The role of GNLY gene polymorphisms in psoriasis pathogenesis

Abstract BACKGROUND: Psoriasis is a systemic inflammatory disorder that involves complex pathogenic interactions between the innate and adaptive immune systems. The most accepted mechanism in the etiopathogenesis of psoriasis is the induction of inflammation with keratinocyte hyperproliferation. Granulysin (GNLY) is a cytolytic antimicrobial peptide (AMP) that is secreted together with granzyme and perforin from the granules of human cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It has been immunohistochemically proven that the expression of granulysin is increased in lesions of psoriasis. OBJECTIVE: This study aimed to investigate the relationship between psoriasis disease and granulysin gene polymorphisms. METHODS: GNLY rs7908 and rs10180391 polymorphisms were studied by PCR-RFLP in 100 psoriasis patients under treatment in the Dermatology Polyclinic of Bulent Ecevit University. In addition, 100 healthy individuals with similar age and sex distribution were used as a control group. RESULTS: In the control group, GNLY rs7908 CC genotype was significantly higher than in psoriasis patients (P= 0.031; OR= 0.305; Cl= 0.305 (0.121 - 0.773). In our study, the genotype distributions in patients and control groups were GNLY rs7908 (SNP) GG (51%, 37%), GC (41%, 44%), CC (8%, 19%); GNLY rs10180391 (SNP) from the CC (41%, 44%), CT (42%, % 41), TT (17%, 15%). STUDY LIMITATIONS: The study only included Turkish patients. CONCLUSION: Our findings showed that GNLY rs7908 CC genotype and C allele had a protective effect against psoriasis and decreased the disease severity (according to PASI score), whereas rs10180391 SNP did not show any effective role in psoriasis pathogenesis.

Guidelines for the management of Stevens–Johnson syndrome/toxic epidermal necrolysis: An Indian perspective.

Background: Stevens–Johnson syndrome and toxic epidermal necrolysis are severe, life‑threatening mucocutaneous adverse drug reactions with a high morbidity and mortality that require immediate medical care. The various immunomodulatory treatments include systemic corticosteroids, cyclosporine, intravenous immunoglobulin, cyclophosphamide, plasmapheresis and tumor necrosis factor‑α inhibitors. Aim: The ideal therapy of Stevens– Johnson syndrome/toxic epidermal necrolysis still remains a matter of debate as there are only a limited number of studies of good quality comparing the usefulness of different specific treatments. The aim of this article is to comprehensively review the published medical literature and frame management guidelines suitable in the Indian perspective. Methods: The Indian Association of Dermatologists, Venereologists and Leprologists (IADVL) assigned the task of preparing these guidelines to its special interest group on cutaneous adverse drug reactions. The group performed a comprehensive English language literature search for management options in Stevens–Johnson syndrome/toxic epidermal necrolysis across multiple databases (PubMed, EMBASE, MEDLINE and Cochrane) for keywords (alone and in combination) and MeSH items such as “guidelines,” “Stevens–Johnson syndrome,” “toxic epidermal necrolysis,” “corticosteroids,” “intravenous immunoglobulin,” “cyclosporine” and “management.” The available evidence was evaluated using the strength of recommendation taxonomy and graded using a three‑point scale. A draft of clinical recommendations was developed on the best available evidence which was also scrutinized and critically evaluated by the IADVL Academy of Dermatology. Based on the inputs received, this final consensus statement was prepared. Results: A total of 104 articles (meta‑analyses, prospective and retrospective studies, reviews [including chapters in books], previous guidelines [including Indian guidelines of 2006] and case series) were critically evaluated and the evidence thus gathered was used in the preparation of these guidelines. Recommendations: This expert group recommends prompt withdrawal of the culprit drug, meticulous supportive care, and judicious and early (preferably within 72 h) initiation of moderate to high doses of oral or parenteral corticosteroids (prednisolone 1‑2 mg/kg/day or equivalent), tapered rapidly within 7‑10 days. Cyclosporine (3‑5 mg/kg/day) for 10‑14 days may also be used either alone, or in combination with corticosteroids. Owing to the systemic nature of the disease, a multidisciplinary approach in the management of these patients is helpful.

