Addition or removal of concomitant methotrexate alters adalimumab effectiveness in rheumatoid arthritis but not psoriatic arthritis.

Objective: Randomized trials have shown that concomitant methotrexate (MTX) augments the effectiveness of tumour necrosis factor (TNF) inhibitors in rheumatoid arthritis (RA), but its benefit in psoriatic arthritis (PsA) has not been demonstrated. The goal of this study was to examine whether the impact of concomitant MTX on therapeutic outcomes in patients with PsA was similar to its effects in RA. Methods: We used data from highly comparable and concurrent observational studies of patients with PsA (N = 1424) or RA (N = 3148) who initiated adalimumab therapy during routine clinical care. The 28-joint Disease Activity Score (DAS28) and patient-reported pain scores were evaluated in patients who received 24 months of continuous treatment with adalimumab monotherapy or adalimumab + MTX and in patients who initiated or stopped concomitant MTX during ongoing adalimumab therapy. Results: Twenty-four months of continuous treatment with adalimumab + MTX was superior to adalimumab monotherapy in RA patients, while no significant difference was observed in patients with PsA. RA patients who added MTX during the study showed significant individual improvements in DAS28 and pain scores at 6 months after the change in therapy, while those who removed MTX had slight increases in disease activity. In contrast, in patients with PsA, neither initiation nor removal of MTX during continuous adalimumab therapy had a significant effect on therapeutic outcomes. Conclusion: Addition of MTX to adalimumab confers further therapeutic benefit in patients with RA, but not in those with PsA, suggesting differences in MTX effects in these two patient populations. Clinicaltrials.gov NCT01078090, NCT01077258, NCT01111240.

Immunotherapeutic maintenance treatment with toll-like receptor 9 agonist lefitolimod in patients with extensive-stage small-cell lung cancer: results from the exploratory, controlled, randomized, international phase II IMPULSE study.

Background: The immune surveillance reactivator lefitolimod (MGN1703), a DNA-based TLR9 agonist, might foster innate and adaptive immune response and thus improve immune-mediated control of residual cancer disease. The IMPULSE phase II study evaluated the efficacy and safety of lefitolimod as maintenance treatment in extensive-stage small-cell lung cancer (ES-SCLC) after objective response to first-line chemotherapy, an indication with a high unmet medical need and stagnant treatment improvement in the last decades. Patients and methods: 103 patients with ES-SCLC and objective tumor response (as per RECIST 1.1) following four cycles of platinum-based first-line induction therapy were randomized to receive either lefitolimod maintenance therapy or local standard of care at a ratio of 3 : 2 until progression or unacceptable toxicity. Results: From 103 patients enrolled, 62 were randomized to lefitolimod, 41 to the control arm. Patient demographics and response patterns to first-line therapy were balanced. Lefitolimod exhibited a favorable safety profile and pharmacodynamic assessment confirmed the mode-of-action showing a clear activation of monocytes and production of interferon-gamma-induced protein 10 (IP-10). While in the intent-to-treat (ITT) population no relevant effect of lefitolimod on progression-free and overall survival (OS) could be observed, two predefined patient subgroups indicated promising results, favoring lefitolimod with respect to OS: in patients with a low frequency of activated CD86+ B cells (hazard ratio, HR 0.53, 95% CI: 0.26-1.08; n = 38 of 88 analyzed) and in patients with reported chronic obstructive pulmonary disease (COPD) (HR 0.48, 95% CI: 0.20-1.17, n = 25 of 103). Conclusions: The IMPULSE study showed no relevant effect of lefitolimod on the main efficacy end point OS in the ITT, but (1) the expected pharmacodynamic response to lefitolimod, (2) positive OS efficacy signals in two predefined subgroups and (3) a favorable safety profile. These data support further exploration of lefitolimod in SCLC.

Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent.

BACKGROUND: Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown. RESULTS: In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment. In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups. In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis. CONCLUSION: Treatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10. The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated.

Abrogation of experimental colitis correlates with increased apoptosis in mice deficient for CD44 variant exon 7 (CD44v7).

