Modified human beta 2-microglobulin (desLys(58)) displays decreased affinity for the heavy chain of MHC class I and induces nitric oxide production and apoptosis.

Beta2-microglobulin (beta2m) is the light chain of major histocompatibility complex class I (MHC-I) molecules, and is a prerequisite for the binding of peptides to the heavy chain and their presentation to CD8+ T cells. beta2m can be modified in vivo and in vitro by proteolytic cleavage by complement C1 and subsequent carboxypeptidase B-like activity--processes that lead to the generation of desLys(58) beta2m (dbeta2m). This work aims to study the effect of dbeta2m on peptide binding to MHC-I, the influence of dbeta2m on the binding of beta2m to the MHC-I heavy chain and the biological activity of dbeta2m. Both beta2m and dbeta2m are able to support the generation of MHC-I/peptide complexes at 18 degrees C, but complexes formed in the presence of dbeta2m destabilize at 37 degrees C. Moreover, a 250 times higher concentration of dbeta2m than of beta2m is needed to displace MHC-I associated beta2m from the cell surface. In addition, only beta2m is able to restore MHC-I/peptide complex formation on acid-treated cells whereas dbeta2m appears to bind preferentially to denatured MHC-I heavy chains. In cell cultures, exogenously added dbeta2m, but not beta2m, induces apoptotic cell death in monocytic leukaemic cell lines but spares other kinds of leukaemic cells. Additionally, the presence of dbeta2m, and to a lesser extent beta2m, enhances IFN-gamma-induced NO production by monocytic leukaemic cells. In conclusion, these data show that dbeta2m is not able to support the formation of a stable tri-molecular MHC-I complex at physiological temperature and that dbeta2m exerts other biological functions compared to beta2m when bound to cells.

Calcium-independent phospholipase A2 regulates retinal pigment epithelium proliferation and may be important in the pathogenesis of retinal diseases.

Calcium-independent phospholipase A2, group VIA (iPLA2-VIA) is involved in cell proliferation. This study aimed to evaluate the role of iPLA2-VIA in retinal pigment epithelium (RPE) cell proliferation and in retinal diseases involving RPE proliferation. A human RPE cell line (ARPE-19) was used to explore this role in vitro. Proliferating ARPE-19 cells had increased expression and activity of iPLA2-VIA. iPLA2-VIA was found in the nuclei of proliferating ARPE-19 cells, whereas in confluent ARPE-19 cells, with limited proliferation, iPLA2-VIA was primarily found in the cytosol. Inhibition of iPLA2-VIA decreased the rate of proliferation, whereas over expression of iPLA2-VIA increased the rate of proliferation. Using an experimental porcine model of RPE proliferation we demonstrated significant nuclear upregulation of iPLA2-VIA in proliferating RPE cells in vivo. We furthermore evaluated the expression of iPLA2-VIA in proliferative vitreoretinopathy (PVR). PVR membranes revealed nuclear expression of iPLA2-VIA in the RPE cells which had migrated and participated in the formation of the membranes. Overall, the present results point to an important role of iPLA2-VIA in the regulation of RPE proliferation suggesting that iPLA2-VIA may be considered as a possible pharmaceutical target in retinal diseases involving RPE proliferation and migration.

Cellular expression or binding of desLys58-beta2 microglobulin is not dependent on the presence of the tri-molecular MHC class I complex.

