Tissue Levels of Stefin A and Stefin B in Hepatocellular Carcinoma

Stefins have been reported to be associated with the progression and metastasis of various malignant tumors. However, the expressions of stefins in hepatocellular carcinoma (HCC) have not been well‐defined. In this study, the protein levels of stefin A and stefin B were assessed by immunohistochemical staining, and the mRNA levels were quantified by real‐time polymerase chain reaction in 85 primary HCC tissues, 85 surrounding non‐cancerous tissues, and 9 normal hepatic tissues. The immunohistochemical staining of cathepsin B and cathepsin D, and the ratio of cathepsins to stefins were assessed. The mRNA expressions of stefin A and stefin B in HCC tissues were significantly higher than surrounding noncancerous tissues and normal hepatic tissues, respectively. A significant positive relationship of stefin A and stefin B was found with node metastasis, tumor size, and Edmondson grade for HCC. Univariate and multivariate analyses revealed that Edmondson grade and stefin B expression were independent factors associated with the risk of lymph node metastasis in HCC. The ratios of cathepsin B to stefin A, cathepsin D to stefin A, cathepsin B to stefin B and cathepsin D to stefin B of the HCC group were significantly higher than that of the surrounding noncancerous group. A significant positive correlation between the ratio of cathepsins to stefins (cathepsin B/stefin A, cathepsin B/stefin B and cathepsin D/stefin B) and node metastasis was demonstrated. We concluded that high expressions of stefin A and stefin B may be an important factor contributing to the development and metastasis of HCC. Anat Rec, 299:428–438, 2016. © 2016 Wiley Periodicals, Inc.     

Myoepithelial cell‐specific expression of stefin A as a suppressor of early breast cancer invasion

Mammography screening has increased the detection of early pre‐invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV‐PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early‐stage tumorigenesis. To characterize the cell‐specific roles of cathepsins in early invasion, we developed a DCIS‐like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial‐specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial‐induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A‐low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA‐074 rescued the DCIS‐like non‐invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138‐patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low‐grade DCIS but reduced in high‐grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Histopathological analysis of cellular localization of cathepsins in abdominal aortic aneurysm wall

An important feature of abdominal aortic aneurysm (AAA) is the destruction of vessel wall, especially elastin and collagen. Besides matrix metalloproteinases, cathepsins are the most potent elastolytic enzymes. The expression of cathepsins with known elastolytic and collagenolytic activities in the individual cells within AAA has not yet been determined. The vessel wall of 32 AAA patients and 10 organ donors was analysed by immunohistochemistry for expression of cathepsins B, D, K, L and S, and cystatin C in all cells localized within AAA. Luminal endothelial cells (ECs) of AAA were positive for cathepsin D and partially for cathepsins B, K and S. Endothelial cells of the neovessels and smooth muscle cells in the media were positive for all cathepsins tested, especially for cathepsin B. In the inflammatory infiltrate all cathepsins were expressed in the following pattern: B > D = S > K = L. Macrophages showed the highest staining intensity for all cathepsins. Furthermore, weak overall expression of cystatin C was observed in all the cells localized in the AAA with the exception of the ECs. There is markedly increased expression of the various cathepsins within the AAA wall compared to healthy aorta. Our data are broadly consistent with a role for cathepsins in AAA; and demonstrate expression of cathepsins D, B and S in phagocytic cells in the inflammatory infiltrate; and also may reveal a role for cathepsin B in lymphocytes.     

