Analysis of granulysin expression in vitiligo and halo-nevus.

Vitiligo and halo nevus are immune-mediated skin diseases that have a similar pathogenesis and involve cellular cytotoxicity mechanisms that are not yet fully understood. In this study, we investigated the expression patterns of the cytolytic molecule granulysin (GNLY) in different cytotoxic cells in skin samples of vitiligo and halo nevus. Skin biopsies were taken from perilesional and lesional skin of ten vitiligo patients, eight patients with halo nevus and ten healthy controls. We analysed the expression of GNLY by immunohistochemistry in CD8+ and CD56+ NK cells. A significantly higher accumulation of GNLY+, CD8+ GNLY+ and fewer CD56+ GNLY+ cells was found in the lesional skin of vitiligo and halo nevus than in the healthy skin. These cells were localised in the basal epidermis and papillary dermis, suggesting that GNLY may be involved in the immune response against melanocytes. Similarly, but to a lesser extent, upregulation of GNLY+ and CD8+ GNLY+ cells was observed in the perilesional skin of vitiligo and halo nevus compared to healthy controls. In this study, we demonstrated for the first time an increased expression of CD8+ GNLY+ T lymphocytes and CD56+ GNLY+ NK cells in lesions of vitiligo and halo nevus, indicating the role of GNLY in the pathogenesis of both diseases.

Serum Granulysin as a Possible Key Marker of Vitiligo Activity and Severity.

Background: Vitiligo is an immune-mediated, chronic skin condition that affects both the innate and adaptive immune systems. Antimicrobial peptide overexpression is one of its defining characteristics. Granulysin (GNLY), an antimicrobial peptide, may play a role in the pathogenesis of various autoimmune diseases. Objectives: To estimate the serum GNLY levels in vitiligo patients and to correlate those levels with the severity and activity of the disease. Materials and Methods: This case-control study included 60 non-segmental vitiligo patients (Group A) and a control group of 60 people who were matched for age and sex, appeared to be in good health, and were not suffering from vitiligo (Group B). The serum granulysin levels of all subjects were measured using an enzyme-linked immunosorbent assay. Results: When compared to the control group, vitiligo patients had significantly higher serum GNLY levels (P = 0.001). When compared to patients with stable disease, those with active vitiligo had significantly higher serum GNLY levels (P = 0.008). Additionally, there was a positive correlation between the serum GNLY levels and the vitiligo area severity index and vitiligo disease activity scores (P = 0.004 and <0.001, respectively). Limitations: Study population was relatively small. Evaluation of serum granulysin before and after treatment could have been more beneficial. Conclusions: Blood granulysin levels could contribute to the pathogenesis of vitiligo. A higher serum granulysin level may also be a trustworthy predictor of the severity and progression of a disease.

Granulysin-mediated reduction of PDZRN3 induces Cx43 gap junctions activity exacerbating skin damage in trichloroethylene hypersensitivity syndrome.

Trichloroethylene (TCE)-induced hypersensitivity syndrome (THS) has been a concern for many researchers in the field of environmental and occupational health. Currently, there is no specific treatment for THS, leaving patients to contend with severe infections arising from extensive skin lesions, consequently leading to serious adverse effects. However, the pathogenesis of severe skin damage in THS remains unclear. This study aims to investigate the specific danger signals and mechanisms underlying skin damage in THS through in vivo and in vitro experiments. We identified that cell supernatant containing 15 kDa granulysin (GNLY), released from activated CD3-CD56+NK cells or CD3+CD56+NKT cells in PBMC induced by TCE or its metabolite, promoted apoptosis in HaCaT cells. The apoptosis level decreased upon neutralization of GNLY in the supernatant by a GNLY-neutralizing antibody in HaCaT cells. Subcutaneous injection of recombinant 15 kDa GNLY exacerbated skin damage in the THS mouse model and better mimicked patients' disease states. Recombinant 15 kDa GNLY could directly induce cellular communication disorders, inflammation, and apoptosis in HaCaT cells. In addition to its cytotoxic effects, GNLY released from TCE-activated NK cells and NKT cells or synthesized GNLY alone could induce aberrant expression of the E3 ubiquitin ligase PDZRN3, causing dysregulation of the ubiquitination of the cell itself. Consequently, this resulted in the persistent opening of gap junctions composed of connexin43, thereby intensifying cellular inflammation and apoptosis through the "bystander effect". This study provides experimental evidence elucidating the mechanisms of THS skin damage and offers a novel theoretical foundation for the development of effective therapies targeting severe dermatitis induced by chemicals or drugs.

