Down syndrome and mouse models.

The past year has seen major advancements in the characterisation of the Ts65Dn mouse model (which is now known to display many features of Down syndrome). A newer model that is trisomic for the region 21 q22.2--previously called 'Down syndrome' region--has been generated and these mice display behavioural and learning defects. Mutations in the genes Minibrain and SOD1 have been implicated in the development of learning defects in Down syndrome and many new genes from human chromosome 21 are being cloned, which should result in the genesis of other models that phenocopy one or more pathologies of the syndrome.

Antiproliferative potencies of interferons on melanoma cell lines and xenografts: higher efficacy of interferon beta.

BACKGROUND: Human melanomas have shown only limited responsiveness to clinical therapy with interferon (IFN). PURPOSE: Our aim was to determine the most effective class of IFN for inhibiting growth of melanoma cells and to establish whether variation exists in response of various cell lines to different IFNs. METHODS: We compared the direct antiproliferative effects of the type I IFN alpha-2b, IFN alpha-4a, and IFN-beta and the type II IFN-gamma on eight melanoma cell lines grown in vitro. We did this comparison by determining the concentration of each IFN that resulted in 50% growth inhibition, using the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium tetrazolium bromide] dye uptake method. We also tested IFN alpha-2a and IFN-beta for their ability to inhibit the growth of xenografts of the LiBr melanoma cell line in vivo in nude mice. Receptor binding was determined using [35S]methionine-labeled IFN alpha-4a, in competition with unlabeled IFN alpha-2b, IFN alpha-4a, and IFN-beta. RESULTS: The melanoma cell lines differed markedly in their sensitivity to the IFNs tested: Five were sensitive to low concentrations (less than 30 pM) of IFN-beta, only one was sensitive to similar concentrations of IFN alpha-2b, and none were sensitive to IFN alpha-4a at concentrations up to 920 pM. For all cell lines, the antiproliferative potency of the type I IFNs was IFN-beta greater than IFN alpha-2b greater than IFN alpha-4a. IFN-gamma was less active than IFN-beta on all except one of the cell lines. Similarly, IFN-beta was more potent than IFN alpha-2a in inhibiting the growth of the LiBr xenograft in nude mice. Labeled IFN alpha-4a bound with high specificity in all four melanoma lines tested, and competitive binding experiments showed that the order of binding affinity (IFN-beta greater than IFN alpha-2b greater than IFN alpha-4a) correlated with the order of antiproliferative potency. CONCLUSION: The finding that melanoma cell lines differ intrinsically in their sensitivity to IFNs may explain differences in clinical response. Our results suggest that IFN-beta may be the most effective IFN in the treatment of melanoma, although confirmation will require clinical trials involving large numbers of patients.

Pharmacokinetics, tissue distribution, and cell localization of [35S]methionine-labeled recombinant human and murine alpha interferons in mice.

The pharmacokinetics, tissue distribution, cell localization, and penetration into tumor xenografts of recombinant [35S]methionine-labeled human alpha interferon (HuIFN-alpha) and murine alpha interferon (MuIFN-alpha) were examined in mice. Both interferons (IFNs) were removed from the blood in a rapid biphasic manner; HuIFN-alpha was cleared faster than MuIFN-alpha. Tissues were analyzed for radioactivity and over 90% of the IFNs was accounted for. The IFNs were detected predominantly in liver, kidney, gastrointestinal tract, pancreas, spleen, and lung. The levels of MuIFN-alpha compared with HuIFN-alpha were greater in the liver, spleen, and lung and less in the kidney, pancreas, and gastrointestinal tract. Heart, brain, testes, thymus, lymph nodes, fat, skin, and skeletal muscle contained much lower but measurable levels of both IFNs. There was penetration of HuIFN-alpha into tumor xenografts. The pharmacokinetics of IFN-alpha were independent of the strain of mouse, BALB/c or CBA, immune deprivation, or the presence of a tumor xenograft. Autoradiography of tissue sections from mice given injections of HuIFN-alpha or MuIFN-alpha indicated focal radioactivity in proximal convoluted tubules in the kidney and diffuse radioactivity in the liver, gastrointestinal tract, and pancrease. MuIFN-alpha, but not HuIFN-alpha, showed intense localization in cells in hepatic sinusoids, marginal zones in the spleen, and pulmonary alveolar walls, suggesting uptake by cells of the monocyte/macrophage lineage in these sites. The study shows the utility of biosynthetic labeling for pharmacokinetic studies of cytokines, clear differences in tissue distribution of IFN-alpha according to its species of origin, and targeting of homologous IFN-alpha to cells of the monocytic lineage.