The monoclonal antibody 332-01 is a newly developed antibody which specifically recognizes human desLys58-beta2 microglobulin (dbeta2m). In the present study, we characterized the binding of 332-01 to peripheral blood mononuclear cells (PBMC), a number of human leukaemic and monocytic cell lines, and beta2m gene-deleted murine lymphocytes. dbeta2m was found to be expressed on non-activated and activated monocytes. When cells were pre-exposed to dbeta2m, 332-01 also bound to non-activated T lymphocytes. dbeta2m was expressed on the monocytic cell lines U937 and TIB-202, and binding was significantly increased when cells were pre-incubated with dbeta2m and when TIB-202 cells were exposed to lipopolysaccharide. dbeta2m was also expressed on T leukaemic Jurkat cells as well as on low HLA-expressing erythroleukaemic K562 cells. beta2m gene-deleted murine splenocytes only bound 332-01 after pre-exposure to dbeta2m. Binding of 332-01 antibody could not be displaced by addition of high concentrations of native beta2m. In conclusion, our data indicate that dbeta2m - in contrast to native beta2m - binds to a hitherto unknown cell surface receptor independent of classical MHC class I molecules. As beta2m has previously been shown to display biological activities such as the induction of both growth promotion and apoptosis, C1 complement activity, shown to mediate cleavage of beta2m, could be involved in these processes.

Veterinarian's dilemma: a study of how Danish small animal practitioners handle financially limited clients.

This study examined the extent to which Danish veterinary practices encounter financially limited clients and how different factors relating to the animal, the client and the veterinarian affect decisions to provide treatment for these clients. 300 small animal practices were invited to participate in an online survey. 195 participated, giving a response rate of 65 per cent. The results show that Danish small animal veterinary practices encounter clients with limited finances regularly: 33.8 per cent of them 3-4 times, 24.6 per cent 5-10 times and 19.5 per cent 1-2 times a month. Only around 9 per cent reported having a written practice policy on handling financially limited clients. Factors affecting decisions to treat include the severity and type of the animal's condition, the medical care needed and the client's expressed emotions. The propensity to treat is significantly higher in female veterinarians and in situations involving unborn animals. The overall conclusion is that small animal veterinary practices often provide treatment to clients who are not able to pay-far beyond what is legally required. This can be considered a major economic and psychological challenge for the practising veterinarians.

Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s.

The major histocompatibility complex (MHC) class I antigens contain a light chain, beta 2-microglobulin, non-covalently associated to the transmembrane heavy alpha-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with beta 2-microglobulin, and we recently found that activated C1s complement was capable of cleaving beta 2-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22,000 and 24,000 g/mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the alpha 2-and alpha 3-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-Ad). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the alpha 1-and alpha 2-domains which represent the binding site for antigenic peptides.

Constitutive STAT3-activation in Sezary syndrome: tyrphostin AG490 inhibits STAT3-activation, interleukin-2 receptor expression and growth of leukemic Sezary cells.

Interleukin-2 (IL-2) is a growth factor which upon binding to high-affinity receptors (IL-2Ralphabetagamma) triggers mitogenesis in T cells. IL-2Ralpha expression is restricted to T cells which have recently encountered antigen, and in healthy individuals the majority (>95%) of peripheral T cells are IL-2Ralpha negative. An aberrant expression of IL-2Ralpha has recently been described in cutaneous T-cell lymphoma (CTCL). Here, we study the regulation of IL-2Ralpha expression and STATs in a tumor cell line obtained from peripheral blood from a patient with Sezary syndrome (SS), a leukemic variant of CTCL. We show that (1) STAT3 (a transcription factor known to regulate IL-2Ralpha transcription) is constitutively tyrosine-phosphorylated in SS tumor cells, but not in non-malignant T cells; (2) STAT3 binds constitutively to a STAT-binding sequence in the promotor of the IL-2Ralpha gene; (3) the Janus kinase inhibitor, tyrphostine AG490, inhibits STAT3 activation, STAT3 DNA binding, and IL-2Ralpha mRNA and protein expression in parallel; and (4) tyrphostine AG490 inhibits IL-2 driven mitogenesis and triggers apoptosis in SS tumor cells. In conclusion, we provide the first example of a constitutive STAT3 activation in SS tumor cells. Moreover, our findings suggest that STAT3 activation might play an important role in the constitutive IL-2Ralpha expression, survival, and growth of malignant SS cells.