Experimental colitis in mice is characterized by infiltration of activated T helper (Th) cells and macrophages into the lamina propria. Particularly, these cells expressed CD44 variant exon 7 (CD44v7)-containing isoforms. Upregulation of CD44v7 isoforms was induced by CD40 ligation, an inflammation-driving interaction between activated Th cells and macrophages. To define the role of CD44v7 in colitis, mice bearing a targeted deletion for exon v7 were generated. In trinitrobenzene sulfonic acid-induced colitis, wild-type mice developed severe signs of persistent inflammation. Mice lacking CD44v7 initially showed unspecific inflammation, then recovered completely. The pathogenic origin was shown to reside in bone marrow-derived CD44v7(+) cells, because adoptive transfer experiments demonstrated an absolute requirement for CD44v7 on hematopoietic cells for maintenance of colitis. Interleukin (IL)-10-deficient mice, which develop a chronic Th1-driven enterocolitis, were crossbred with CD44v6/v7 null mice. In IL-10 x CD44v6/v7 double deficient mice, intestinal inflammation developed only weakly and at an older age. Analysis of cell death in the inflamed lesions revealed that mononuclear cells in the CD44v7 null infiltrates had higher rates of apoptosis than those from wild-type mice. Thus, the region encoded by CD44v7 appears to be essential for survival of effector lymphocytes, resulting in persistence of inflammation.

The level of Z-DNA in metabolically active, permeabilized mammalian cell nuclei is regulated by torsional strain.

Permeabilized nuclei from mammalian cells encapsulated within agarose microbeads in an isotonic buffer are active in transcription and replication (Jackson, D. A., and P. R. Cook. 1985. EMBO (Eur. Mol. Biol. Organ.) J. 4:913-918). Their DNA is intact and the nuclei are accessible to macromolecules. Myeloma nuclei prepared in this way were used to probe the extent of DNA negative supercoiling and the effects of altering torsional strain by binding radioactively labeled monoclonal antibodies to Z-DNA. Control experiments used monoclonal antibodies against a nonhistone chromosomal protein, HMG-17. On increasing the amount of anti-HMG-17 added, a binding plateau was reached encompassing a 200-fold range of antibody concentration. On binding anti-Z-DNA antibody, a similar broad plateau of constant binding was found encompassing a 100-fold range of antibody concentration. The latter result was taken as a measure of preexisting Z-DNA in the nuclei. Additional anti-Z-DNA antibody binding can be "induced" in the presence of much higher concentration of antibody, apparently by perturbing the B-DNA/Z-DNA equilibrium. On inhibiting topoisomerase I with camptothecin, an elevated antibody binding plateau was found, suggesting that elastic torsional strain in the DNA is responsible for stabilizing the preexisting Z-DNA. This interpretation is supported by the fact that addition of small, nicking amounts of DNase I leads to a complete loss of antibody binding in the Z-DNA plateau region but not in the region of "induced" Z-DNA.

Selectivity in signal transduction determined by gamma subunits of heterotrimeric G proteins.

Various heterotrimeric guanine nucleotide-binding proteins have been identified on the basis of the individual subtypes of their alpha subunits. The beta gamma complexes, composed of beta and gamma subunits, remain tightly associated under physiological conditions and have been assumed to constitute a common pool shared among various guanosine triphosphate (GTP)-binding (G) protein heterotrimers. Particular alpha and beta subunit subtypes participate in the signal transduction processes between somatostatin or muscarinic receptors and the voltage-sensitive L-type calcium channel in rat pituitary GH3 cells. Among gamma subunits the gamma 3 subtype was found to be required for coupling of the somatostatin receptor to voltage-sensitive calcium channels, whereas the gamma 4 subtype was found to be required for coupling of the muscarinic receptor to those channels.

Subacute sclerosing panencephalitis: isolation of suppressed measles virus from lymph node biopsies.

Measles virus was isolated in mixed cultures of lymph node cells and HeLa cells. The agent was isolated by cocultivation from biopsy specimens of two of five patients with subacute sclerosing panencephalitis. The virus was identified by hemagglutination-inhibition, immunofluorescent, and neutralization tests. Biopsies from controls did not show evidence of measles virus.

[Chronic inflammatory intestinal diseases. Pathophysiology and therapy]. / Chronisch entzündliche Darmerkrankungen. Pathophysiologie und Therapie.