Effect of hsa-microRNA-218 on granulysin expression / 中国免疫学杂志

Objective:To elucidate the effect of hsa-microRNA-218(hsa-mir-218)on exogenous granulysin (GLS) expression in 293T cells.Methods:Total RNA was extracted from THP-1 cells induced by phorbol 12-myristate 13-acetatefor (PMA), and GLS gene was amplified by RT-PCR, and then cloned into pDsRed-Express-C1 to construct the GLS expression vector pDsRed-GLS.Then 293T cells were co-transfected with pDsRed-GLS and pGenesil-mir-218 (pGenesil-mir-control) and laser confocal microscopy was per-formed 36 h later to detect their co-expression .Total RNA and protein were extracted 48 h post transfection , and RT-PCR and Western blot were performed to detect the effect of hsa-mir-218 on exogenous GLS expression .Results:The GLS expression vector pDsRed-GLS was constructed successfully and laser confocal microscopy indicated that it was co -expressed with the interference vector .Compared with that of cells transfected with pGenesil-mir-control, Western blot showed a markedly decrease of GLS protein expression (50%) in the cells transfected with pGenesil-mir-218.However, GLS mRNA expression remained unchanged .Conclusion: hsa-mir-218 nega-tively regulates GLS expression at a post-transcriptional level , and this provides an experimental basis for future study of mechanism of GLS expression regulated by mir-218 .

Lamotrigine-induced toxic epidermal necrolysis confirmed by in vitro granulysin and cytokine assays

Lamotrigine is a recent medication which is prescribed for various neuropsychiatric conditions. It is generally well-tolerated, but recent pharmacoepidemiological evidence suggests that lamotrigine is associated with risks of developing severe cutaneous drug reactions like toxic epidermal necrolysis (TEN). However, there still remains the diagnostic challenge regarding how to confirm the drug causality in suspected cases. In most cases so far, lamotrigine causality has not been objectively demonstrated, which was possibly due to high risk of oral challenge tests or the lack of useful in vitro drug assays. Here we report a case of lamotrigine-induced TEN, of which the drug causality was confirmed by in vitro granulysin and cytokine assays.

Cloning, expression and bioactivity analysis of human granulysin / 基础医学与临床

Objective To obtain recombinant human granulysin using prokaryotic expression system. Methods Total RNA was extracted from cultured PBMC. Granulysin gene segments were obtained with granulysin-specific primers by RT-PCR and then inserted into pET32a(+) plasmid. After identification by DNA sequence, pET-GN-LY9K and pET-GNLY15K were transferred to E. Coli Rosetta (DE3). The fusion protein was identified by SDS-PAGE and Western blot. The bioactivity of granulysin fusion protein was measured by MTT assay. Results The prokaryotic expression vectors pET-GNLY9K and pET-GNLY15K were successfully constructed. The corresponding protein was highly expressed in E. Coli. Recombinant protein was specifically bound by anti-granulysin antibody. GNLY9K fusion protein significantly inhibited the growth of A549 cells in a dose-dependent manner, while GN-LY15K had little effect on the growth of A549. Conclusion Granulysins with different mw were successfully expressed using prokaryotic expression system, which might be helpful for the further study of granulysin.

Cloning,expression and bioactivity analysis of human granulysin / 基础医学与临床

Objective To obtain recombinant human granulysin using prokaryotic expression system.MethodsTotal RNA was extracted from cultured PBMC. Granulysin gene segments were obtained with granulysin-specific primers by RT-PCR and then inserted into pET32a(+) plasmid. After identification by DNA sequence,pET-GNLY9K and pET-GNLY15K were transferred to E. coli Rosetta (DE3). The fusion protein was identified by SDS-PAGE and Western blot.The bioactivity of granulysin fusion protein was measured by MTT assay.Results The prokaryotic expression vectors pET-GNLY9K and pET-GNLY15K were successfully constructed.The corresponding protein was highly expressed in E.coli. Recombinant protein was specifically bound by anti-granulysin antibody. GNLY9K fusion protein significantly inhibited the growth of A549 cells in a dose-dependent manner,while GNLY15K had little effect on the growth of A549.Conclusion Granulysins with different mw were successfully expressed using prokaryotic expression system,which might be helpful for the further study of granulysin.

Granulysin / 医学研究生学报

Granulysin is a cytotoxic granule protein,colocalizes with perforin and granzymes in the cytolytic granules of human CTL and NK cells. The released granulysin was found in the sera of healthy individuals and patients. Granulysin exhibits potent cytotoxic activity against tumors and a broad panel of microbes, including bacteria, fungi, and parasites. The serum levels of granulysin is well associated with diverse activities of NK cells and CTL in physiological and pathological settings and could be a useful novel serum marker for acute rejection of grafts and the overall status of host cellular immunity.