In vivo activation of STAT3 in cutaneous T-cell lymphoma. Evidence for an antiapoptotic function of STAT3.

A characteristic feature of neoplastic transformation is a perpetual activation of oncogenic proteins. Here, we studied signal transducers and activators of transcription (STAT) in patients with mycosis fungoides (MF)/cutaneous T-cell lymphoma (CTCL). Malignant lymphocytes in dermal infiltrates of CTCL tumors showed frequent and intense nuclear staining with anti-PY-STAT3 antibody, indicating a constitutive activation of STAT3 in vivo in tumor stages. In contrast, only sporadic and faint staining was observed in indolent lesions of patch and plaque stages of MF. Moreover, neoplastic lymphocytes in the epidermal Pautrier abscesses associated with early stages of MF did not express activated STAT3. To address the role of STAT3 in survival/apoptosis, CTCL tumor cells from an advanced skin tumor were transfected with either wild-type STAT3 (STAT3wt) or dominant-negative STAT3 (STAT3D). Forced inducible expression of STAT3D triggered a significant increase in tumor cells undergoing apoptosis, whereas forced expression of STAT3wt or empty vector had no effect. In conclusion, a profound in vivo activation of STAT3 is observed in MF tumors but not in the early stages of MF. Moreover, STAT3 protects tumor cells from apoptosis in vitro. Taken together, these findings suggest that STAT3 is a malignancy factor in CTCL.

Induction of cytotoxic CD8+CD56+ T cells from human thymocytes by interleukin-15.

CD8(+) CD56(+) cells isolated from human peripheral blood lymphocytes have been shown recently to represent a population of cytotoxic active T cells. However, it is not known if these cells are intrathymically or extrathymically developed or how these cells are influenced by growth factors. In the present study, we investigated the effects of interleukin-2 (IL-2) and IL-15 on human thymocytes with respect to development of CD8(+) CD56(+) T cells. Freshly isolated thymocytes contain few CD8(+) CD56(+) cells, but the number of these cells increases significantly when thymocytes are grown in the presence of IL-15 or IL-2. However, IL-15 induced a significantly higher fraction of CD8(+) CD56(+) cells compared with IL-2. Thus, although IL-2 and IL-15 are known to have a number of redundant functions, we here demonstrate that IL-15 is superior to IL-2 in inducing CD8(+) CD56(+) T cells from cultures of thymocytes. The majority of the IL-15-grown CD8(+) CD56(+) cells were CD45R0(+), representing a memory phenotype, and showed high expression of the IL-15R-complex and high numbers of CD69(+) cells. Moreover, cytotoxic activity was confined to this cell population.

A simple method for the preparation and purification of C1 complement cleaved beta 2-microglobulin from human serum.

A simple method is described for the preparation of proteolytically processed forms of beta 2-microglobulin suitable for structural and biological studies. PEG 6000 was added to the serum of healthy individuals to precipitate the C1 complement complex from C1 esterase inhibitor (C1-inh). After dissolving the precipitate containing the C1 complement in Tris-HCl buffer, pH 7.6, efficient conversion of added beta 2-microglobulin to desLys58 beta 2-microglobulin was observed. Addition of a specific carboxypeptidase B inhibitor (Plummers inhibitor) could partly prevent the deletion of Lys-58 from cleaved beta 2-microglobulin, whereby Lys58-cleaved beta 2-microglobulin was obtained. The proteolytically processed forms were subsequently purified by G-75 Sephadex gel filtration followed by chromatofocusing. A yield of 10-40% of proteolytically processed beta 2-microglobulin was obtained. Only one component was seen by SDS-PAGE stained with Coomassie Brilliant Blue.

Human retinal pigment epithelial cell-induced apoptosis in activated T cells.