Human ERG is a proto-oncogene with mitogenic and transforming activity.

The ETS related gene, ERG, is one of 20 or more genes belonging to the ETS family of transcription factors. Translocation of the ERG gene t(21;22) results in the chimeric fusion transcript seen in approximately 10% of Ewings sarcomas. In addition, recent studies have shown that a reciprocal translocation t(21;16) of ERG gives rise to two aberrant transcripts seen in some forms of acute myeloid leukaemia. In vitro studies have linked the up regulation of ERG expression with stromal cell independence in erythroleukemic clones and shown that the ERG related genes ETS1 and ETS2 have a mitogenic and transforming activity when overexpressed in NIH3T3 cells. Interestingly ERGB/FLI-1, which is also involved in Ewings sarcoma translocations and shares a very high sequence identify with ERG has been reported to be unable to transform NIH3T3 cells. In this study we investigate the effects of overexpression of ERG on cell proliferation, factor dependence, growth in soft agar and tumorigenesis in nude mice. An ERG expression construct with the human ERG2 cDNA driven by the sheep metallothionein la promoter (sMTERG) was transfected into NIH3T3 cells. Clonal cell lines overexpressing ERG were established. The cell lines became morphologically altered, grew in low serum and serum free media and gave rise to colonies in soft agar suspension. Furthermore, we demonstrate that after subcutaneous injection these clones grow as solid tumors in nude mice. These data demonstrate that c-ERG is a proto-oncogene capable of transforming NIH3T3 cells. Therefore, overexpression or inappropriate expression of ERG may contribute to oncogenesis.

Monoclonal antibodies against subunits of yeast mitochondrial H+-ATPase.

Fourteen stable lines of myeloma-spleen cell hybrids producing antibodies against the mitochondrial H+-ATPase have been isolated. One reacted with the alpha-subunit of the enzyme complex (Mr 56000), nine with the beta-subunit (Mr 54000), and four with a 25 kDa subunit which has not been previously characterized. These antibodies are inhibitory or stimulatory or have no effect upon the enzyme activity. Two of the monoclonal anti-beta-subunit antibodies were found to be particularly effective in immunoprecipitating intact H+-ATPase complex.

Characterization of epitopes of the yeast mitochondrial H+-ATPase complex recognized by monoclonal antibodies.

Nine monoclonal antibodies which react with the beta subunit of the yeast mitochondrial H+-ATPase and three which react with a 25 kDa subunit of the enzyme complex (P25) have been characterized. Competitive binding studies indicated the presence of at least four antigenic regions on the beta subunit of the enzyme complex. One antigenic region of the beta subunit is recognized by two monoclonal antibodies RH 57.1 and RH 45.5 which inhibit the ATPase activity to different degrees. Antibody RH 48.6 appears to bind to a second region on the beta subunit and has no effect on the ATPase activity. A third region of the beta subunit is recognized by antibodies RH 51.4 and RH 72.1. RH 51.4 has no effect on the ATPase activity, whereas RH 72.1 stimulates ATPase activity. Antibody RH 32.4 which has no effect on the ATPase activity appears to bind to the fourth epitope of the beta subunit. All three monoclonal anti-P25 antibodies, RH 66.3, RH 41.2 and RH 37.0, apparently bind to the same antigenic region on this subunit. Two of the monoclonal anti-beta antibodies RH 48.6 and RH 51.4 were found to be very effective in immunoprecipitating the whole H+-ATPase complex in a solid phase system. However, the other monoclonal antibodies (and also a polyclonal antiserum) appear to induce the dissociation of one or more of the H+-ATPase subunits by their binding to the epitopes on the beta or the P25 subunits.