Involvement of M1-Activated Macrophages and Perforin/Granulysin Expressing Lymphocytes in IgA Vasculitis Nephritis.

We investigated the polarisation of CD68+ macrophages and perforin and granulysin distributions in kidney lymphocyte subsets of children with IgA vasculitis nephritis (IgAVN). Pro-inflammatory macrophage (M)1 (CD68/iNOS) or regulatory M2 (CD68/arginase-1) polarisation; spatial arrangement of macrophages and lymphocytes; and perforin and granulysin distribution in CD3+ and CD56+ cells were visulaised using double-labelled immunofluorescence. In contrast to the tubules, iNOS+ cells were more abundant than the arginase-1+ cells in the glomeruli. CD68+ macrophage numbers fluctuated in the glomeruli and were mostly labelled with iNOS. CD68+/arginase-1+ cells are abundant in the tubules. CD56+ cells, enclosed by CD68+ cells, were more abundant in the glomeruli than in the tubuli, and co-expressed NKp44. The glomerular and interstitial/intratubular CD56+ cells express perforin and granulysin, respectively. The CD3+ cells did not express perforin, while a minority expressed granulysin. Innate immunity, represented by M1 macrophages and CD56+ cells rich in perforin and granulysin, plays a pivotal role in the acute phase of IgAVN.

Assessment of serum granulysin and cathepsin-L levels in vitiligo patients

SUMMARY OBJECTIVE: Cellular and humoral immunity plays a role in the pathogenesis of vitiligo. T lymphocytes and natural killer cells involved in cellular immunity carry out their cytotoxic activities through perforin/granzyme-dependent granule exocytosis, in which granulysin and cathepsin-L are also involved. The aim of this study was to investigate the possible role of serum granulysin and cathepsin-L in the etiopathogenesis of vitiligo and their association with disease activity and severity. METHODS: This randomized, prospective case-control study was conducted with 46 vitiligo patients admitted to the hospital for vitiligo between January and November 2021 and 46 healthy volunteers of similar age and gender. Serum levels of granulysin and cathepsin-L were measured by the enzyme-linked immunosorbent assay method. RESULTS: The mean serum levels of granulysin and cathepsin-L were statistically significantly higher in vitiligo patients compared with the control group (p=0.048 and p=0.024, respectively). There was no statistically significant correlation between serum granulysin and serum cathepsin-L levels and disease severity in the patient group (r=0.30, p=0.062 and r=0.268, p=0.071, respectively). Disease activity also showed no significant association with serum granulysin and cathepsin-L levels (p=0.986 and p=0.962, respectively). CONCLUSION: Although granulysin and cathepsin-L are molecules involved in the pathogenesis of vitiligo, the use of these molecules may not be helpful in assessing disease activity and severity. It may be helpful to conduct comprehensive and prospective studies to find new molecules to fill the gap in this area.

Clinical characteristics and immune profiles of patients with immune-mediated alopecia associated with COVID-19 vaccinations.