PURPOSE: The immune privilege of the eye has been thought to be dependent on physical barriers and absence of lymphatic vessels. However, the immune privilege may also involve active immunologic processes, as recent studies have indicated. The purpose of the present study was to investigate whether human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which block alloactivation, had no inhibitory effect on RPE-mediated T-cell apoptotic responses in MHC class II-specific CD4+ T-cell lines. CONCLUSIONS: Retinal pigment epithelial cells express FasL and induce TCR-independent apoptosis in activated human T cells through Fas-FasL interaction. Retinal pigment epithelial cells may constitute an immunologic functional barrier against potentially harmful T cells.

Binding of human beta 2-microglobulin to murine EL4 thymoma cells upregulates MHC class I heavy-chain epitopes, inhibits IL-2 secretion and induces resistance to killing by natural killer cells.

A variety of murine tumor cell lines was studied for its binding of exogeneously added human beta 2-microglobulin (h beta 2m). Three T lymphomas and one IL-2-dependent T-cell line (HT-1) bound substantial amounts of h beta 2m, whereas P815 mastocytoma cells, an Abelson virus-infected pre-B cell line (ABLS-8), X63 B-lymphoma cells and YAC cells did not bind h beta 2m. In two of the T lymphomas, EL4 and BW5147, binding of h beta 2m led to an increase in major histocompatibility complex class I (MHC-I) heavy-chain epitope expression as measured by anti-H-2K/D antibody binding and FACS analysis. EL4 cells which had bound h beta 2m decreased their rate of constitutive IL-2 secretion and became resistant to activated natural killer (NK) cell killing. The present data suggest the binding of h beta 2m to mouse T cells leads to conformational changes of MHC-I heavy chains which influence both effector and target functions of the cell.

Human p53(264-272) HLA-A2 binding peptide is an immunodominant epitope in DNA-immunized HLA-A2 transgenic mice.

C57BL/10 mice transgenic for HLA-A2 were immunized with either a full-length DNA-construct of the tumor suppressor p53 or with a minigene encoding the p53-derived immunodominant peptide p53(264)LLGRNSFEV272 (L9V). Vaccination with the full-length p53 construct induced potent cytotoxic activity of splenocytes against L9V-pulsed target cells after in vivo re-stimulation. Vaccination with the L9V-encoding minigene likewise induced specific anti-L9V cytotoxicity in vitro. Subsequent experiments revealed that peptide-pulsed dendritic cells were the most efficient cell types for in vitro re-stimulation. In concordance with this, immunization with L9V-pulsed dendritic cells also induced a potent and specific anti-L9V cytotoxic response in vitro. These data show that HLA-A2/peptide-specific cytotoxic immunity can be generated in vivo against the same immunodominant epitope by immunizing either with full-length DNA or with a DNA minigene encoding the immunodominant peptide epitope.

Beta-2-microglobulin excretion: an indicator of long term nephrotoxicity during cis-platinum treatment?

To evaluate the value of beta-2-microglobulin as an indicator of acute and long-term cis-platinum-induced nephrotoxicity, 51Cr-EDTA clearance and serum concentration and urinary excretion of beta-2-microglobulin were measured in 18 patients treated with a regimen including cis-platinum. Before treatment all values were within the normal range. During treatment 51Cr-EDTA clearance decreased from 108 to 90 ml/min/1.73 m2 (P less than 0.02). The decrease was irreversible, while a transient 2 to 5-fold increase in beta-2-microglobulin excretion in the urine was seen during treatment. Serum beta-2-microglobulin remained unchanged. The decrease in 51Cr-EDTA clearance was not correlated to either the peak increase in the beta-2-microglobulin excretion or to the time of occurrence of the peak (R = 0.3). Thus, it is not possible to predict the long-term nephrotoxicity of cis-platinum by measuring the beta-2-microglobulin excretion during treatment.

Phase I study of the cisplatin analogue 1,1-diamminomethylcyclohexane sulfatoplatinum (TNO-6) (NSC 311056).