Expression of Cathepsin B,D and L Protein in Juvenile Idiopathic Arthritis

Juvenile idiopathic arthritis (JIA) is the most common childhood autoimmune rheumatic disease and like rheumatoid arthritis (RA), it is characterized by inflammation and the progressive destruction of joints. In RA, cathepsins as proteinases play a major role in destroying synovial tissue and cartilage matrix. So far no data on cathepsin expression in pannus tissue of JIA patients exist. The aim of this study was to characterize the expression levels of cathepsins B, D, H, and L in JIA and to compare them with those in RA. Synovectomy tissue from 16 JIA and 12 RA patients was investigated for cathepsin expression levels by Western blot analysis. Expression of cathepsins B, D and L was on comparable levels in the synovectomy tissue of JIA and RA patients. The following graduation of expression was determined: cathepsin D>cathepsin L>cathepsin B. Cathepsin H was neither found to be expressed in JIA nor in RA patients. The expression levels of cathepsins in pannus tissue showed no clear difference between patients with systemic JIA and patients with monoarticular JIA. In summary, the comparable expression of cathepsins B, D and L in RA and JIA synovectomy tissue suggests that they may play a similarly important role in destroying synovial tissue and cartilage matrix in the course of JIA and RA.     

Expression of cathepsin B,D and L protein in juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common childhood autoimmune rheumatic disease and like rheumatoid arthritis (RA), it is characterized by inflammation and the progressive destruction of joints. In RA, cathepsins as proteinases play a major role in destroying synovial tissue and cartilage matrix. So far no data on cathepsin expression in pannus tissue of HA patients exist. The aim of this study was to characterize the expression levels of cathepsins B, D, H, and L in HA and to compare them with those in RA. Synovectomy tissue from 16 HA and 12 RA patients was investigated for cathepsin expression levels by Western blot analysis. Expression of cathepsins B, D and L was on comparable levels in the synovectomy tissue of HA and RA patients. The following graduation of expression was determined: cathepsin D > cathepsin L > cathepsin B. Cathepsin H was neither found to be expressed in HA nor in RA patients. The expression levels of cathepsins in pannus tissue showed no clear difference between patients with systemic JIA and patients with monoarticular JIA. In summary, the comparable expression of cathepsins B, D and L in RA and JIA synovectomy tissue suggests that they may play a similarly important role in destroying synovial tissue and cartilage matrix in the course of HA and RA.     

Expression of cysteine proteinases cathepsins B and K and of cysteine proteinase inhibitor cystatin C in giant cell tumor of tendon sheath.

The expression of cysteine proteinases cathepsins B and K and of the endogenous inhibitor of cysteine proteinases, cystatin C, was investigated in tissue specimens of patients with giant cell tumor of tendon sheath (GCTTS). Expression of both enzymes was examined by immunohistochemistry in tissue specimens of 14 patients with GCTTS. Applying double-labeling techniques, the coexpression of cathepsin B and its major endogenous inhibitor cystatin C was additionally studied. Cells expressing the respective proteins were further characterized with the macrophage markers HAM56 and anti-CD68 (clone PG-M1). Cathepsin B could be detected in numerous HAM56-positive mononuclear cells (MC), but only in very few giant cells (GC). In contrast, cathepsin K was predominantly identified in GC that were also strongly immunoreactive for cystatin C and CD68. Coexpression of cathepsin B and cystatin C occurred only in a few MC. The strong expression of both cathepsin B and K suggests that in GCTTS, bone erosion might be mediated not only by pressure of the proliferative tissue, but also by matrix-degrading cysteine proteinases. Because previous studies showed that osteoclasts express high levels of CD68, cathepsin K, and cystatin C but not of cathepsin B, our study contributes to the view that GC of GCTTS and osteoclasts are closely associated.     

Placental Interleukin‐15 Expression in Recurrent Miscarriage

Citation Toth B, Haufe T, Scholz C, Kuhn C, Friese K, Karamouti M, Makrigiannakis A, Jeschke U. Placental Interleukin‐15 expression in recurrent miscarriage. Am J Reprod Immunol 2010; 64: 402–410 Problem Cytokines play a fundamental role at the feto–maternal interface. A dysregulation of the cytokine profile could be linked with fetal rejection. This study investigated differences in the expression of the Th1 cytokine interleukin‐15 (IL‐15) of normal and disturbed pregnancy. Method of study Analysis of IL‐15 expression by immunohistochemistry and real‐time RT‐PCR in placental tissue from normal pregnancies, spontaneous miscarriage and recurrent miscarriage (RM) was performed. Results Expression of IL‐15 was upregulated on mRNA and protein level in the placenta of miscarriage patients, especially in patients with RM. Immunohistochemistry and immunofluorescence double staining revealed extravillous trophoblast cells (EVT) as IL‐15‐expressing cells. Staining results were confirmed by real‐time PCR (TaqMan). Conclusion Increased expression of IL‐15 in the decidua of patients with RM may be linked to disturbed implantation and vascularization with consecutive placental and fetal rejection.     