BACKGROUND: The clinical characteristics and pathomechanism for immune-mediated alopecia following COVID-19 vaccinations are not clearly characterized. OBJECTIVE: We investigated the causality and immune mechanism of COVID-19 vaccines-related alopecia areata (AA). STUDY DESIGN: 27 new-onset of AA patients after COVID-19 vaccinations and 106 vaccines-tolerant individuals were enrolled from multiple medical centers for analysis. RESULTS: The antinuclear antibody, total IgE, granulysin, and PARC/CCL18 as well as peripheral eosinophil count were significantly elevated in the patients with COVID-19 vaccines-related AA compared with those in the tolerant individuals (P = 2.03 × 10-5-0.039). In vitro lymphocyte activation test revealed that granulysin, granzyme B, and IFN-γ released from the T cells of COVID-19 vaccines-related AA patients could be significantly increased by COVID-19 vaccine excipients (polyethylene glycol 2000 and polysorbate 80) or spike protein (P = 0.002-0.04). CONCLUSIONS: Spike protein and excipients of COVID-19 vaccines could trigger T cell-mediated cytotoxicity, which contributes to the pathogenesis of immune-mediated alopecia associated with COVID-19 vaccines.

Clinical, Biochemical, Genetic, and Therapeutic Profile of Patients with Epidermal Necrolysis: A Descriptive Study.

Background: Epidermal necrolysis (SJS/TEN) is a rare but acute severe drug reaction associated with high morbidity and mortality rates. Aims: To describe the clinical, molecular, biochemical, and therapeutic profile of these patients. Methods: A total of 24 acute SJS/TEN patients were recruited during their hospital stay and detailed clinical history and treatment course recorded. Blood samples collected were subjected to DNA and serum separation for molecular and biochemical analysis. Results: Of 24 patients, 18 (75%) were females and six (25%) were males with six SJS, six SJS-TEN overlap, and 12 TEN cases. The inciting drugs were non-steroidal anti-inflammatory (87.50%; n = 21) followed by antibiotics (66.67%; n = 16), antiepileptics (37.50%; n = 9), and others (37.50%; n = 9). Seventeen patients (77.2%) showed skin eruptions within 7 days after drug intake. Different co-morbidities were observed in 22 (91.6%) and 20 (83.3%) patients showed ocular manifestations. Length of hospital stay ranged from 8 to 55 days, 20 (83.3%) patients were treated with corticosteroids, and four (16.6%) received antimicrobial therapy. Interleukin polymorphisms revealed significantly low frequency of IL-4 in the patients, HLA-A locus typing revealed higher frequency of HLA-A*3301 (20.8%), HLA-A*02 (25%), HLA-A*2402 (14.6%), and sera showed raised levels of granulysin and sFas L in the patients compared to controls. Conclusions: The preliminary study illustrates the clinical, molecular, and biochemical features of acute SJS/TEN and provides a better understanding that helps to improve patient care at an earlier stage. It also highlights the use of corticosteroids and antimicrobial therapy for effective treatment of patients.

Use of the Human Granulysin Transgenic Mice To Evaluate the Role of Granulysin Expression by CD8 T Cells in Immunity To Mycobacterium tuberculosis.

The cytotoxic granules of human NK and CD8 T cells contain the effector molecule granulysin. Although in vitro studies indicate that granulysin is bactericidal to Mycobacterium tuberculosis and human CD8 T cells restrict intracellular M. tuberculosis by granule exocytosis, the role of granulysin in cell-mediated immunity against infection is incompletely understood, in part because a granulysin gene ortholog is absent in mice. Transgenic mice that express human granulysin (GNLY-Tg) under the control of human regulatory DNA sequences permit the study of granulysin in vivo. We assessed whether granulysin expression by murine CD8 T cells enhances their control of M. tuberculosis infection. GNLY-Tg mice did not control pulmonary M. tuberculosis infection better than non-Tg control mice, and purified GNLY-Tg and non-Tg CD8 T cells had a similar ability to transfer protection to T cell deficient mice. Lung CD8 T cells from infected control and GNLY-transgenic mice similarly controlled intracellular M. tuberculosis growth in macrophages in vitro. Importantly, after M. tuberculosis infection of GNLY-Tg mice, granulysin was detected in NK cells but not in CD8 T cells. Only after prolonged in vitro stimulation could granulysin expression be detected in antigen-specific CD8 T cells. GNLY-Tg mice are an imperfect model to determine whether granulysin expression by CD8 T cells enhances immunity against M. tuberculosis. Better models expressing granulysin are needed to explore the role of this antimicrobial effector molecule in vivo. IMPORTANCE Human CD8 T cells express the antimicrobial peptide granulysin in their cytotoxic granules, and in vitro analysis suggest that it restricts growth of Mycobacterium tuberculosis and other intracellular pathogens. The murine model of tuberculosis cannot assess granulysin's role in vivo, as rodents lack the granulysin gene. A long-held hypothesis is that murine CD8 T cells inefficiently control M. tuberculosis infection because they lack granulysin. We used human granulysin transgenic (GNLY-Tg) mice to test this hypothesis. GNLY-Tg mice did not differ in their susceptibility to tuberculosis. However, granulysin expression by pulmonary CD8 T cells could not be detected after M. tuberculosis infection. As the pattern of granulysin expression in human CD8 T cells and GNLY-Tg mice seem to differ, GNLY-Tg mice are an imperfect model to study the role of granulysin. An improved model is needed to answer the importance of granulysin expression by CD8 T cells in different diseases.