The pathogenesis and therapy of chronic inflammatory intestinal diseases are characterized by an obvious discrepancy. There is extensive agreement that the pathogenesis is substantially based on a disruption of the barrier of the intestinal mucous membrane against luminal bacteria. This has been demonstrated in recent years by evidence from various disciplines, in particular from genetics, microbiology, morphology and innate immunology. However, there is also the evidence-based therapy which, as in the past, is aimed against the effectors of the adaptive immune system. In this case the therapy with biologicals is more aggressive and takes the risk of a series of undesired side-effects. This dichotomy of pathological knowledge and therapeutic innovation is not only medically unsatisfactory but also makes it difficult to present a consistent picture of these symptoms. Despite this an attempt will be made to bridge these inconsistencies and to demonstrate possible future developments which will lead to a final causal therapy. An extended version of this article appears in our newly published book "Colitis ulcerosa und Morbus Crohn".

CD44v7 ligation downregulates the inflammatory immune response in Crohn's disease patients by apoptosis induction in mononuclear cells from the lamina propria.

Deletion of exon CD44v7 abrogates experimental colitis by apoptosis induction in intestinal mononuclear cells. Here we show that CD44v7 expression was upregulated upon CD40 ligation in human mononuclear cells, and examined whether ligation of CD44v7 also affects activation and apoptosis in lamina propria mononuclear cells (LPMC) from Crohn's disease (CD) patients. Thirty five patients with chronic inflammatory bowel disease (IBD), fourteen controls and four patients with diverticulitis were evaluated. CD44v7 was upregulated predominantly in the inflamed mucosa of CD patients. Furthermore, incubation with an anti-CD44v7 antibody induced apoptosis in LPMC isolated from inflamed mucosa of CD patients, but not from non-inflamed mucosa, from patients with ulcerative colitis (UC) or from normal controls. CD40 ligation and simultaneous incubation with anti-CD44v7 significantly downregulated CD80 in dendritic cells, thus inhibiting a critical second signal for naive T-cell activation. The apoptotic signal was mediated via the intrinsic mitochondrial pathway with decreased Bcl-2 and increased 7A6 (a mitochondrial membrane protein) expression. It was Fas independent and required caspases-3 and -9 activation. The process is highly specific for macrophage activation via CD40. These findings point to a novel mechanism of apoptosis induction in CD patients mediated by CD44v7 ligation.

Absence of specific alternatively spliced exon of CD44 in macrophages prevents colitis.

CD44 is a transmembrane molecule appearing in numerous isoforms generated by insertions of alternatively spliced variant exons (CD44v) and having various binding partners. CD44v7 on T cells was proposed to promote colitis by preventing T-cell apoptosis. Here we demonstrate that Cd44v7-deficient T cells - like Cd44 wild-type (Cd44WT) T cells - provoked disease in two different colitis models: the model induced by CD4+CD45RBhigh T-cell transfer into Rag2-deficient mice and a new model based on ovalbumin (OVA)-specific T-cell transfer into Rag-sufficient, OVA-challenged mice. In contrast, CD44v7 absence on macrophages in recipient mice prevented colitis. Prevention was associated with the downregulation of signal transducer and activator of transcription 3 (STAT3)-activating and Foxp3-counteracting interleukin-6 (IL-6), lower numbers of phospho-STAT3-containing lymphocytes, and higher Foxp3+ T-cell counts in the colon. Consequently, the protected colons showed lower IL-12, IL-1ß expression, and decreased interferon-γ levels. Importantly, stimulation of T cells by Cd44v7-deficient macrophages induced upregulation of Foxp3 in vitro, while cotransfer of Cd44WT macrophages into Cd44v7-deficient mice reduced Foxp3+ T-cell counts and caused colitis. Accordingly, the CD44v7 ligand osteopontin, whose levels were elevated in Crohn's disease, specifically induced IL-6 in human monocytes, a cytokine also increased in these patients. We suggest macrophage-specific targeting of the CD44v7 pathway as a novel therapeutic option for Crohn's disease.

The TLR9 agonist MGN1703 triggers a potent type I interferon response in the sigmoid colon.

