Protection from articular damage by passive or active anti-tumour necrosis factor (TNF)-α immunotherapy in human TNF-α transgenic mice depends on anti-TNF-α antibody levels.

Active anti-tumour necrosis factor (TNF)-α immunization with the kinoid of TNF-α (TNF-K) induces polyclonal anti-TNF-α antibodies and ameliorates arthritis in human TNF-α (hTNF-α) transgenic mice (TTg). We compared the efficacy of TNF-K to that of infliximab (IFX) and of TNF-K and IFX co-administration, and evaluated whether the titres of anti-hTNF-α antibodies induced by immunization were a determinant of TNF-K efficacy. Forty-eight TTg mice received one of the following treatments: TNF-K immunization (TNF-K group); weekly IFX throughout the study duration (IFXw0-15); TNF-K plus weekly IFX for 4 weeks (TNF-K + IFX); and weekly IFX for 4 weeks (IFXw0-4); PBS. Animals were killed at week 16. Anti-hTNF-α antibody titres and clinical and histological scores were compared. All TNF-K immunized mice (TNF-K and TNF-K + IFX) produced anti-hTNF-α antibodies. Titres were higher in TNF-K versus TNF-K + IFX (P < 0·001) and correlated inversely with histological inflammation (R = -0·78; P = 0·0001) and destruction (R = -0·67; P = 0·001). TNF-K + IFX had higher histological inflammation and destruction versus TNF-K (P < 0·05). A receiver operating characteristic (ROC) analysis of anti-hTNF-α antibody titres identified the criterion cut-off value to discriminate most effectively between the TNF-K and TNF-K + IFX groups. Mice with high versus low titres had less histological inflammation and destruction (P < 0·05). In a model of TNF-α-dependent arthritis, protection from articular damage by TNF-K correlates with the titres of induced anti-hTNF-α antibodies. The co-administration of TNF-K and a short course of infliximab does not result in less articular damage versus solely TNF-K, due probably to lower anti-hTNF-α antibody production. These results are relevant for future development of active anti-TNF-α immunization in human disease.

Early and long-lasting protection from arthritis in tumour necrosis factor alpha (TNFalpha) transgenic mice vaccinated against TNFalpha.

OBJECTIVE: To evaluate the effect in mice with arthritis of active anti-tumour necrosis factor (TNF)alpha immunotherapy based on a keyhole limpet haemocyanin-human TNFalpha heterocomplex (hTNFalpha kinoid or TNFK) adjuvanted in incomplete Freund adjuvant. Immunotherapy was evaluated also with methotrexate. METHODS: Human TNFalpha-transgenic mice received TNFK with or without methotrexate. Follow-up ranged from 6 weeks (short term) to 17 weeks (long term). Arthritis was evaluated clinically and histologically. Monitoring included titration of anti-hTNFalpha antibodies by ELISA and neutralisation assay. RESULTS: Vaccination with TNFK was associated with rapid-onset, long-lasting protection. Long-term results showed significantly milder arthritis in vaccinated animals than in control animals at the peak of the disease. Vaccination was followed by resolution of the clinical evidence of arthritis, contrasting with severe progressive arthritis in the control group. Histological improvements with decreased inflammation and destruction were noted in all immunised groups, even after the shortest follow-up (6 weeks). High titres of neutralising anti-hTNFalpha antibodies were detected as early as the fifth week post immunisation and persisted over time. Methotrexate given concomitantly with the vaccine did not influence either the effect on arthritis or the anti-hTNFalpha antibody titres. CONCLUSION: Anti-cytokine induction of autoimmune protection against chronic hTNFalpha overproduction is an efficient alternative to TNFalpha blockade in experimental arthritis and can be achieved using a TNFK vaccine.

Disassembly of nuclear inclusions in the dividing cell--a novel insight into neurodegeneration.

Spinocerebellar ataxias and Huntington's disease are examples of neurodegenerative diseases caused by a trinucleotide repeat expansion. One hallmark of such diseases is the formation of inclusion bodies (IBs) within neuronal tissue. Although these inclusions may play a pivotal role in the disease process, the reasons underlying their specific accumulation remain obscure. By studying intranuclear IBs in dividing cells we demonstrate for the first time that inclusions such as those of ataxin-1 disperse during mitosis, thus reducing the nuclear aggregate burden. IBs reform in the interphase nucleus. By high-resolution confocal microscopy we also show that inclusions comprise ordered structures capable of homotypic interactions. Unlike those of a non-pathologic protein, ataxin-1 inclusions were shown to be capable of non-specific protein sequestration. Our studies indicate that the specific accumulation of inclusions in terminally differentiated cells such as neurons is a direct consequence of their inability to divide and therefore provides a key to explaining their persistence in neurodegenerative disease.

Isolation, sequencing and expression of RED, a novel human gene encoding an acidic-basic dipeptide repeat.