Successful Treatment of Primary Cutaneous Anaplastic Large Cell Lymphoma with Denileukin Diftitox.

Primary cutaneous anaplastic large cell lymphoma (PCALCL) is a rare variant of cutaneous T cell lymphoma (CTCL) characterized by CD30-expressing large atypical cells with kidney-shaped nuclei called hallmark cells. Since PCALCL is a rare variant of CTCL, the treatment of PCALCL is still controversial. In this report, a case of PCALCL successfully treated with denileukin diftitox as second-line therapy is described. Interestingly, the administration of denileukin diftitox decreased CD8+ T cells, CD25+ cells, granulysin-bearing lymphocytes, and CD163+ macrophages. The present case suggests that denileukin diftitox might induce an anti-lymphoma effect as well as modulate the tumor microenvironment.

Conjugation of the 9-kDa Isoform of Granulysin with Liposomes Potentiates Its Cytotoxicity.

Nine kDa granulysin (GRNLY) is a human cytolytic protein secreted by cytotoxic T lymphocytes (CTL) and NK cells of the immune system whose demonstrated physiological function is the elimination of bacteria and parasites. In previous studies by our group, the anti-tumor capacity of recombinant granulysin was demonstrated, both in vitro and in vivo. In the present work, we developed lipid nanoparticles whose surfaces can bind recombinant granulysin through the formation of a complex of coordination between the histidine tail of the protein and Ni2+ provided by a chelating lipid in the liposome composition and termed them LUV-GRNLY, for granulysin-bound large unilamellar vesicles. The objective of this formulation is to increase the granulysin concentration at the site of contact with the target cell and to increase the cytotoxicity of the administered dose. The results obtained in this work indicate that recombinant granulysin binds to the surface of the liposome with high efficiency and that its cytotoxicity is significantly increased when it is in association with liposomes. In addition, it has been demonstrated that the main mechanism of death induced by both granulysin and LUV-GRNLY is apoptosis. Jurkat-shBak cells are resistant to GRNLY and also to LUV-GRNLY, showing that LUV-GRNLY uses the mitochondrial apoptotic pathway to induce cell death. On the other hand, we show that LUV-GRNLY induces the expression of the pro-apoptotic members of the Bcl-2 family Bim and especially PUMA, although it also induced the expression of anti-apoptotic Bcl-xL. In conclusion, we demonstrate that binding of GRNLY to the surfaces of liposomes clearly augments its cytotoxic potential, with cell death executed mainly by the mitochondrial apoptotic pathway.

Can granulysin provide prognostic value in primary breast cancer?