Isolation and characterisation of monoclonal antibodies against hydrophobic membrane subunit 9 of the yeast mitochondrial H+-ATPase.

Five stable lines of myeloma-spleen cell hybrids, producing antibodies against the proteolipid subunit 9 of the yeast mitochondrial H+-ATPase F0-sector, have been isolated by immunizing mice with a proteolipid preparation in the presence of sodium dodecyl sulphate. One of these monoclonal antibodies also reacted with subunit 8 of the enzyme complex indicating a shared epitope. The antibodies did not react with the holo-H+-ATPase, suggesting that their epitopes are shielded by other subunits of the enzyme complex.

The chromosome 21 transcription factor ETS2 transactivates the beta-APP promoter: implications for Down syndrome.

The gene that codes for beta-amyloid precursor protein (beta-APP), a protein centrally involved in senile plaque formation in Down syndrome (DS) and Alzheimer's disease (AD), is located on chromosome 21. In DS beta-APP expression is three- to fourfold higher than what is expected from the 1.5-fold increased gene load, suggesting that other genes on chromosome 21 directly or indirectly can further up-regulate beta-APP. Here we show that the chromosome 21 transcription factor ETS2 transactivates the beta-APP gene via specific Ets binding sites in the beta-APP promoter and, in this respect, cooperates with the transcription factor complex AP1. We further show that brains and primary neuronal cultures from Ets2 transgenic mice, as well as 3T3 fibroblasts that overexpress ETS2, display molecular abnormalities also seen in DS, such as elevated expression of beta-APP protein, an increase in presenilin-1 and increased beta-amyloid production. We conclude that ETS2 is a transcriptional regulator of beta-APP and that overexpression of ETS2 in DS may play a role in the pathogenesis of the brain abnormalities in DS and possibly AD.

Functional analysis of interferon-alpha subtypes using monoclonal antibodies to interferon-alpha 4a--subtype reactivity, neutralisation of biological activities and epitope analysis.

A panel of monoclonal antibodies (mAb) to a major human interferon-alpha (IFN-alpha) subtype, -alpha 4a, have been produced, characterised and used for studies of structure/function relationships of IFN-alpha subtypes. The mAb were tested for effects on receptor binding of IFN-alpha 4a, reactivity with other major subtypes -alpha 1, -alpha 2b and -alpha 14 by competitive ELISA and western immunoblotting, and for neutralisation of antiviral and antiproliferative activities of the four subtypes. The mAb could be grouped according to reactivity with IFN-alpha subtypes, group I (designated I-4-A) reacted with -alpha 4a and -alpha 2b, group II (I-4-C and I-4-F) reacted with -alpha 4a and -alpha 1, group III (I-4-D), I-4-G and I-4-H) reacted with -alpha 4a only, whereas group IV (I-4-I) reacted with -alpha 4a, -alpha 1 and -alpha 2b. No mAb reacted with IFN-alpha 14. Sequence comparisons of reactive and non-reactive IFN-alpha subtypes, and reactivity patterns with IFN-alpha fragments obtained by Lys-C digestion indicated that the epitopes were located in the N-terminal region (group I), in two regions of the middle of the molecule (group III and IV) and in the C-terminal region (group II). Binding of mAb to any of these four distinct epitopes neutralised the biological activities of IFN-alpha 4a, and in all cases, except I-4-A, inhibited receptor binding. Only the group III mAb bind to an epitope proposed to be in the vicinity of residues 30-40 which are implicated, from in vitro mutagenesis studies, in receptor binding. Binding of mAb to the other 3 epitopes neutralises biological activities by indirect mechanisms. These results emphasise the antigenic diversity between highly homologous IFN-alpha subtypes, which may have a wider functional significance. Individual mAb will have practical applications in the purification and detection of several IFN-alpha subtypes and so facilitate their further characterisation. By virtue of their different mechanisms of neutralisation, this panel of mAb will be useful in further studies of receptor interaction and signal transduction by IFN-alpha, and illustrate principles which are relevant to immunochemical studies of the receptor interactions of other cytokines.

Subtype-specificity of antipeptide antibodies raised against unique sequences of human interferons-alpha.