A novel human gene RED, and the murine homologue, MuRED, were cloned. These genes were named after the extensive stretch of alternating arginine (R) and glutamic acid (E) or aspartic acid (D) residues that they contain. We term this the 'RED' repeat. The genes of both species were expressed in a wide range of tissues and we have mapped the human gene to chromosome 5q22-24. MuRED and RED shared 98% sequence identity at the amino acid level. The open reading frame of both genes encodes a 557 amino acid protein. RED fused to a fluorescent tag was expressed in nuclei of transfected cells and localised to nuclear dots. Co-localisation studies showed that these nuclear dots did not contain either PML or Coilin, which are commonly found in the POD or coiled body nuclear compartments. Deletion of the amino terminal 265 amino acids resulted in a failure to sort efficiently to the nucleus, though nuclear dots were formed. Deletion of a further 50 amino acids from the amino terminus generates a protein that can sort to the nucleus but is unable to generate nuclear dots. Neither construct localised to the nucleolus. The characteristics of RED and its nuclear localisation implicate it as a regulatory protein, possibly involved in transcription.

Inhibitor (IK) of IFN-gamma induced HLA class II antigens expression also inhibits HLA class II constitutive expression in the human Raji B cell line.

The expression of major histocompatibility complex (MHC) class II antigens is constitutive in professional antigen presenting cells (APCs) but can also be induced by interferon-gamma (IFN-gamma) on the majority of the non professional APCs (e.g. fibroblasts). We have recently characterised a new factor called IK which is an efficient inhibitor of IFN-gamma induction of MHC class II antigens expression. Here, we demonstrate a novel role for IK in MHC class II expression since over-expression of this protein by stable transfection into human B cells led to a total disappearance of constitutive MHC class II mRNA expression. The class II transactivator (CIITA) is necessary for both constitutive and IFN-gamma induced MHC class II expressions. Examination of CIITA mRNA in IK stably transfected clones revealed a marked reduction of CIITA mRNA transcription. Taken together these results demonstrate that the IK protein plays a key role in the constitutive expression of MHC class II antigens and that inhibition induced by IK is upstream of CIITA in this regulatory pathway.

A new cytokine (IK) down-regulating HLA class II: monoclonal antibodies, cloning and chromosome localization.

The role of HLA class II Antigens in the control of the immune response is determined not only by the genetic polymorphism of these molecules, but also by their density on the cell surface. It is therefore essential to identify the signals that modulate HLA Class II gene activity in normal and neoplastic cells. We have purified a cytokine (IK factor, 19 kDa) secreted by the leukemic cell line K562 and several cancer cells, which inhibits HLA Class II antigen induction by IFN-gamma. We produced specific mAbs which antagonize the biological effect of IK in colon carcinoma Colo 205 cells induced to express HLA-DR molecules by IFN-gamma. Moreover, in Colo 205, HLA-DR can also be induced by the protein synthesis inhibitor Cycloheximide (0.1 micrograms ml-1); and addition of IK factor almost completely abolishes HLA class II expression. We have also performed the cloning and the sequencing of a specific cDNA. This probe recognizes a 2.1 Kb mRNA in different cell types. The nucleotide sequence exhibits no homologies with known cytokines. IK gene localization shows that it maps on chromosome 2p15-p14. The transient transfection of the cDNA in COS cells induces the secretion of a biologically active 19 kDa protein which is recognized in Western blot by 1C5B11 blocking mAb. This paper reports the characterization of a new cytokine down-regulating HLA class II Antigens, whose analysis will help to better understand HLA class II gene regulation and the mechanism of escape from immunorecognition in cancer cells.

IL-2 production by myofibroblasts from post-radiation fibrosis in breast cancer patients.

The origin of cell activation in post-radiation fibrosis and its chronic extension are still poorly understood. Since local IL-2 cancer treatment sometimes triggers intraperitoneal fibrosis we have analyzed three myofibroblastic cell strains from post-radiation skin fibrosis (FPR7, FPR10 and FPR15) for their interactions with IL-2. In these cells we have observed the surface expression of the two chains of the IL-2R (IL-2R alpha beta), the presence of the 0.9 kb transcript specific for the IL-2 gene and, by flow cytometry with anti-IL-2 mAbs, the presence of IL-2 immunoreactive material inside the cells up to 8 days after subculture. The FPR cell lines secreted IL-2, as determined by ELISA. The secreted IL-2 is biologically active since it sustains the proliferation of the IL-2-dependent murine lymphoid cell line CTLL2 and preincubation with anti-IL-2 blocking mAbs completely abolishes this activity. Overnight incubation of FPR cells with polyclonal anti-IL-2 antibodies leads to a decreased expression of the membrane adhesion molecules ICAM-1 and CD44, suggesting the existence of an autocrine/paracrine loop involved in the surface expression of these antigens. By contrast, in normal adult skin fibroblasts we did not detect IL-2 gene activation. In vivo, IL-2 secretion by post-radiation fibrosis fibroblasts and the subsequent up-regulation of ICAM-1 and CD44 may represent key events during the process that leads to radiation fibrosis.