BACKGROUND: Granulysin (GNLY) is a cytolytic and proinflammatory molecule which also acts as an immune alarmin. The multifunctional nature of this molecule has made it challenging to define its full potential as a biomarker in breast cancer. AIM: To evaluate the prognostic value of intratumoral GNLY in primary breast cancer patients and its association with established clinicopathological parameters. PATIENTS AND METHODS: The study included 69 node-negative breast cancer patients with known clinicopathological parameters, all of whom had not received any prior hormonal or chemotherapeutic systemic therapy that would interfere with the course of disease. The median follow-up period was 144 months. Steroid hormone receptor status was determined by ligand-binding assay and HER2 status by chromogenic in situ hybridisation (CISH). Intratumoral GNLY mRNA levels were determined by RT-qPCR. Prognostic performance was evaluated by the receiver operating characteristic (ROC), Cox proportional hazards regression and Kaplan-Meier analysis. Classification of patients into GNLYlow and GNLYhigh subgroups was performed by the use of the outcome-oriented cut-off point categorisation approach. RESULTS: There was a significant difference between GNLY values of patients without any recurrences and those with local or distant recurrences (Mann-Whitney test, p = 0.05 and p = 0.02, respectively). None of the tested parameters showed prognostic significance for local and distant recurrences when combined. When distant metastases and local recurrences were separated as events, the best prognostic performance was observed for GNLY as compared with any clinicopathological parameter (AUC=0.24 and p = 0.04 for local events; AUC=0.71 and p = 0.03 for distant events). Local recurrence incidence was 0% for the GNLYhigh subgroup and 19% for the GNLYlow subgroup; however distant recurrence incidence was 24% for the GNLYhigh subgroup but only 3% for the GNLYlow subgroup (Kaplan-Meier analysis). A significant positive correlation was found between intratumoral ER and GNLY levels, and a significant negative correlation between tumour grade and GNLY levels. CONCLUSION: High levels of granulysin prognosticate low risk of local recurrence but a high risk of distant metastasis in primary, untreated, breast cancer patients.

The uterine natural killer cell, cytotoxic T lymphocyte, and granulysin levels are elevated in the endometrium of women with nonstructural abnormal uterine bleeding.

OBJECTIVE: To examine the expression of uterine natural killer (uNK) cells and cytotoxic T lymphocytes (CTLs) in endometrial biopsies from reproductive-age women with and without nonstructural abnormal uterine bleeding (AUB) and evaluate the expression of granulysin within these cell populations and potential modulation of matrix metalloproteinase (MMP) expression. DESIGN: Experimental study, retrospective design. SETTING: Academic research laboratory. PATIENT(S): Patients with nonstructural AUB with no other gynecological pathologies and control patients without AUB. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Immunohistochemical analysis of granulysin, CD56 (uNK cell marker), and CD8 (CTL marker) expression as well as granulysin messenger ribonucleic acid (mRNA) expression levels in endometrial biopsy samples. Assessment of granulysin regulation of human endometrial stromal cell MMP-1 and MMP-3 mRNA expression. RESULT(S): The numbers of uNK cells and CTLs were significantly greater in endometrial biopsy tissue from women with AUB than those from controls. In accord with the increased expression of uNK cells and CTLs, granulysin expression was significantly greater in endometrial biopsies from patients with AUB than in from controls and colocalized to both cell types but not endometrial stromal or epithelial cells. The increased granulysin protein expression was associated with the increased granulysin mRNA expression in adjacent serial sections from these same samples. The treatment of the human endometrial stromal cell line t-HESC with granulysin resulted in a significant increase in MMP-1 and MMP-3 mRNA expression. CONCLUSION(S): In the current study, immunohistochemistry showed an increased expression of uNK cells, CTLs, and granulysin among subjects with AUB compared with that of subjects without AUB, leading to conclusions that disturbances in the balance of immune cells and an increase in granulysin expression may have implications in the pathophysiology of AUB and include enhanced MMP-1 and MMP-3 expression.

Preclinical Studies of Granulysin-Based Anti-MUC1-Tn Immunotoxins as a New Antitumoral Treatment.