The cDNA cloning of human granulysin Mr 15000 and Mr 9000 active segments from CTL activated by allogenic antigen / 重庆医科大学学报

Objective; To clone, sequence and analyze the coding sequences of the Mr 15000 and Mr 9000 active segments of natural granulysin derived from human CTLs activated by allogenic antigen ,in order to establish the basis for further purifying and investigating the immune - impairing mechanism of granulysin. Methods; The coding sequences of the Mr 15000 and Mr 9000 granulysin gene were amplified from the total RNA of activated CTLs of healthy person after reverse transcription by Nested - PCR, inserted into pET32a ( + ) vectors and then transformed into E. Coli TOP10, respectively. The recons were identified by PCR, endonuclease digestion and sequencing. Results: The whole coding sequences of the Mr 15000 and Mr 9000 active segments were successfully cloned as expected. The accurate pET32a - Mr 15000 and pET32a - Mr 9000 recons were obtained through Colony - PCR, endonuclease digestion and sequencing. There lied polymorphism on the 119 th amino acid of the product encoded by GLS gene. Conclusion; The coding sequences of the Mr 15000 and Mr 9000 active segments of human granulysin can be obtained and cloned by the methods mentioned above and can be used for subsequent research.

Evaluation synergism of GLS/IL-12 recombinant M.s megmatis to anti-tuberculosis chemotherapy in murine model of M.tuberculosis infection / 重庆医科大学学报

Objective:To evaluate the synergism ofrecombinant M.smegmatis(MS)encoding human granulysin and murine IL-12 to anti-tuberculosis chemotherapy in murine M.tuberculosis M.tuberculosis infection.Methods:Balb/c mice were infected with normal saline, recombinant MS,INH(Isoniazid)+PZA(pyrazinamide),recombinant MS+INH+PZA respectively.The numbers of viable bacteria in the lung and the spleen were counted.The levels of IL12 and IFN-?in serum and IFN-?and TNF-?released from spleen lymphocytes stimulated with PPD were detected with ELISA.Lungs and spleens were prepared for pathological analysis.Results:The recombinant MS group,INH+PZA group,and recombinant MS+INH+PZA group showed significantly reduced number of colony forming units(log CFU/g) compared with saline group.The increase of IL-12 and IFN-?in serum were found in recombinant MS group.IFN-?and TNF-?released from spleen lymphocytes stimulated with PPD in recombinant MS group was higher than that in other groups(P

Eukaryotic expression and identification of recombinant shuttle plasmid for co-expression of human granulysin active peptide and murine IL-12 / 第三军医大学学报

Objective To construct an eukaryotic co-expression plasmid pBM9 carrying human granulysin active peptide and murine IL-12 and determine its expression.Methods The primer pairs including granulysin leader peptide DNA sequence were designed to amplify granulysin from the plasmid pZM03 carrying granulysin gene by polymerase chain reaction(PCR).PCR product was directly cloned into an eukaryotic co-expression plasmid pBudCE4.1 to construct plasmid pBudCE4.1-S9K.pBudCE4.1-S9K plasmid was identified by DNA sequencing.Murine IL-12 gene was subcloned into pBudCE4.1-S9K to construct eukaryotic co-expression plasmid pBudCE4.1-S9K/mIL-12.Mycobacteria replicon Orim from plasmid pZM03 was subcloned into NotⅠsite of pBudCE4.1-S9K/mIL-12 to construct eukaryotic co-expression shuttle plasmid pBM9.pBM9 was tansfected into RAW264.7 cells.RT-PCR was used to detect the mRNA expressions of granulysin and IL-12.The protein expression of them were observed by immunocytochemical method and ELISA respectively.Results RT-PCR identified the expressions of granulysin and mIL-12 in transfected cells and culture supernatant.Immunocytochemical method and ELISA verified the protein expressions.Conclusion The recombinant shuttle plasmid pBM9 is successfully constructed and expressed in vitro,which laid a foundation of gene therapy for tumors with granulysin and mIL-12.

Effect of granulysin (GLS) and IL-12 genes' expression products on proliferation and apoptosis of melanoma B16 cell / 重庆医科大学学报

Objective:To dectect the effect of granulysin and IL-12 genes'expression products on proliferation and apoptosis of melanoma B16 cell in vitro.Methods:Co-expression plasmid including granulysin peptide and murine interleukin 12(mIL-12)genes was transfected into melanoma B16 cell with Lipofectamin TM2000 and its expression products were detected by RT-PCR.Growth suppression was detected with MTT colorimetric assay,and cell apoptotic alterations were evaluated by Hoechst 33258 staining,AO/EB staining,and Annexin V-FITC flow cytometry(FCM).Results:GLS peptite and IL-12 genes could be expressed in B16 cells.Expression products inhibited the proliferation of melanoma cells under MTT observaton.Cells apoptosis with nuclear chromatin condensation,fragmentation and cell membrane change were observed under Hoechst 33258 staining and AO/EB staining.FCM analysis showed the apoptotic rates in test group was 21.02%,which was higher than that in control in control group(15.57%).Conclusion:Expression products of granulysin and mIL-12 genes can not only inhibit proliferation but also induce apoptosis of murine melanoma cell line B16 in vitro.