A strategy for the production of human interferon-alpha (IFN-alpha) subtype-specific antibodies, based on immunizing rabbits with short unique synthetic peptides coupled to protein carriers, has been validated. These peptides correspond to amino acid residues 99-111 of IFN-alpha 1, 50-57 and 103-116 of IFN-alpha 2, and 37-50 of IFN-alpha 4. The antipeptide antibodies [anti-IFN alpha 1(99-111), anti-IFN alpha 2(50-57C), anti-IFN alpha 2(103-116) and anti-IFN alpha 4(C37-50)] were tested by ELISA and Western blotting for their reactivity with immunoaffinity-purified recombinant human IFN-alpha 1, -alpha 2b and -alpha 4a. The anti-IFN alpha 1(99-111) and anti-IFN alpha 2(50-57C) reacted with their corresponding IFN-alpha and did not crossreact with the other IFN subtypes. The anti-IFN alpha 2(103-116) reacted with IFN-alpha 2b and also crossreacted slightly with the other subtypes. The anti-IFN alpha 4(C37-50) reacted well with IFN-alpha 4a, crossreacted with significantly lower affinity with IFN-alpha 1 and did not bind IFN-alpha 2b. Residues 104-107 and 108-111 are the major components of the epitopes recognized by anti-IFN alpha 1(99-111) and anti-IFN alpha 2(103-116), respectively, as determined by ELISA against overlapping octapeptides.

Constitutive and virus-induced interferon production by peripheral blood leukocytes.

The production of interferon-alpha (IFN-alpha) by normal human peripheral blood mononuclear cells (PBMNC) was studied using polyclonal antipeptide antibodies designed to react either with all IFN-alpha subtypes or with individual subtypes IFN-alpha 2 or IFN-alpha 4. In this study, we demonstrate the detection of intracellular IFN-alpha in PBMNC using immunofluorescence staining and flow-cytometric analysis. Virtually all cells of the PBMNC population were shown to produce IFN-alpha reactive with all three antisera after stimulation with Sendai virus. The immunofluorescence studies also demonstrated that IFN-alpha is produced by PBMNC in the absence of known viral stimulation but is not secreted in detectable levels. Double-labeling with specific monoclonal antibodies to T and B lymphocytes confirmed that the entire populations of these two cell types produce IFN-alpha, both constitutively and after virus induction. Polymorphonuclear cells (PMNC) isolated from Ficoll-Paque pellets were also shown to contain intracellular IFN-alpha, both before and after virus induction. The finding that all PBMNC produce IFN-alpha constitutively suggests that IFN-alpha may have important regulatory functions in situations other than during overt viral infections.

Differential expression and effects of gp130 cytokines and receptors in prostate cancer cells.

High levels of circulating interleukin-6 (IL6), and possibly neuroendocrine (NE) differentiation, correlate with advanced prostate cancer (PCa). IL6 has many overlapping biological effects with the related gp130 cytokines LIF and OSM that can be explained by the shared usage of the signalling receptor, gp130. We set out to determine whether LIF and OSM can substitute for IL6 in PCa, particularly in relation to neuroendocrine differentiation. Expression analysis of the gp130 cytokines and receptors by RT-PCR, Southern blotting and immunohistochemistry showed that they are widely expressed in LNCaP, DU145 and PC3 cells, but not in normal prostate epithelial PZ-HPV-7 cells. IL6, but not LIF or OSM inhibited proliferation, induced NE differentiation and tyrosine phosphorylation of STAT3 in LNCaP cells. The data suggests that IL6 has a unique role in the progression of PCa.

Binding of interferon-alpha and -beta to a component of the human type I interferon receptor expressed in simian cells.

The type I interferons (IFNs) are a family of homologous cytokines that compete for receptor binding. Past experiments with a cloned human IFN-alpha receptor component (designated herein as HuIFNAR-1) transfected into different cell backgrounds have given contradictory results in terms of binding and signalling after exposure of cells to different human type I IFNs. In order to investigate the binding specificity of human type I IFN subtypes to HuIFNAR-1, a cDNA encoding HuIFNAR-1 was transfected into simian COS cells. HuIFNAR-1 expression in COS cells, which was confirmed by Northern blot analysis, resulted in increased binding of 125I-labelled HuIFN-alpha 2 and -beta. These data support the participation of this receptor component in ligand binding, probably in association with other receptor components.