Two granulysin (GRNLY) based immunotoxins were generated, one containing the scFv of the SM3 mAb (SM3GRNLY) and the other the scFv of the AR20.5 mAb (AR20.5GRNLY). These mAb recognize different amino acid sequences of aberrantly O-glycosylated MUC1, also known as the Tn antigen, expressed in a variety of tumor cell types. We first demonstrated the affinity of these immunotoxins for their antigen using surface plasmon resonance for the purified antigen and flow cytometry for the antigen expressed on the surface of living tumor cells. The induction of cell death of tumor cell lines of different origin positive for Tn antigen expression was stronger in the cases of the immunotoxins than that induced by GRNLY alone. The mechanism of cell death induced by the immunotoxins was studied, showing that the apoptotic component demonstrated previously for GRNLY was also present, but that cell death induced by the immunotoxins included also necroptotic and necrotic components. Finally, we demonstrated the in vivo tumor targeting by the immunotoxins after systemic injection using a xenograft model of the human pancreatic adenocarcinoma CAPAN-2 in athymic mice. While GRNLY alone did not have a therapeutic effect, SM3GRNLY and AR20.5GRNLY reduced tumor volume by 42 and 60%, respectively, compared with untreated tumor-bearing mice, although the results were not statistically significant in the case of AR20.5GRNLY. Histological studies of tumors obtained from treated mice demonstrated reduced cellularity, nuclear morphology compatible with apoptosis induction and active caspase-3 detection by immunohistochemistry. Overall, our results exemplify that these immunotoxins are potential drugs to treat Tn-expressing cancers.

Upregulated serum granulysin levels in women with antiphospholipid antibody-associated recurrent miscarriage are downregulated by heparin treatment.

Purpose: Granulysin is a cytotoxic protein that simultaneously activates innate and cellular immunity. The authors aimed to evaluate whether granulysin is associated with the antiphospholipid antibody syndrome and whether heparin changes the granulysin levels. Methods: A cohort study was performed with women with antiphospholipid antibody-positive recurrent pregnancy loss (RPL). The authors examined granulysin levels under RPL and evaluated the changes in serum granulysin levels before and 1 week after the commencement of heparin treatment. Results: Serum granulysin levels before heparin treatment were significantly higher in women who tested positive for one or more types of antiphospholipid antibodies (2.75 ± 1.03 vs. 2.44 ± 0.69, p = 0.0341 by Welch's t test), particularly anti-phosphatidylethanolamine antibodies (IgG: 2.98 ± 1.09 vs. 2.51 ± 0.86, p = 0.0013; IgM: 2.85 ± 1.09 vs. 2.47 ± 0.77, p = 0.0024 by Welch's t test). After heparin treatment for 1 week, serum granulysin levels were significantly reduced (p = 0.0017 by the paired t test). The miscarriage rate was significantly higher in women whose serum granulysin levels were not reduced by heparin treatment (p = 0.0086 by Fisher's exact probability test). Conclusion: The results suggest that heparin may reduce the incidence of miscarriage by suppressing serum granulysin levels.

Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis in the Era of Systems Medicine.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are severe mucocutaneous bullous disorders characterized by widespread skin and mucosal necrosis and detachment, which are most commonly triggered by medications. Despite their rarity, these severe cutaneous adverse drug reactions will result in high mortality and morbidity as well as long-term sequela. The immunopathologic mechanisms is mainly cell-mediated cytotoxic reaction against keratinocytes leading to massive skin necrolysis. Subsequent studies have demonstrated that immune synapse composed of cytotoxic T cells with drug-specific human leukocyte antigen (HLA) class I restriction and T cell receptors (TCR) repertoire is the key pathogenic for SJS/TEN. Various cytotoxic proteins and cytokines such as soluble granulysin, perforin, granzyme B, interleukin-15, Fas ligand, interferon-γ, tumor necrosis factor-α have been as mediators involved in the pathogenesis of SJS/TEN. Early recognition and immediate withdrawal of causative agents, and critical multidisciplinary supportive care are key management of SJS/TEN. To date, there is yet to be a sufficient consensus or recommendation for the immunomodulants of the treatment in SJS/TEN. Systemic corticosteroids remain one of the most common treatment options for SJS/TEN, though the efficacy remain uncertain. Currently, there is increasing evidence showing that cyclosporine and TNF-α inhibitors decrease the mortality of SJS/TEN. Further multicenter double-blinded, randomized, placebo-controlled trials are required to confirm the efficacy and safety.