Effects of Ternatolide on expression granulysin mRNAin the peripheral blood lymphocytes patients with mycobacterium tuberculosis / 中国药理学通报

AIM To study the effects of ternatolide(tern) on expression of granulysin mRNA in the activated peripheral blood lymphocytes(PBLS) of the adults and children with pulmonary Mycobacterium tuberculosis(PMTB). METHODS Using competitive quantitative reverse transcription polymerase chain reaction(QC TR PCR)analyze granulysin gene expression in the PBLs. RESULTS The dosage of 100 mg?L -1 , 200mg?L -1 of ternatolide significantly enhanced the granulysin mRNA experession in PBLs in vitro stimulated phytohemagglutinin (PHA) from adults and children with PMTB (P0 05). CONCLUSION The molecular mechanism of tern. against intracellular pathogens might be associated with the inducting highly level on the expression of granulysin mRNA, a anti bacterial peptide in activated human cytotoxic lymphocytes.

Construction and expression of recombinant bacille calmette-guerin carrying murine interleukin-12 / 重庆医科大学学报

Objective:To construct recombinant bacille calmette-guerin (BCG) carrying murine interleukin-12 (mIL-12) genes,and to identify the ability of recombinant BCG for submitting plasmids to eukaryotic cells. Methods:Co-expression shuttle plasmid of pBM12, including mIL-12 gene and mycobacterial replicon of Orim,was built using molecular biological approach.The pBM12 was transfected to BCG competence by electroporation to construct recombinant BCG. The plasmid of recombinant BCG was extracted for PCR identification. The ability of submitting plasmids to eukaryotic cells was determined by RT-PCR. Results:The plasmid pBM12 may be transduced into BCG with electroporation. The specific bands for mIL-12 of the recombinant BCG were detected by PCR. The specific bands for 1632 bp mIL-12 were amplified from transfected macrophages after 96 hours byRT-PCR. Conclusion:The recombinant BCG carrying pBM12 was constructed successfully. The recombinant BCG can submit the carried genes to mice macrophages.

The distribution and expression of recombinant M.smegmatis in mice receiving intranasal immunization / 重庆医科大学学报

Objective: To observe the distribution and expression of recombinant Mycobacterium smegmatis carrying the plasmid encoding human granulysin(GLS) and murine IL-12 by intranasal immunization in mice.Methods: BALB/C mice were intranasally immunized with recombinant M.smegmatis carrying the plasmid encoding green fluorescent protein(GFP).The distribution of GFP and recombinant Mycobacterium smegmatis in the lungs and spleens was detected 14 days after the first immunization.BALB/C mice were intranasally immunized three times with recombinant M.smegmatis carrying the plasmid encoding GLS and IL-12.28 days after the first immunization,the expression of GLS in tissue,levels of IL-12 in serum and SIgA in BALF(bronchoalveolar lavage fluid)were detected with immunohistochemistry and ELISA respectively.Results: The distribution of GFP and recombinant Mycobacterium smegmatis in lung and spleen was detected.The expression of GLS,the increased of IL-12 in serum and the specific SIgA in BALF were found.Conclusion: The distribution of recombinant Mycobacterium smegmatis carrying the plasmid encoding of GFP in lung and spleen is detected.The specific anti-bacterium SIgA is generated.GLS and IL-12 are expressed in BALB/C mice after intranasal immunization with recombinant Mycobacterium smegmatis carrying the plasmid encoding GLS and IL-12.The results lay the foundation for new vaccine study.

Cytotoxic effects of granulysin on Candida albicans / 第二军医大学学报

Objective:To study the cytotoxic effects of granulysin on Candida albicans.Methods: Candida albicans were cultured with different concentrations of granulysin peptides and the colonies of Candida albicans on Sabouraud dextrose agar were calculated.Broth microdilution method was used to determine the minimum inhibitory concentrations(MIC) of granulysin and fluconazole on Candida albicans.Results: When granulysin peptides 2 and 3(corresponding to G2 and G3 peptides) were at 20 ?g/ml,the average colonies of Candida albicans decreased from 838 and 927 to 203 and 218,respectively;G1,G4 and G5 did not reduce the average colony of Candida albicans even at 40 ?g/ml.Three strains of Candida albicans were sensitive to granulysin,with their MICs being 26,22,29 ?g/ml,and their MICs to fluconazole were 4,20 and 128 ?g/ml.Conclusion: Granulysin has cytotoxic effects on Candida albicans and is one of the natural anti-fungi proteins in human body;it has a promising future for anti-fungi drug development.