Toll-like receptor 9 (TLR9) agonists are being developed for treatment of colorectal and other cancers, yet the impact of these drugs on human intestines remains unknown. This, together with the fact that there are additional potential indications for TLR9 agonist therapy (e.g., autoimmune and infectious diseases), led us to investigate the impact of MGN1703 (Lefitolimod) on intestinal homeostasis and viral persistence in HIV-positive individuals. Colonic sigmoid biopsies were collected (baseline and week four) from 11 HIV+ individuals on suppressive antiretroviral therapy, who received MGN1703 (60 mg s.c.) twice weekly for 4 weeks in a single-arm, phase 1b/2a study. Within sigmoid mucosa, global transcriptomic analyses revealed 248 modulated genes (false discovery rate<0.05) including many type I interferon (IFN)-stimulated genes. MGN1703 increased the frequencies of cells exhibiting MX1 (P=0.001) and ISG15 (P=0.014) protein expression. No changes were observed in neutrophil infiltration (myeloperoxidase; P=0.97). No systematic effect on fecal microbiota structure was observed (analysis of similarity Global R=-0.105; P=0.929). TLR9 expression at baseline was inversely proportional to the change in integrated HIV DNA during MGN1703 treatment (P=0.020). In conclusion, MGN1703 induced a potent type I IFN response, without a concomitant general inflammatory response, in the intestines.

Cyclophosphamide pulse therapy followed by azathioprine or methotrexate induces long-term remission in patients with steroid-refractory Crohn's disease.

BACKGROUND: In patients with steroid-refractory Crohn's disease, the therapeutic goal is to achieve both rapid remission and maintenance of clinical response. AIM: To evaluate the long-term benefit in patients treated with cyclophosphamide pulse therapy and azathioprine or methotrexate, a combination shown to be effective in a recent pilot study. METHODS: Sixteen patients with acute steroid-refractory Crohn's disease participated in a prospective open-labelled uncontrolled pilot study between December 1998 and June 2003. All had a median number of 4 monthly pulses of intravenous cyclophosphamide (750 mg) and were followed until relapse of the disease. RESULTS: Thirteen of 16 patients (81%) achieved remission within 8 weeks after two pulses of cyclophosphamide in combination with azathioprine or methotrexate, with a Crohn's Disease Activity Index decrease from 294 to 111 (median). Remission sustained for 19 months (median, range: 1-45). Moreover, eight patients with pyoderma gangrenosum and erythema nodosum who responded to cyclophosphamide have maintained their remission for up to 30 months. CONCLUSIONS: In steroid refractory patients with Crohn's disease, cyclophosphamide is highly effective to induce remission. This uncontrolled study indicates that cyclophosphamide-induced remission is long-lasting under standard immunosuppressive therapy.

Angiotensin II activates nuclear transcription factor kappaB through AT(1) and AT(2) in vascular smooth muscle cells: molecular mechanisms.

Nuclear factor-kappaB (NF-kappaB) regulates many genes involved in vascular physiopathology. We have previously observed in vivo NF-kappaB activation in injured vessels that diminished by angiotensin-converting enzyme inhibition. In the present work, we investigated the effect of angiotensin II (Ang II) on NF-kappaB activity in rat vascular smooth muscle cells, evaluating the molecular mechanisms and the specific receptor subtype involved. Ang II increased NF-kappaB DNA binding (5-fold, 10(-)(9) mol/L at 1 hour; electrophoretic mobility shift assay), nuclear translocation of p50/p65 subunits, and cytosolic inhibitor kappaBalpha (IkappaBalpha) degradation. Ang II elicited NF-kappaB-mediated transcription (transfection of a reporter gene) and expression of NF-kappaB-related genes (monocyte chemoattractant protein-1 and angiotensinogen). AT(1) (DUP753) and AT(2) (PD123319 and CGP42112) receptor antagonists inhibited Ang II-induced NF-kappaB DNA binding in a dose-dependent manner ( approximately 85% for each one; 10(-)(5) mol/L at 1 hour). The AT(2) agonist p-aminophenylalanine(6)-Ang II augmented NF-kappaB binding (4.6-fold, 10(-)(9) mol/L at 1 hour), p65 nuclear levels, and transcription of an NF-kappaB reporter gene. AT(1) antagonist markedly inhibited NF-kappaB-mediated transcription and gene expression. Some differences between AT(1)/AT(2) intracellular signals were found. Antioxidants and ceramide inhibitors, but not protein kinase C inhibitors, diminished NF-kappaB activation elicited by both Ang II and the AT(2) agonist, while tyrosine kinase inhibitors only decreased Ang II-induced NF-kappaB activity. Our results demonstrate that Ang II activates NF-kappaB via AT(1) and AT(2), although NF-kappaB-mediated transcription occurred mainly through AT(1). Both receptors share some signaling pathways (oxygen radicals and ceramide); however, tyrosine kinases only participate in AT(1)/NF-kappaB responses. These data provide novel insights into Ang II actions, suggesting a potential implication of the AT(2) in the pathobiology of vascular cells.