The cisplatin derivative TNO-6 was evaluated for clinical toxicity in a phase I trial. TNO-6 was given daily for 5 days every 3 weeks as a 30-min IV infusion without hydration. In all, 39 patients with advanced cancer were treated at doses of 2.5-9.0 mg/m2. No dose-limiting nephrotoxicity occurred, but evidence of mild, reversible tubular damage was found. Dose-limiting toxicity was hematologic with both thrombopenia and leukocytopenia, which with high dose levels reached WHO grade 4. Hematologic toxicity was most pronounced for pretreated patients. No antitumor activity was seen. The recommended dose for phase II trials will be 9.0 mg/m2 for previously untreated and 8.0 mg/m2 for pretreated patients.

Modification of beta 2-microglobulin in serum from patients with small cell carcinoma of the lung--correlation with the clinical course.

A beta 2-microglobulin (beta 2m) 'modifying activity' has been demonstrated by crossed radioimmunoelectrophoresis of serum. The activity could be estimated by planimetry and expressed in arbitrary units (A.U.). Elevated values of beta 2m 'modifying activity' (greater than 0.30 A.U.) has been demonstrated in 49 of 54 patients with small cell lung cancer. The values returned to normal (less than 0.30 A.U.) in eight of the ten patients achieving complete remission (CR) and in three of seven patients achieving partial remission (PR) after chemotherapy. The decrease was more pronounced (median 0.56 versus 0.16, p less than 0.01) in patients achieving CR compared with patients achieving PR. Relapse was accompanied by rising values in 11 of 15 patients monitored during chemotherapy. In nine of these patients abnormally high values of beta 2m 'modifying activity' was demonstrated more than 1 month before clinical or radiological evidence of disease progression. Total serum beta 2m was measured by radioimmunoassay. Elevated values (greater than 200 nmol/l) was found in 14 of 48 patients with small cell lung cancer. No correlation with the clinical course was found in patients monitored during chemotherapy. Estimation of total beta 2m is of no clinical value in small cell lung cancer. Estimation of beta 2m 'modifying activity, provides clinically relevant information, but is too laborious for routine clinical application. The biochemical process underlying this phenomenon should be studied further to allow development of a more simple test.

Congophilicity (Congo red affinity) of different beta2-microglobulin conformations characterized by dye affinity capillary electrophoresis.

The amyloidogenic protein beta-microglobulin was characterized by affinity capillary electrophoresis (CE). CE could separate conformational variants of beta2-microglobulin and with the amyloid-specific dye Congo red as a buffer additive it was possible to measure different Congo red-affinities of native and abnormally folded beta2-microglobulin. We find that native beta2-microglobulin has an intermediate affinity for Congo red at pH 7.3 and that binding involves electrostatic interactions. The conformational variant of beta2-microglobulin that appears in acetonitrile solutions binds Congo red more strongly. Affinity CE using Congo red as a buffer additive is a new, simple, fast, and quantitative micromethod for the characterization of soluble conformational intermediates of amyloidogenic proteins.

Des-Lys58-beta 2m and native beta 2m in rheumatoid arthritis serum and synovial fluid.

OBJECTIVE: Levels of beta 2-microglobulin and modified beta 2-microglobulin (Des-Lys58-beta 2m) were measured in serum and synovial fluids from patients with rheumatoid arthritis (RA) and other inflammatory joint disorders using rabbit antisera prepared against the beta 2m peptide VEHSDLSFS encompassing residues 49-57 and absorbed with the C-terminal beta 2m peptide (87-97) LSQPKIVKWDR: These antisera which did not react with native beta 2m were employed to quantitate Des-Lys58-beta 2m in serum and SF. Native beta 2m was measured using a direct ELISA method. RESULTS: Removal of serum rheumatoid factor by adsorption to monomeric IgG columns did not change serum levels of beta 2m or Des-Lys58-beta 2m. Native beta 2m was found in all of 20 RA sera, but only rarely in SLE sera. No serum beta 2m was found in 20 patients with ankylosing spondylitis or 25 normal controls. Significant elevations of Des-Lys58-beta 2m were found in 80% of 21 SF from RA patients and in 43% of 41 SF from other subjects with various forms of inflammatory arthritis. In RA and other disorders such as gout or pseudogout, levels of Des-Lys58-beta 2m were higher in synovial fluid than in serum during an acute episode of synovitis. Both native beta 2m and Des-Lys58-beta 2m showed minimal neutrophil and T cell chemotactic activity. CONCLUSION: Des-Lys58-beta 2m present in many inflammatory SF may contribute to the inflammatory reaction in many forms of connective tissue disease by its known amplification of T cell cytotoxicity.

