Patent Application: US-43819907-A

Abstract:
use of ifi16 protein , fragments or peptides thereof for differential diagnosis of the limited cutaneous form of scleroderma in a subject suspected of or at risk of having an autoimmune disease and the corresponding method of diagnosis and kit .

Description:
the present invention will now be described in detail in relation to some preferred embodiments by way of non - limiting examples . fig1 . increased expression of ifi16 in sle and ssc lesions compared with skin from healthy controls . expression of ifi16 in skin from healthy control ( a ), sle lesion ( b ) and ssc lesion ( c ) ( original magnification × 10 ). d , enlarged section of derma from panel c showing ifi16 staining in inflammatory cells ( original magnification × 20 ). fig2 . a , igg titers against human recombinant ifi16 in patients with ssc ( 82 ), sle ( 100 ), primary sjs ( 20 ), ra ( 50 ), cu ( 38 ) and hcv infection ( 40 ) and from healthy controls ( 80 ). boxes show values within 25 th and 75 th percentiles , the horizontal bar represents the median , 80 % of values are between the extremities of the vertical bars ( 10 th - 90 th percentiles ), and extreme values are represented by individual symbols . differences between the groups have been evaluated by one - way anova and bonferroni multiple comparisons tests after logarithmic transformation . values under the boxes represent the percentage of subjects with igg titers above the cut off value , calculated at the 95 th percentile of the control population . statistical significance : * p & lt ; 0 . 001 vs controls . b , igg titers against human recombinant ifi16 in patients with lc - ssc ( 57 ), dc - ssc ( 25 ), and healthy controls ( 80 ). graphical representation and statistical analysis as in panel a . statistical significance : * p = 0 . 02 vs dc - ssc and controls . fig3 . a , schematic representation of ifi16 and a series of recombinant ifi16 fragments . the two hin200 domains ( domain a and domain b ) are indicated . recombinant peptides corresponding to ifi16 aa 1 - 204 ( ifi16 n - term ) and aa 525 - 726 ( ifi16 c - term ) are indicated by a box . b , reactivity to full length ifi16 by immunoblotting . recombinant ifi16 ( ifi16 ) or control peptide ( pet30a ) were separated by sds - page , transferred to nitrocellulose membranes and then incubated with 1 : 100 dilution of patients sera that were positive ( lanes 1 - 12 , with decreasing titers ) or negative ( lanes 13 - 14 ) using elisa . c , reactivity to ifi16 n - terminal ( ifi16 n - term ), c - terminal ( ifi16 c - term ) fragments or control peptide ( pet30a ). nitrocellulose membranes with transferred recombinant proteins were incubated with patients sera that were reactive using immunoblotting to full length ifi16 . fig4 . igg titers against human recombinant ifi16 in patients with multiple sclerosis ( ms , n = 163 ) and healthy individuals ( ctrl , n = 64 ) as normal controls examined by elisa with recombinant ifi16 protein . the horizontal bar represents the median . the present inventors showed , for the first time , enhanced expression levels of the interferon - inducible protein ifi16 in the epidermis and in the dermal inflammatory infiltrate from both sle and ssc lesions by immunohistochemistry . additionally , the present inventors confirmed that anti - ifi16 autoantibody titers above the 95 th percentile of the controls are significantly elevated in patients with sle and sjs , but not in those with other autoimmune diseases compared with controls . interestingly , the present inventors found comparable prevalence of these autoantibodies in ssc as well . furthermore , the association of anti - ifi16 autoantibody levels in ssc patients with a range of clinical and laboratory parameters was assessed by univariate analysis . the results obtained demonstrated that anti - ifi16 autoreactivity was not associated with either disease duration or disease severity , but with the limited cutaneous form of ssc ( lc - ssc ). ifi16 expression was , in fact , greatly increased and found to be ubiquitously expressed in all layers of the epidermis and in the dermal inflammatory infiltrate in the lesional skin from both sle and ssc patients . patients with sle , sjs and ssc exhibited significantly ( one - way anova p & lt ; 0 . 0001 ) higher anti - ifi16 antibody levels of the igg isotype than normal controls ( sle p & lt ; 0 . 002 , sjs p & lt ; 0 . 001 and ssc p & lt ; 0 . 