Mogens H. Nissen

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-2Rαβγ) triggers mitogenesis in T cells. IL-2Rα expression is restricted to T cells which have recently encountered antigen, and in healthy individuals the majority (>95%) of peripheral T cells are IL-2Rα negative. An aberrant expression of IL-2Rα has recently been described in cutaneous T-cell lymphoma (CTCL). Here, we study the regulation of IL-2Rα 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-2Rα 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-2Rα gene; (3) the Janus kinase inhibitor, tyrphostine AG490, inhibits STAT3 activation, STAT3 DNA binding, and IL-2Rα 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-2Rα 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.

Age-related macular degeneration: Epidemiology and optimal treatment

Age-related macular degeneration (AMD) is a common macular disease affecting elderly people in the Western world. It is characterised by the appearance of drusen in the macula, accompanied by choroidal neovascularisation (CNV) or geographic atrophy. The disease is more common in Caucasian individuals than in pigmented races. In predominantly Caucasian populations, the age-standardised prevalence of AMD in at least one eye is 7760 cases per million. The age-standardised cumulated 1-year incidence of AMD in at least one eye is 1051 cases per million individuals. AMD is the most important single cause of blindness among Caucasian individuals in developed countries. Blindness resulting from AMD rarely occurs before age 70, and most cases occur after age 80. The age-standardised 1-year incidence of legal blindness resulting from AMD is 212 cases per million. Two-thirds of AMD cases have CNV (exudative cases); the remainder has only geographic atrophy. In cross-sectional population-based studies about 45% of eyes with AMD have visual acuity reduced to 20/200 or worse. This is true both for exudative AMD and pure geographic atrophy. Age and genetic predisposition are known risk factors for AMD. Smoking is probably also a risk factor.Preventive strategies using macular laser photocoagulation are under investigation, but their efficacy in preventing visual loss is as yet unproven. There is no treatment with proven efficacy for geographic atrophy. Optimal treatment for exudative AMD requires a fluorescein angiographic study and a physician capable of interpreting it. For CNV not involving the foveal centre, the only evidence-based treatment is laser photocoagulation. For AMD cases with subfoveal CNV, good visual acuity, and predominantly classic fluorescence pattern on fluorescein angiography, photodynamic therapy with verteporfin is the treatment of choice. Photodynamic therapy is also effective in eyes with pure occult CNV and evidence of recent disease progression. For new subfoveal CNV with poor vision and recurrent CNV, laser photocoagulation can be considered.

Enteric bacterial antigens activate CD4+ T cells from scid mice with inflammatory bowel disease

Scid mice transplanted with CD4+ T cells from congenic donor mice develop a chronic and lethal inflammatory bowel disease (IBD) 2–3 months post‐transplantation. In the present study we have investigated the response of CD4+ T cells from scid mice with colitis against fecal extracts. Our results show that in contrast to CD4+ T cells from normal BALB/c mice, CD4+ T cells from scid mice with colitis proliferate strongly in response to antigen‐presenting cells (APC) pulsed with fecal extracts. The IBD‐associated T cells did not respond to either extracts from food antigens or fecal extracts from germ‐free mice, which indicates that they recognize bacterial antigens in the fecal extracts. CD4+ T cells isolated from the colonic lamina propria of scid mice 3 weeks post transplantation also responded vigorously to fecal extracts, demonstrating that reactive CD4+ T cells are present in the gut mucosa of transplanted scid mice prior to clinical manifestations of IBD. CD4+ T cells activated by fecal extracts produced high amounts of IL‐2 and IFN‐γ, intermediate amounts of IL‐4 and low amounts of IL‐10, consistent with a Th1 profile. The proliferative reactivity towards fecal extracts was restricted by MHC class II molecules and dependent on antigen processing, as the response could be blocked by anti‐MHC class II antibodies or a short fixation of the APC. This study demonstrates that class II‐restricted CD4+ Th1 cells, which recognize enteric bacterial antigens, infiltrate the gut mucosa and spleen of transplanted scid mice prior to and during the course of colitis.

Efficient assembly of recombinant major histocompatibility complex class I molecules with preformed disulfide bonds.

The expression of major histocompatibility class I (MHC‐I) crucially depends upon the binding of appropriate peptides. MHC‐I from natural sources are therefore always preoccupied with peptidescomplicating their purification and analysis. Here, we present an efficient solution to this problem. Recombinant MHC‐I heavy chains were produced in Escherichia coli and subsequently purified under denaturing conditions. In contrast to common practice, the molecules were not reduced during the purification. The oxidized MHC‐I heavy chain isoforms were highly active with respect to peptide binding. This suggests that de novo folding of denatured MHC‐I molecules proceed efficiently if directed by preformed disulfide bond(s). Importantly, these molecules express serological epitopes and stain specific T cells; and they bind peptides specifically. Several denatured MHC‐I heavy chains were analyzed and shown to be of a quality, which allowed quantitative analysis of peptide binding. The analysis of the specificity of the several hundred human MHC haplotypes, should benefit considerably from the availability of pre‐oxidized recombinant MHC‐I.