Granulysin expression and granulysin-mediated apoptosis in the peripheral blood of osteoarthritis patients.

Osteoarthritis (OA) is a chronic joint disease caused by mechanical damage and metabolic factors that support the development of low-grade inflammation. Increased levels of T helper 1 pro-inflammatory cytokines in the serum of OA patients may support granulysin (GNLY) mediated cytotoxicity, which in-turn may contribute to the pathogenesis of OA. In the present study, GNLY expression and cytotoxic/apoptotic mechanisms mediated by GNLY in the peripheral blood of OA patients were assessed. A total of 40 non-obese women (median age of 64 years old) with knee OA, and 40 controls (median age 62 years old) were enrolled in the study. GNLY, IFN-γ and IL-4 expression levels were investigated in peripheral blood lymphocytes (PBLs) using flow cytometry, immunocytochemistry and/or confocal microscopy. Natural killer (NK) GNLY-mediated apoptosis through NK effectors against K-562 targets was analyzed using the PKH-26 18-h cytotoxicity assay. Serum GNLY levels were assessed using ELISA. The percentage of GNLY+PBLs was higher in the OA patients than that in the controls due to the increase in the proportions of GNLY+ cells in the natural killer (NK), T and natural killer T (NKT) subsets. GNLY localization inside exocytotic lysosomal-associated membrane protein-1+ granules was ~40% in both groups. However, the intensity of GNLY labeling in PBLs was higher in OA patients than in the controls, and it was supported by the increased expression of IFN-γ relative to IL-4 in NK and T cells from OA patients. The serum GNLY concentration was <0.3 ng/ml in both groups. RC8 anti-GNLY mAb by itself was unable to significantly alter early apoptosis, whereas RC8 anti-GNLY mAb combined with anti-perforin mAb significantly reduced NK-mediated early apoptosis of K-562 targets in the OA patients, whilst not exerting a notable effect in the controls. Anti-perforin mAb by itself did not affect apoptosis significantly. These results suggest that in women with knee OA, GNLY expression in the PBL subsets and GNLY-mediated early apoptosis of K-562 targets are increased compared with the controls and accompanied by intracellular dominance of IFN-γ over IL-4 in NK cells.

The altered HLA-DQ expression in peripheral blood T cells of chronic hepatitis B patients characterizes the function of T cells.

OBJECTIVE: This study aimed to clarify the expression of HLA-DQ and granulysin in peripheral blood T-cell subsets in patients with chronic hepatitis B virus (CHB) and to evaluate their significance in assisting CHB diagnosis and immune status assessment. METHODS: Peripheral blood from 34 CHB patients, 36 inactive HBsAg carriers and 33 healthy controls were collected, and HLA-DQ and granulysin in a series of T-cell subsets were analysed by flow cytometry. The ability to secrete IL-10 and IFN-γ and the functional T-cell subsets were measured in Treg and CD4 cells expressing HLA-DQ or not. Correlation analyses were further conducted between HLA-DQ/granulysin-related subsets and clinical indicators of HBV infection, and ROC curves were built to evaluate diagnosis efficiency of HLA-DQ-related subsets. RESULTS: HLA-DQ+ percentages in circulating CD4 T cells were downregulated in CHB patients. The proportions of HLA-DQ + Tfh in CHB were upregulated while HLA-DQ+ percentages in Treg were decreased. In terms of function, the IFN-γ secretion ability of CD4 + T cells and IL-10 secretion in Tregs were stronger in HLA-DQ+ than HLA-DQ- subsets. HLA-DQ + CD4 + T cells and HLA-DQ + Treg were negatively correlated with HBV-DNA, while HLA-DQ + Tfh and Tfc cells were positively correlated with HBV-DNA and ALT. HLA-DQ + Treg/Tfh/Tfc could help to distinguish CHB from inactive HBsAg carriers. CONCLUSION: HLA-DQ on T cells can characterize the function of T-cell subsets and analysis of HLA-DQ can help to evaluate immune status and assist in diagnosis of CHB.