Leukocyte activation in the decidua of chromosomally normal and abnormal fetuses from women with recurrent abortion

As part of our continuing programme to investigate immunological causes of unexplained recurrent pregnancy losses, we studied subpopulations of white blood cells and their activation status in decidua of women with a history of recurrent spontaneous abortion (RSA). We differentiated specifically between normal karyotyped male fetuses and abnormal karyotyped fetuses with trisomy 16 because trisomy 16 is not compatible with life and is thus a non-controversial cause of spontaneous miscarriage. Leukocytes were counted in paraffin-embedded decidua after immunohistological staining for CD45 (LCA), CD3, CD56, CD68, CD69 and CD25. Numbers of activated versus non-activated T lymphocytes, NK cells and macrophages were compared in decidua from women with: (i) unexplained RSA who had a normal male karyotype (n = 17) miscarriage; (ii) unexplained RSA who had a trisomy 16 (n = 21) miscarriage; and (iii) normal gestationally age-matched first trimester pregnancies following elective termination procedures (n = 20). Significantly more activated leukocytes were detected in the decidua of women with unexplained RSA who had a normal male karyotype compared to the other groups (P < 0.0001). In addition, numbers of cells comprising the major leukocyte subpopulation, CD56+ NK cells, appeared reduced in the decidua of women with unexplained RSA compared to decidua from women having elective terminations. Increased numbers of activated leukocytes in the decidua of women with a history of unexplained recurrent pregnancy loss who had a normal karyotyped pregnancy provide evidence that cellular immunity may be involved in unexplained recurrent pregnancy loss.     

Gene deletion of cystatin C aggravates brain damage following focal ischemia but mitigates the neuronal injury after global ischemia in the mouse

Cystatin C is distributed in all human tissues and fluids with a particular abundance in the cerebrospinal fluid. Cystatin C is a strong endogenous inhibitor of lysosomal cysteine proteases, such as cathepsin B, L, H and S, that are involved in various biological processes such as degradation of cellular proteins and regulation of enzymes, as well as in pathological processes. Pharmacological inhibition of cathepsins has been shown to reduce neuronal damage after brain ischemia, suggesting that cystatin C is an endogenous neuroprotectant. Cystatin C has also amyloidogenic properties and is co-localized with beta-amyloid in degenerated neurons in Alzheimer's disease, suggesting a role in neuronal degeneration. To test the hypothesis that endogenous cystatin C is neuroprotective during brain ischemia, global and focal brain ischemia was induced in mice with the cystatin C gene knocked out. Following focal ischemia, larger brain infarcts were found in cystatin C knockout mice, probably due to a reduced inhibition of the cathepsins during ischemia. In contrast, brain damage after global ischemia was diminished in cystatin C knockout mice, suggesting that cystatin C has an aggravating effect on selective neuronal damage after global ischemia.     

The expression of cathepsins in osteoclast-like giant cells of an anaplastic thyroid carcinoma with tracheal perforation

Anaplastic carcinoma of the thyroid gland (ACT) is a rapidly growing neoplasm with a very poor prognosis. In this study, we examined an ACT with osteoclast-like giant cells expressing matrix--degrading cysteine proteinases and their endogeneous inhibitor cystatin C. Bronchoscopic evaluation of a 50-year-old man suffering from hoarseness, dysphagia, and dyspnea revealed an irregular tumor mass infiltrating into the trachea and the cricothyroid ligament region. On histological examination, a necrotizing undifferentiated anaplastic carcinoma with osteoclast-like giant cells was detected. An immunohistochemical study of the tumor tissue was performed using a panel of 15 antibodies, including double labeling techniques. Most of the osteoclast-like multinucleated giant cells (MGC) expressed CD68 and cathepsin K. Colocalization of cathepsin B and its endogenous inhibitor cystatin C occurred in the majority of MGC. Mononuclear cells (MC) were positive for cathepsin B, cystatin C, and CD 68, but only faintly for cathepsin K. Expression of cathepsins B and K in the MGC of the ACT might contribute to the invasive behavior of this tumor, thus promoting metastatic ability and destruction of the cartilagenous trachea.     