Cloning of an interferon regulatory factor 2 isoform with different regulatory ability.

Interferons (IFNs) are a family of multifunctional proteins involved in immune activation, regulation of cell growth and antiviral response. They exert their functions by induction of several IFN-stimulated genes, including IFN regulatory factors (IRFs), a family of transcriptional regulators. One of these factors, IRF-2, was initially cloned as an antagonistic counterpart to IRF-1 with oncogenic potential. Here we describe a second isoform of IRF-2, termed IRF-2s, cloned from human and murine cells. This isoform lacks two amino acids located C-terminal of the DNA-binding domain, which is conserved in all IRF family members, leading to a change in the predicted secondary structure. Both isoforms have similar binding affinities to known target sequences in electrophoretic mobility shift assays. Using reporter gene constructs with the type IV promoter region of the MHC class II transactivator (CIITA), which is the essential factor for IFN-gamma-induced MHC class II expression, we show that the short isoform IRF-2s exhibits a weaker activation ability compared to IRF-2. Thus, our data present the first evidence of two IRF-2 isoforms with different regulatory ability.

Purification of class A, B, and C DNA-dependent RNA polymerases from chicken embryos.

Crude nuclei were isolated from trunks of 13-day-old chicken embryos under conditions which prevent leakage of RNA polymerases from nuclei. RNA polymerases were solubilized by subsequent incubation in alkaline buffer and sonication at high salt concentration. Purification of RNA polymerases A, B, and C was achieved by conventional column chromatographic procedures. RNA polymerase B was freed from an UTP:polynucleotidyl exotransferase by chromatography on a tRNA-Sepharose column. Purified RNA polymerase A contained six putative subunits with molecular weights 190 000 (A1), 117 000 (A2), 57 000 (A3), 50 000 (A4), 25 000 (A5), 19 000 (A6); RNA polymerase B contained eight putative subunits with molecular weights 98 000 (B2'), 86 000 (B2''), 155 000 (B3), 44 000 (B4), 31 000 (B5), 28 000 (B6), 26 000 (B7), 19 000 (B8); RNA polymerase C contained nine putative subunits with molecular weights 170 000 (C1), 117 000 (C2), 84 000 (C3), 60 000 (C4), 49 000 (C5), 36 000 (C6), 33 000 (C7), 22 000 (C8), 19 000 (C9).

Identification of transcriptionally induced Z-DNA segments in the human c-myc gene.

Using monoclonal antibodies against Z-DNA three AluI restriction fragments of the human c-myc gene were previously found to form Z-DNA in agarose-embedded, metabolically active permeabilized nuclei. The formation of Z-DNA in these fragments was dependent on negative supercoiling generated by transcription of the gene. Here we show which sequence elements of these three AluI restriction fragments adopt the Z conformation upon negative supercoiling. The three fragments (Z1, Z2 and Z3) were inserted in a suitable plasmid vector. Z-DNA forming elements were detected by comparing DEPC reactivity in relaxed circular and supercoiled plasmid DNA. Z1 and Z3 each contained one major Z-DNA forming region 20-25 nucleotides long, whereas Z2 contained two discrete regions 90 nucleotides apart one about 35 nucleotides the other about 20 nucleotides long.

Identification of processes that influence negative supercoiling in the human c-myc gene.

DNA elements with sequences suitable for Z-DNA formation are found frequently at various positions in chromatin. Z-DNA formation in these sequences depends largely on the level of local negative supercoiling. We can use binding of a Z-DNA specific antibody at low concentrations in metabolically active permeabilized nuclei to detect naturally occurring Z-DNA formation. Previously we identified three sequence elements in the human c-myc gene that adopt the Z-DNA conformation in the transcribed gene. The three elements are found far upstream (Z1), close to the main transcription start site (Z2) and in the first intron (Z3). Here we measure the persistence of Z-DNA at these three sites under the influence of various metabolic inhibitors. This provides some insight into the varying levels of negative supercoiling. alpha-Amanitin, an inhibitor of transcription, reduced the persistence of Z-DNA in all three elements. Aphidicolin, an inhibitor of replication, increased the persistence of Z-DNA in one element without significantly influencing the other two elements. When camptothecin an inhibitor of topoisomerase I was added in the presence of alpha-amanitin, the persistence of Z-DNA was extended in all three elements. However, in the presence of aphidicolin no effect of camptothecin on Z-DNA formation was observed.