Bcl-2, Bax, and c-Fos expression correlates to RPE cell apoptosis induced by UV-light and daunorubicin.

PURPOSE: The aim of this study was to determine the role of Bcl-2, Bcl-X L, Bax, and c-Fos in regulation of apoptosis, induced by ultraviolet-light A (UV-A) and daunorubicin (DNR), in retinal pigment epithelium (RPE) cells grown on bovine extracellular matrix (ECM)-coated or uncoated plastic dishes. METHODS: Apoptosis in confluent RPE cells cultured on ECM-coated or uncoated dishes was induced by UV-A or DNR. Apoptosis was detected by 7-amino-actinomycin D labeling followed by flow cytometry and by terminal deoxy-transferase mediated X-dUTP nick end labeling (TUNEL). Cellular expression of Bcl-2, Bcl-X L, Bax, and c-Fos was determined by the use of antibodies and flow cytometry, Western blot analysis, and immunocytochemical staining. RESULTS: Both UV-A and DNR induce apoptosis in human RPE cells in vitro. Human fetal RPE cells grown on ECM-coated dishes were significantly more resistant to UV-A or DNR induced apoptosis than cells grown on uncoated dishes. RPE cells grown on ECM-coated dishes expressed higher Bcl-2 levels and lower Bax levels compared to cells grown on uncoated dishes. However, Bcl-X L and c-Fos levels were comparable in the two cultures. After UV-A or DNR treatment, Bcl-2, Bcl-X L, Bax, and c-Fos levels were differently regulated in cells grown on ECM-coated dishes compared to cells grown on uncoated dishes. CONCLUSION: A significant protection against apoptosis of RPE cells grown on ECM compared to cells grown on uncoated plastic dishes was found after exposure to UV-A or DNR. This protection was found to be proportionally correlated to the anti-apoptotic protein Bcl-2 and inversely correlated to the expression of Bax. Furthermore a sustained induction and expression of c-Fos was found to correlate to a higher percentage of apoptotic cells of RPE cells grown on plastic. These findings demonstrate that ECM is of great importance for RPE cell survival during noxious stimuli and points out the essential role for a healthy Bruch's Membrane (BM) for RPE survival.

Complement component C1r mediated cleavage of the heavy chain of the major histocompatibility class I antigens.

Apart from cleaving C1s, we demonstrate for the first time that: 1) at concentrations found in serum, the activated forms of the complement components C1r in addition to C1s can cleave the heavy chain of MHC class I antigens, 2) the cleavage by C1r and C1s is seemingly dependent upon a native configuration of the MHC class I antigen, since heat denaturation of the HLA antigens reduce the cleavage. The proteolytic fragments following C1 cleavage were characterized by precipitation with Con A-Sepharose, anti-MHC class I and anti-beta 2-microglobulin antibodies. The proteolysis of the alpha-chain of MHC class I was shown to take place between the alpha 2- and alpha 3- domains as estimated by the Con A-Sepharose precipitation pattern on SDS-PAGE. The alpha 1/alpha 2 fragment was still shown to interact with beta 2-microglobulin as shown by immunoprecipitation.