Detailed mapping of the ERG-ETS2 interval of human chromosome 21 and comparison with the region of conserved synteny on mouse chromosome 16.

We have carried out a detailed annotation of 550 kb of genomic DNA on human chromosome 21 containing the ERG and ETS2 genes. Comparative genomic analysis between this region and the interval of conserved synteny on mouse chromosome 16 indicated that the order and orientation of the ERG and ETS2 genes were conserved and revealed several regions containing potential conserved noncoding sequences. Four pseudogenes including those for small protein G, laminin receptor, human transposase protein and meningioma-expressed antigen were identified. A potentially novel gene (C21orf24) with alternative mRNA transcripts, consensus splice donor and acceptor sites, but no coding potential nor murine orthologue, was identified and found to be expressed in a range of human cell lines. We have identified four novel splice variants that arise from a previously undescribed 5' exon of the human ERG gene. Comparison of the cDNA sequences enabled us to determine the complete exon-intron structure of the ERG gene. We have also identified the presence of noncoding RNAs in the first and second introns of the ETS2 gene. Our studies have important implications for Down syndrome as this region contains multiple mRNA transcripts, both coding and potentially noncoding, that may play as yet undescribed roles in the pathogenesis of this disorder.

Generation and characterization of recombinant unmodified and phosphorylatable murine IFN-alpha1 in the methylotropic yeast Pichia pastoris.

In order to generate reagents to study the murine type I interferon (IFN) system, recombinant murine IFN-alpha1 (rMuIFN-alpha1) protein was expressed in the methylotropic yeast Pichia pastoris. rMuIFN-alpha1 with a phosphate acceptor site engineered at the C-terminus (rMuIFN-alpha1P) to enable radiolabeling by gamma(32)P-ATP and cAMP-dependent protein kinase was also generated. Proteins of 20, 25 (MuIFN-alpha1) and 25.5 (MuIFN-alpha1P), kDa were detected in the yeast growth medium, had type I IFN activity, and were recognized by antimurine L929 cell IFN antibodies. The MuIFN-alpha1 proteins produced in P. pastoris were a mixture of glycosylated and unglycosylated forms, with sugars of approximately 5 kDa added via N-linked glycosylation. The recombinant proteins were highly purified using a single RP-HPLC elution step, and their authenticity was confirmed by amino-terminal amino acid sequencing. The MuIFN-alpha1 and MuIFN-alpha1P protein preparations had specific antiviral activities of 1.3 x 10(7) and 4.7 x 10(6) IU/mg protein, respectively. MuIFN-alpha1P could be radiolabeled to a high specific radioactivity (0.6-2 x 10(8) cpm/microg protein) with gamma(32)P-ATP and cAMP-dependent protein kinase without significantly altering its biologic activity or electrophoretic properties. Binding experiments on COS-7 cells transiently transfected with MuIFNAR-2 and IFNAR-2 demonstrated specific and dose-dependent binding of gamma(32)P-ATP-MuIFN-alpha1P to cell surface type I IFN receptors.

Type I interferons mediate the lipopolysaccharide induction of macrophage cyclin D2.

Lipopolysaccharide (LPS) is a powerful macrophage-activating agent and antimitogen. We recently showed that LPS unexpectedly induces cyclin D2 in macrophages. Since LPS stimulates macrophages to produce autocrine-acting cytokines, we examined whether LPS induction of cyclin D2 was mediated by one such type of cytokine, type I interferons (IFN). We report that bone marrow-derived macrophages (BMM) lacking a component of the type I interferon receptor (IFNAR-1) do not express cyclin D2 mRNA or protein in response to LPS stimulation (0.01-1 microg/ml for 7-30 h). Consistent with this result, addition of anti-IFN-alpha/beta neutralizing antibodies reduced levels of LPS-stimulated cyclin D2 in normal BMM. Furthermore, IFN-alpha alone induced cyclin D2 mRNA and protein in normal BMM. Thus, we have identified a new role for type I IFN in macrophages, namely, as essential mediators of LPS-stimulated cyclin D2 expression.