Differential expression of cysteine and aspartic proteases during progression of atherosclerosis in apolipoprotein E-deficient mice

Several groups of proteolytic enzymes are able to degrade components of the extracellular matrix. During atherosclerosis, matrix remodeling is believed to influence the migration and proliferation of cells within the plaque. In the present study, gene expression of several proteases and their inhibitors was analyzed during the development of atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. Quantitative real-time polymerase chain reaction was used to study gene expression of proteases after 10 and 20 weeks in ApoE-/- and C57BL/6 mice and in atherosclerotic lesions and nonaffected regions of the same ApoE-/- mouse. Some of the differentially expressed proteolytic enzymes were studied by immunohistochemistry. The matrix metalloproteinase (MMP)-9 and its inhibitor TIMP-1 were differentially expressed and the expression increased with time. Urokinase-type plasminogen activator showed no major expression. In contrast, cathepsins B, D, L, and S all showed strong and increased expression in ApoE-/- mice compared to C57BL/6 mice whereas the expression of their inhibitor, cystatin C, did not differ between the two mouse strains. The expression of cathepsins was mainly localized to the lesions and not to nonaffected regions of the aorta of ApoE-/- mice. Furthermore, cathepsin expression was similar to the expression of the macrophage marker macrosialin (CD68) although expression of cathepsins B, D, and L could be demonstrated in healthy C57BL/6 mice and in nonaffected vessel segments of atherosclerotic ApoE-/- mice. Cathepsin S mRNA expression was restricted to lesions of ApoE-/- mice. Furthermore, cathepsin S was the only cathepsin that was expressed in the media and absent in lipid-rich regions. All cathepsins studied showed intimal expression, the degree and localization of which differed between individual cathepsins. In conclusion, increased expression of several cathepsins in atherosclerotic lesions suggests that these proteases may participate in the remodeling of extracellular matrix associated with the atherosclerotic process.     

Cystein Proteinase Inhibitor Stefin A as an Indicator of Efficiency of Tumor Treatment in Mice

The concentration of stefin A (cystatin A in mice) was measured in animals with experimental tumors (LS lymphosarcoma, HA-1-hepatoma, and Lewis lung carcinoma) during effective antitumor therapy. In mice with these tumors serum concentrations of stefin A increased, while the concentration of cystatin C (extracellular cystein proteinase inhibitor) decreased. The concentration of stefin A in tumor tissue in Lewis lung carcinoma was higher than in LS lymphosarcoma and HA-1-hepatoma ascitic cells, which can be explained by the degree of their malignancy. The content of stefin A in tumor tissue was similar to that in the liver and spleen of tumor-bearing animals, while its concentration in the liver and spleen of tumor-bearing animals was lower than in intact mice. The level of stefin A is an important marker of malignancy and an indicator of the efficiency of antitumor therapy.     

IL-6-STAT3 controls intracellular MHC class II alphabeta dimer level through cathepsin S activity in dendritic cells