Improved Purification of Human Granzyme A/B and Granulysin Using a Mammalian Expression System.

Cytotoxic lymphocytes release proteins contained within the cytoplasmic cytolytic granules after recognition of infected or tumor target cells. These cytotoxic granular proteins (namely granzymes, granulysin, and perforin) are key immunological mediators within human cellular immunity. The availability of highly purified cytotoxic proteins has been fundamental for understanding their function in immunity and mechanistic involvement in sepsis and autoimmunity. Methods for recovery of native cytotoxic proteins can be problematic leading to: 1) the co-purification of additional proteins, confounding interpretation of function, and 2) low yields of highly purified proteins. Recombinant protein expression of individual cytolytic components can overcome these challenges. The use of mammalian expression systems is preferred for optimal post-translational modifications and avoidance of endotoxin contamination. Some of these proteins have been proposed for host directed human therapies (e.g. - granzyme A), or treatment of systemic infections or tumors as in granulysin. We report here a novel expression system using HEK293T cells for cost-effective purification of high yields of human granzymes (granzyme A and granzyme B) and granulysin with enhanced biological activity than previous reports. The resulting proteins are free of native contaminants, fold correctly, and remain enzymatically active. Importantly, these improvements have also led to the first purification of biologically active recombinant human granulysin in high yields from a mammalian system. This method can be used as a template for purification of many other secreted cellular proteins and may lead to advances for human medicine.

GNLY gene polymorphism: A potential role in understanding psoriasis pathogenesis.

BACKGROUND: Psoriasis is a chronic inflammatory illness that affects 2%-3% of the world's population. Granulysin (GNLY), along with granzyme and perforin, is a cytolytic antimicrobial peptide (AMP) released from the granules of both natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). By immunohistochemistry, it has been shown that GNLY expression increases in psoriasis lesions. AIMS: Our work aimed to reveal the potential effect of GNLY gene polymorphism and its association with psoriasis pathogenesis. METHODS: This case-control research included 25 patients with psoriasis vulgaris and 25 healthy people who were age and gender matched. History taking and medical examination were done for all patients. Venous blood samples were withdrawn, DNA was extracted, and real-time PCR was done. RESULTS: There was an insignificant difference between psoriasis cases and controls as regards gene sequence (p > 0.05). The polymorphism of GNLY gene regarding genotype distribution G/G was detected in seven patients with psoriasis (28%) and in only two controls (8%). This clarifies that GNLY rs 7908 G/G gene polymorphism may be a strong diagnostic tool for psoriasis. CONCLUSIONS: In patients with psoriasis, the GNLY rs7908 CC genotype alongside with C allele played a preventative function and reduced disease severity (as shown by PASI score). Detection of GG genotype points out that a psoriatic case will run a progressive course, will have a severe pattern in the future, and will develop multiple site affection.

GRANULYSIN PEPTIDE AND GENE POLYMORPHISM IN THE PATHOGENESIS OF HASHIMOTO THYROIDITIS.

Background: Hashimoto thyroiditis (HT) is an autoimmune disease and the most common cause of hypothyroidism. The widespread lymphocyte infiltration in the thyroid gland and intolerance of the body against its thyroid antigens leads to the destruction of thyroid cells and impaired thyroid function. Granulysin (GNLY) is a cytolytic antimicrobial peptide that has been associated with a wide range of diseases such as various infections, cancer, transplantation, and skin problems. However, there are a few studies investigating the relationship between HT and granulysin. Aim: Our study aims to investigate whether granulysin levels and GNLY gene polymorphism contribute to the damaged immune response leading to HT. Material and Methods: 100 unrelated patients diagnosed with HT and 140 healthy individuals were included in our study. Frequencies of GNLY rs10180391 and rs7908 gene polymorphisms were determined using PCR- RFLP method and serum granulysin levels were determined using ELISA. Results: There is no statistical significance between patient and control groups in terms of genotype and allele frequencies of GNLY gene polymorphisms and serum levels of granulysin. Conclusion: In conclusion, granulysin and GNLY gene polymorphisms do not appear to relate to HT disease.