/ml,P
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BACKGROUND: Elafin is a serine proteinase inhibitor first discovered in keratinocytes from psoriatic epidermis. The molecular structure of elafin contains two different functional domains; one for the proteinase inhibitor, which is directed against elastase and proteinase-3, and the other for transglutaminase substrate. As this unique structural characteristic implies, elafin would be expected to have two distinct biologic functions in tissues. But, the roles and biological features of elafin have not been extensively studied in the skin. OBJECTIVES: This study was designed to demonstrate the immunohistochemical localization of elafin in inflammatory and keratinizing skin disorders, and to elucidate its biological functions. MATERIALS AND METHODS: The pre-elafin sequence was amplified by PCR using a human epidermal cDNA library and expression construct which was ligated into an expression vector. Then, the expressed proelafin sequence was purified and injected intradermally into rabbits to raise a polyclonal antibody. The skin biopsy samples of various skin diseases and normal controls were used for immunohistochemical staining to detect elafin expression. RESULTS: 1)Expression of elafin was observed in infected epidermis, and is believed to be involved in the defense mechanism of the skin. 2)In dermal and subcutaneous inflammatory diseases, epidermal elafin expression was influenced by the location of the inflammation. 3)Elafin was expressed in bullous dermatoses accompanied by acantholysis or spongiosis of epidermal cells. 4)Expression of elafin was upregulated in papulosquamous skin diseases which was characterized by epidermal hyperplasia and abnormal differentiation. 5)Elafin was expressed in keratinizing skin diseases that was accompanied by abnormal differentiation of epidermal cells. 6)Expression of elafin was demonstrated in skin tumors that showed proliferation of suprabasal cells. The intensity of expression is not related to the degree of malignancy, but to the degree of differentiation of tumor cells. CONCLUSION: Induction of elafin expression may play an important role in protecting the skin components against tissue damage. Elafin expression is related to the abnormal proliferation and differentiation of suprabasal cells. This means that elafin may be used as a marker of these conditions.
Subject(s)
Humans , Rabbits , Acantholysis , Biopsy , Elafin , Epidermis , Gene Library , Hyperplasia , Immunohistochemistry , Inflammation , Keratinocytes , Molecular Structure , Pancreatic Elastase , Polymerase Chain Reaction , Serine Proteases , Skin Diseases , Skin Diseases, Papulosquamous , Skin Diseases, Vesiculobullous , SkinABSTRACT
BACKGROUND: Elafin is a serine proteinase inhibitor first discovered in keratinocytes from hyperproliferative human epidermis. In addition to the proteinase inhibiting domain, elafin contains multiple transglutaminase substrate domains which enable cross-linking to extracellular and cell envelope proteins. Several characteristics of elafin suggest potential anti-microbial activity. Elafin is absent in normal skin at protein level, but is induced in inflammatory and infectious dermatoses which threat the epidermal integrity by vesicopustule formation and neutrophilic cell infiltration. Cutaneous fungal infection is one of the well-known examples of diseases characterized by such condition. The purpose of this study was to check out the possibility that elafin may be involved in the pathomechanism of fungal infection. METHODS: The biopsy samples taken from 10 cases of superficial fungal infections, 10 cases of deep and systemic mycoses, 2 cases of slide culture specimens of Candida species, were used for the immunohistochemical tissue staining for elafin expression. Polyclonal anti-elafin was used in 1:300 dilution. As control, biopsy smaples of normal skin, ichthyosis, psoriasis were used for the staining for elafin expression. RESULTS: In the normal and ichthyotic epidermis, elafin expression was virtually negative. In superficial mycoses except candidiasis, elafin was expressed in the spinous layer of infected epidermis, and fungal structures in the stratum corneum were stained with elafin antisera. In the cases of dermatophytosis of ichthyosis patients, while fungal hyphae were stained with elafin antisera, epidermal cell did not express elafin. In candidial esophagitis, elafin was expressed in the esophgeal mucosa, but spores were not stained with elafin anti-sera. In slide culture of Candida species, spores were not stained with elafin antisera, also. In cases of systemic and deep mycoses, fungal hyphae and spores were stained with elafin antisera and epidermis adjacent to severe dermal inflammatory reaction showed elafin expression. CONCLUSION: Elafin may have certain role in systemic and cutaneous fungal infection to contribute to high resistance of the epidermis against proteolysis and fungal infections, and it is shown that elafin or elafin-like protein may also be produced and utilized by fungi themselves.
Subject(s)
Humans , Biopsy , Candida , Candidiasis , Elafin , Epidermis , Esophagitis , Fungal Structures , Fungi , Hyphae , Ichthyosis , Immune Sera , Immunohistochemistry , Keratinocytes , Mucous Membrane , Mycoses , Neutrophils , Proteolysis , Psoriasis , Serine Proteases , Skin , Skin Diseases , Spores , TineaABSTRACT
Objective:To construct the highly efficient expression recombinant adenovirus that expresses elafin protein for further study.Methods:The elafin gene was amplified by RT-PCR from lung tissues,then the fragment was inserted into the multiple clone site of pAdTrack-CMV. The linearized shuttle plasmid was homogenously recombined with pAdEasy-1 in BJ5183. The candidate clone was further analyzed by restriction endonuclease digestion, PCR, and sequence scanned. Then the recombined adenovirus plasmid was digested with Pac Ⅰ and transfected into 293 cells for packaging and amplifying. Infection titer and rate were monitored by green fluorescent protein(GFP) expression. The expression of elafin protein was detected by Western blot and ELISA to appraise the function of elafin protein for oppressing the activity of elastic protease.Results:Digestion with restriction endonuclease, PCR, Western blot and ELISA confirmed that elafin gene was cloned into the adenovirus vector successfully.Conclusion:The recombined adenovirus Ad-elafin is constructed successfully, which will be benefit for future study.