We found IL-6-STAT3 pathway suppresses MHC class II (MHCII) expression on dendritic cells (DCs) and attenuates T cell activation. Here, we showed that IL-6-STAT3 signaling reduced intracellular MHCII alphabeta dimmer, Ii, and H2-DM levels in DCs. IL-6-mediated STAT3 activation decreased cystatin C level, an endogenous inhibitor of cathepsins, and enhanced cathepsin activities. Importantly, cathepsin S inhibitors blocked reduction of MHCII alphabeta dimer, Ii, and H2-DM in the IL-6-treated DCs. Overexpression of cystatin C suppressed IL-6-STAT3-mediated increase of cathepsin S activity and reduction of MHCII alphabeta dimer, Ii, and H2-DM levels in DCs. Cathepsin S overexpression in DCs decreased intracellular MHCII alphabeta dimer, Ii, and H2-DM levels, LPS-mediated surface expression of MHCII and suppressed CD4(+) T cell activation. IL-6-gp130-STAT3 signaling in vivo decreased cystatin C expression and MHCII alphabeta dimer level in DCs. Thus, IL-6-STAT3-mediated increase of cathepsin S activity reduces the MHCII alphabeta dimer, Ii, and H2-DM levels in DCs, and suppresses CD4(+) T cell-mediated immune responses.     

Cathepsin B in gingival crevicular fluid of adult periodontitis patients: Identification by immunological and enzymological methods

Cathepsin B (EC 3.4.22.1), a typical lysosomal cysteine proteinase was identified immunologically with anti-human cathepsin B antibody in inflammatory exudate, gingival crevicular fluid (GCF) of adult periodontitis patients. The sensitive enzyme immunoassay (EIA) system initially developed, was rarely influenced by the presence of endogenous, cysteine proteinase inhibitors, cystatin(s), indicating that it is possible to quantify the gross amount of cathepsin B including free enzyme forms and enzyme-inhibitor complex forms using this EIA system. The cathepsin B levels in, GCF as determined by EIA and the activity measured with Z-Arg-Arg-MCA showed positive and significant correlation with various clinical parameters. Immunoblotting analysis revealed that the molecular form was a 29 kDa mature enzyme. More than 95% of Z-Arg-Arg-MCA hydrolytic activity in each GCF sample was inhibited by CA-074, specific inhibitor of cathepsin B. These results strongly suggested that the gross amount of cathepsin B in GCF as well as its activity level is closely associated with the severity of the disease and that cathepsins B play an important role in the pathogenesis of periodontitis.accepted by W. B. van den Berg     

Multiple thrombophilic gene mutations rather than specific gene mutations are risk factors for recurrent miscarriage

PROBLEM: Recurrent miscarriage is a heterogeneous condition. While the role of acquired thrombophilia has been accepted as an etiology of recurrent miscarriage, the contribution of specific inherited thrombophilic genes to this disorder has remained controversial. We compared the prevalence of 10 thrombophilic gene mutations among women with a history of recurrent miscarriages and fertile control women. METHOD OF STUDY: A total of 150 women with a history of two or more recurrent pregnancy losses and 20 fertile control women with no history of pregnancy losses had buccal swabs taken for DNA analyses of 10 gene mutations [factor V G1691A, factor V H1299R (R2), factor V Y1702C, factor II prothrombin G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, HPA1 a/b (L33P), MTHFR C677T, MTHFR A1298C]. The prevalence of these mutations was compared between women experiencing recurrent miscarriage and controls. RESULTS: No differences in the frequency of specific gene mutations were detected when women with recurrent miscarriage were compared with control women. However, the prevalence of homozygous mutations and total gene mutations among patients with recurrent miscarriage was significantly higher than among controls. Homozygous mutations were found in 59% of women with a history of recurrent pregnancy loss contrasted to 10% of control women. More than three gene mutations among the 10 genes studied were observed in 68% of women with recurrent miscarriage and 21% of controls. CONCLUSION: Inherited thrombophilias are associated with recurrent miscarriage. This association is manifest by total number of mutations rather than specific genes involved.     