Comparison of different ELISAs for the detection of monoclonal antibodies to human interferon-alpha. Implications for antibody screening.

Three enzyme-linked immunosorbent assays (ELISA) were compared in the initial screening of some 400 hybridoma supernatants for antibodies to a recombinant human interferon-alpha subtype, 4a (IFN-alpha 4a). In these assays, (i) the antigen was coated directly to polystyrene microtitre plates (ELISA-PS), (ii) the antigen was coated directly to nitrocellulose (ELISA-NC), or (iii) the antigen and mouse antibody were reacted in solution and the resulting complex immobilized to a solid support precoated with polyclonal rabbit anti-IFN-alpha antibody (ELISA-SW). The ELISA-PS detected eight antibodies, the ELISA-NC 15 and the ELISA-SW 18. The interferon specificity of the MAbs detected by each of the ELISAs was confirmed by neutralization of IFN-alpha antiviral activity and Western immunoblotting analysis. The results suggest that in ELISAs, the presentation of an antigen and its recognition by antibodies is substantially influenced by the method used in the immobilization of antigen and the type of solid support used. The ELISA-SW proved optimal for screening hybridoma supernatants for antibodies to IFN-alpha 4a, and is recommended for screening for antibodies to other soluble antigens.

Improved conditions for the production of monoclonal antibodies to carcinogen-modified DNA, for use in enzyme-linked immunosorbent assays (ELISA).

The methodology for the production of monoclonal antibodies to chemical carcinogen-modified DNA has been improved to provide high yields of hybridomas, using guanine-imidazole ring-opened aflatoxin B1-modified DNA as an example (iro-AFB1 DNA). The percentage of immunised mice which responded to iro-AFB1 DNA-protein immunisation and the number of specific hybridomas produced was dependent on the level of modification of DNA. One in three BALB/c mice had detectable (but low) antibody titre when 0.3% modified iro-AFB1 DNA was used and this yielded 2 specific hybridomas, whereas all mice responded at reasonable titres and 6 specific hybridomas were obtained when 3% modified iro-AFB1 DNA was used. Other factors found to improve the number and titre of mice responding to immunisation and the yield of hybridomas were: KLH greater than BSA as carrier protein, C57 BL/6 X BALB/c F1 greater than BALB/c mice for antibody production, fusion success and ascites growth. The conditions limiting the sensitivity and reproducibility of an enzyme-linked immunosorbent assay (ELISA) using these monoclonal antibodies with beta-galactosidase-linked sheep F(ab')2 anti-mouse IgG as the second antibody were also tested. Present experience with AFB1 and other carcinogens indicates that these methods should be applicable to the production of monoclonal antibodies to DNA modified by a wide variety of chemical carcinogens.

Molecular detection of interferon-alpha expression in multiple sclerosis brain.

The demonstration of intermittent interferonaemia in patients with multiple sclerosis prompted a molecular analysis of brain tissue for expression of interferon-alpha genes. A sensitive method was developed based on the polymerase chain reaction. Primer sets were used that could amplify all interferons-alpha or two particular subtypes, interferon-alpha 2 and interferon-alpha 4. The procedure was successful in detecting expression of interferons-alpha in brain and non-brain tissues in most patients with multiple sclerosis. However, expression was demonstrable also in a similar proportion of patients with other neural diseases, and patients with other illnesses. The data indicate that there can be constitutive expression of interferons-alpha in brain tissue, but the possibility that this becomes amplified in multiple sclerosis was not revealed by this study.

Decreased natural killer cell activity in endometriosis patients: relationship to disease pathogenesis.

A number of reports have measured NK cell activity in patients with endometriosis with varied results. Therefore we have examined the NK activity of PBL from 44 gynecological patients undergoing laparoscopy. This analysis has demonstrated a significant reduction in NK activity only in more severe stages of endometriosis (stages III and IV) relative to patients with milder disease and controls. These data indicate that decreased NK activity is unlikely to be a primary etiological factor in the development of endometriosis but may indicate that decreased NK activity is related to the development of the more frequent and/or larger lesions characteristic of severe endometriosis. These data could indicate potential for immunotherapy of patients with advanced endometriosis by the upregulation of NK activity in vivo.