Expression of interferon regulatory factor 4 in chronic myeloid leukemia: correlation with response to interferon alfa therapy.

PURPOSE: Mice experiments have established an important role for interferon regulatory factor (IRF) family members in hematopoiesis. We wanted to study the expression of interferon regulatory factor 4 (IRF4) in various hematologic disorders, especially chronic myeloid leukemia (CML), and its association with response to interferon alfa (IFN-alpha) treatment in CML. MATERIALS AND METHODS: Blood samples from various hematopoietic cell lines, different leukemia patients (70 CML, 29 acute myeloid leukemia [AML], 10 chronic myelomonocytic leukemia [CMMoL], 10 acute lymphoblastic leukemia, and 10 chronic lymphoid leukemia patients), and 33 healthy volunteers were monitored for IRF4 expression by reverse transcriptase polymerase chain reaction. Then, with a focus on CML, the IRF4 level was determined in sorted cell subpopulations from CML patients and healthy volunteers and in in vitro-stimulated CML cells. Furthermore, IRF4 expression was compared in the CML samples taken before IFN-alpha therapy and in 47 additional CML samples taken during IFN-alpha therapy. IRF4 expression was then correlated with cytogenetic response to IFN-alpha. RESULTS: IRF4 expression was significantly impaired in CML, AML, and CMMoL samples. The downregulation of IRF4 in CML samples was predominantly found in T cells. In CML patients during IFN-alpha therapy, a significant increase in IRF4 levels was detected, and this was also observed in sorted T cells from CML patients. The increase seen during IFN-alpha therapy was not due to different blood counts. In regard to the cytogenetic response with IFN-alpha, a good response was associated with high IRF4 expression. CONCLUSION: IRF4 expression is downregulated in T cells of CML patients, and its increase is associated with a good response to IFN-alpha therapy. These data suggest IRF4 expression as a useful marker to monitor, if not predict, response to IFN-alpha in CML.

P53 and induction of apoptosis as a target for anticancer therapy.

p53 is the most frequently mutated gone in human cancer cells. Its wild-type gone encodes for a protein with pivotal functions: (i) interaction with key players in the cell cycle leading to cell cycle arrest; (ii) induction of programmed call death, or apoptosis. P53 may be seen as another member of the family of proteins involved in resistance to anticancer therapy, since mutations/deletions involving the p53 gene lead frequently to resistance of radiation/cytotoxic drug treatment. Consequently, patients with p53-mutated tumors may harbor a worse prognosis. On the other hand, reintroducing wild-type P53 may lead to an adequate function of the cellular cell cycle and/or apoptosis program, thus enabling efficient anti-cancer therapy even in the presence of mutated P53. Two options are being discussed: (i) gene therapy approaches; (ii) modulating mutated P53 with yet unknown molecules.

Proliferation-dependent induction of apoptosis by the retinoid CD437 in p53-mutated keratinocytes.

6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-Naphthalene carboxylic acid (CD437) is a synthetic retinoid with strong apoptogenic properties in various neoplastic cell lines. CD437 was shown to induce apoptosis in malignant human keratinocytes but not in normal keratinocytes. We demonstrate that CD437 is also capable of inducing apoptosis in the non-tumorigenic keratinocyte cell line HaCaT that carries UV-type mutations on both alleles of the p53 gene. The concentration-dependent induction of apoptosis was restricted to proliferative HaCaT cells, whereas no effect was seen in differentiating post-mitotic cells. The apoptotic elimination of the proliferative cells was accompanied by rapid upregulation of c- jun, downregulation of c- fos, and activation of the AP-1 complex, which normally only occur during the differentiation process of post-mitotic keratinocytes. Pharmacological impairment of this precocious AP-1 activation reduced the rate of apoptosis induced by CD437. The potent, selective, and p53-independent apoptosis-inducing efficacy of CD437 is of utmost importance for the prophylaxis and treatment of skin cancer caused by mutational inactivation of the p53 gene.