Global analysis of differentially expressed genes in early gestational decidua and chorionic villi using a 9600 human cDNA microarray

The global gene expression profiles of the decidua and chorionic villi of early human pregnancies were analysed by using cDNA microarray technology. Decidual and villous placental tissues were obtained from first trimester abortus and mRNA was extracted for cDNA microarray analysis. The human cDNA microarray [9600 clones, including known regulatory genes and expressed sequence tags (EST)] with colorimetric detection was used to identify differentially expressed genes between early gestational decidua and villi. According to cDNA microarray analysis, we have identified 641 genes with highly expressed mRNA in both decidua and villi, 49 genes with higher expressions in decidua, and 75 genes with higher expression in chorionic villi. These differentially expressed genes were further grouped into categories by their putative functions, including: cell growth-related factors, hormones/cytokines, cell adhesion molecules, signal transduction molecules, apoptosis-related factors, cytoskeleton/extracellular matrix proteins, and EST. Immunohistochemical stainings of cathepsin L, leukaemia inhibitory factor-receptor, and proliferative cell nuclear antigen showed results consistent with the microarray data. Identification of the differentially expressed genes between decidua and villi by microarray provide a global profiling of the gene expression pattern. This work adds to our understanding of placentation by reporting the gene expression profiles during first trimester human pregnancies using cDNA microarray.     

Complement as a predictor of further miscarriage in couples with recurrent miscarriages

BACKGROUND: The clinical significance of complement is unclear in patients with unexplained recurrent miscarriage, though low levels of complement 3 (C3) and/or complement 4 (C4) are reported to be associated with the antiphospholipid syndrome (aPS). We therefore investigated whether C3 and C4 have a predictive value for subsequent miscarriages. METHODS: In total, 215 patients with a history of two consecutive first-trimester miscarriages and no abnormal chromosomes in either partner, no uterine anomalies and no antiphospholipid (aPL) antibodies were examined. Blood tests for C3, C4, total haemolytic complement (CH50), immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) were performed before subsequent pregnancy. Patients were then followed up without treatment, and their pregnancy outcomes were compared with their previous blood test results. RESULTS: From 215 pregnant patients, 45 subsequently miscarried, whereas the remainder had a live birth. There was no relation with serum CH50, IgG, IgA and IgM levels, but C3 and C4 levels in patients with subsequent miscarriage were significantly higher than in those whose pregnancy was successful. CONCLUSION: In patients with two previous miscarriages, C3 and C4 levels were higher in those women who had a third miscarriage, than in women that went on to have a live birth.     

Synovial giant cells in rheumatoid arthritis: expression of cystatin C, but not of cathepsin B.

This study was designed to investigate the expression of the matrix degrading proteinase cathepsin B and its endogenous inhibitor cystatin C in rheumatoid arthritis (RA) with special regard to multinucleated synovial giant cells (SGC). We applied an immunohistochemical double-labeling technique. SGC strongly expressed cystatin C and CD68, but were negative for cathepsin B. This staining pattern occurred in osteoclasts as well. Our findings support the idea that in RA matrix destruction by cathepsin B is not mediated by SGC or osteoclasts, but by mononuclear synoviocytes.     

Increased expression of elastolytic cysteine proteases, cathepsins S and K, in the neointima of balloon-injured rat carotid arteries

The matrix-degrading activity of several proteases are involved in the accelerated breakdown of extracellular matrix associated with vascular remodeling during the development of atherosclerosis and vascular injury-induced neointimal formation. Previous studies have shown that the potent elastolytic cysteine proteases, cathepsins S and K, are overexpressed in atherosclerotic lesions in human and animal models. However, the role of these cathepsins in vascular remodeling remains unclear. In the present study, the expressions of cathepsin S and K and their inhibitor cystatin C were examined during arterial remodeling using a rat carotid artery balloon-injury model. The increase in both cathepsin S and K mRNA levels was observed from day 1 and day 3 through day 14 following the induction of balloon injury, respectively. Western blotting analysis revealed that both cathepsin S and K protein levels also increased in the carotid arteries during neointima formation, coinciding with an increase elastolytic activity assayed using Elastin-Congo red, whereas, no significant change in the expressions of cystatin C mRNA and protein was observed during follow-up periods after injury. Immunohistochemistry, Western blot, and in situ hybridization showed that the increase of cathepins S and K and the decrease of cystatin C occurred preferentially in the developing neointima. These findings suggest that cathepsin S and K may participate in the pathological arterial remodeling associated with restenosis.