Identification and design of p53-derived HLA-A2-binding peptides with increased CTL immunogenicity.

The replacement of a suboptimal amino acid in a primary anchor position with an optimal residue improves human leucocyte antigen (HLA) binding and immunogenicity, while maintaining cytotoxic T lymphocyte (CTL) specificity. Using a neural network capable of performing quantitative predictions of peptide binding to HLA‐A2 molecules, we identified three p53 protein‐derived nonamer peptides with intermediate binding owing to suboptimal amino acids in the P2 anchor position. These peptides were synthesized along with the corresponding analogs, where the natural P2 residue had been replaced with the optimal leucine residue. All three modified peptides bound to and more efficiently stabilized HLA‐A2 molecules than the corresponding nonmodified peptides. The HLA‐A2 transgenic mice were used for immunization. Two of the epitopes were more immunogenic in their modified than in their natural versions. The CTLs raised against the modified peptides efficiently killed the target cells pulsed with the corresponding native peptide. In terms of sensitizing the targets cells for the CTL killing, the modified peptides were more efficient than native peptides. Finally, the CTLs induced by modified peptide killed HLA‐A2 transgenic mouse fibrosarcoma cells transfected with human p53 DNA. The data suggest that modified self‐peptides derived from overexpressed tumour‐associated proteins can be used in vaccine development against cancer, and that quantitative predictions of HLA binding is of value in the rational selection and improvement of target epitopes recognized by CTLs.

Congophilicity (Congo red affinity) of different β2-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.

Interferon-alpha induces transient suppressors of cytokine signalling expression in human T cells.

The suppressors of cytokine signalling (SOCS) proteins comprise a newly identified family of negative feedback regulators of cytokine signalling. SOCS expression is differentially induced upon cytokine stimulation in different cell types. Here we show that interferon-α (IFNα) is a potent inducer of SOCS expression in human T cells, as high expression of CIS, SOCS-1, SOCS-2, and SOCS-3 was detectable after IFNα stimulation. After 4 h of stimulation, CIS, SOCS-1, and SOCS-3 expression had returned to baseline levels, whereas SOCS-2 expression had not declined. In contrast, after IL-2 induction neither CIS, SOCS-1, nor SOCS-2 expression levels declined after 6 h. In conclusion, we provide the first evidence that IFNα induces SOCS expression in human T cells. Moreover, we show that IFNα and IL-2 induce distinct patterns of expression kinetics, suggesting that dynamic changes in cytokine sensitivity might be mediated via induction of SOCS expression with different kinetics in T cells.

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 Bruchs Membrane (BM) for RPE survival.

Maturation of Dendritic Cells by Recombinant Human CD40L-Trimer Leads to a Homogeneous Cell Population with Enhanced Surface Marker Expression and Increased Cytokine Production

Dendritic cells (DC) have been shown to be potent inducers of specific cytotoxic T‐cell responses both in vivo and in vitro. Furthermore, exposure to cytokines such as tumour necrosis factor (TNF)‐α or CD40 triggering changes DC phenotype and cytokine production and may enhance the T‐cell activating capacity of the DC. We studied DC phenotype and cytokine production as well as the T‐cell proliferation and cytotoxic T lympocyte (CTL) activation induced by DC generated in vitro. In addition, the effect of exposure to recombinant human CD40L‐trimer (huCD40LT) on these parameters was investigated. Effective differentiation of monocytes derived from freshly isolated peripheral blood mononuclear cells (PBMC) was obtained with granulocyte macrophage‐colony stimulating factor (GM‐CSF) and interleukin (IL)‐4. The DC expression of human leucocyte antigen (HLA) molecules, CD80, CD83, and CD86 was markedly enhanced by exposure to huCD40LT even compared to TNF‐α exposure. Only a moderate cytokine production was observed initially, while TNF‐α addition or CD40 triggering, especially, induced enhanced production of IL‐6 and IL‐12 p40. Surprisingly, comparable induction of T‐cell proliferation by a DC allostimulus or through the presentation of PPD, and influenza M1‐peptide specific CTL activity was obtained with nonmaturated (CD83−) and maturated (CD83+) DC. In conclusion, a final maturation of monocyte‐derived DC through huCD40LT resulted in a highly homogeneous cell population with enhanced surface marker expression and high production of pro‐inflammatory cytokines. In addition, the induction of responses to allo or recall antigens presented by huCD40LT maturated DC was comparable to the responses obtained with the DC maturated through TNF‐α exposure.