0005 , respectively ). anti - ifi16 titers above the 95 th percentile of the controls were observed in 26 % of sle , 50 % of sjs and 21 % of ssc respectively . by contrast , anti - ifi16 prevalence was 4 % in ra , 5 % in cu and 10 % in hcv patients respectively . the most striking data came from logistic regression analysis of the three serological autoimmune markers ( anti - ifi16 , anti - centromere and anti - topoisomerase i autoantibodies ), strongly associated with the cutaneous form of ssc in the patients . this analysis showed that all three serological markers investigated were independent predictors of the cutaneous form of scleroderma , and their combination was able to explain 62 % of the associated variability . without being bound to any specific theory , the present inventors believe that ifi16 reactivity in patient with ssc is an important clue to the development of lc - ssc in anti - centromere and anti - topoisomerase i negative patients . in addition , the finding of anti - ifi16 positivity allowed the present inventors to detect lc - ssc patients among the subgroup negative for both anti - centromere and anti - topoisomerase i reactivity , indicating that determination of ifi16 reactivity in patient with ssc is a valuable tool for the differential diagnosis of lc - ssc in the double negative patients . prominent ifi16 expression has been seen in vascular endothelial cells and in stratified squamous epithelia such as skin . its expression is normally restricted to the basal proliferative layer , suggesting a possible role in the control of skin homeostasis . transduction of ifi16 into the human umbilical vein endothelial cells ( huvec ) by recombinant viruses efficiently suppressed the formation of capillary - like structures in vitro and cell - cycle progression associated with cell death ( 8 ). in addition , type i ifn released by plasmacytoid dendritic cells accumulated in cutaneous sle lesions mediates inflammation and expression of interferon - inducible genes . the present invention shows , then , that ifi16 is expressed to a higher level in ssc and sle lesions in both epithelial and inflammatory cells , and autoantibody titers against it are significantly elevated in both diseases . thus , the disease model the present inventors propose is : i ) ifi16 expression in lesional skin is enhanced by local type i ifn production or other pro - inflammatory stimuli ; ii ) ifi16 release , as a consequence of increased cell death , leads to the breakdown in tolerance to this self - antigen as confirmed by the generation of specific anti - ifi16 autoantibodies ; iii ) an additional pathogenic role of ifi16 is suggested by the observation that its endothelial expression triggers apoptosis , up - regulates the expression of genes encoding adhesion molecules ( icam - 1 , e - selectin ) and chemokines ( il - 8 , mcp - 1 ) ( s . l ., unpublished data ) and efficiently suppresses formation of capillary - like structures in vitro . finally , although the root causes of these autoimmune diseases are not yet known , with this disease model the present inventors contribute , and advance , the understanding of the cell and molecular mechanisms that impact on , and underlie , ssc and sle . moreover , antibody titer against ifi16 protein is an important serologic marker for the laboratory testing for the differential diagnosis of the limited cutaneous form of ssc . 290 patients were included in this study , classified as follows : 100 sle , 20 primary sjs , 82 ssc , ra , 38 cu and 163 ms . sera from 80 sex and age matched healthy subjects were collected from blood banks and represented the control group . 40 patients with chronic hcv infection ( kindly provided by dr . mario pirisi , university of piemonte orientale , novara ) were also included as additional controls . ssc and primary sjs sera were from spedali civili , brescia . sle , ra and cu sera were obtained from istituto auxologico italiano , milan . all ssc patients ( 73 women and 9 men , age 21 - 80 , mean age 57 ) were classified as lc - ssc or dc - ssc according to the criteria of leroy et al ( 9 ). patients were regularly assessed using a published consensus core set of variables ( 10 ). disease severity was assessed using the preliminary medsger scale ( 11 ). the most severe involvement was considered for a global severity score . disability index was evaluated by health assessment questionnaire ( haq ) ( 12 ). skin biopsies from 6 patients with ssc and 8 patients with sle were available for immunohistochemistry . they were all taken for diagnostic purposes in stages of active skin disease . control biopsies were taken from unaffected skin of patients undergoing surgery . informed consent for participating in this study was obtained from all donors . the entire coding sequence of the b isoform of human ifi16 ( seq id . no . : 1 , histagged ifi16 ; mhhhhhhssglvprgsgmketaaakferqhmdspdlgtddddkamadigsslmsv kmgkkyknivllkglevindyhfrmvksllsndlklnlkmreeydkiqiadlmee kfrgdaglgklikifediptledlaetlkkeklkvkgpalsrkrkkevdatspap stsstvktegaeatpgaqkrkkstkekagpkgskvseeqtqppspagagmstamg rspspktslsappntsstenpktvakcqvtprrnvlqkrpvivkvlsttkpfeye tpemekkimfhatvatqtqffhvkvlntslkekfngkkiiiisdyleydsllevn eestvseagpnqtfevpnkiinraketlkidilhkqasgnivygvfmlhkktvnq kttiyeiqddrgkmdvvgtgqchnipceegdklqlfcfrlrkknqmsklisemhs fiqikkktnprnndpksmklpqeqrqlpypseasttfpeshlrtpqmppttpsss fftkksedtiskmndfmrmqilkegshfpgpfmtsigpaeshphtpqmppstpss sflttlkprlktepeevsiedsaqsdlkevmvlnatesfvyepkeqkkmfhatva tenevfrvkvfnidlkekftpkkiiaianyvcrngflevypftlvadvnadrnme ipkglirsasvtpkinqlcsqtkgsfvngvfevhkknvrgeftyyeiqdntgkme vvvhgrlttinceegdklkltcfelapksgntgelrsvihshikviktrknkkdi lnpdssmetspdfff ) was subcloned from pbluescript in the pet30a expression vector ( novagen , madison , usa ), containing an n - terminal histidine tag . the sequences encoding n - terminal ( seq id no . : 2 , histagged - ifi16 n - term , amino acid residues 1 - 200 , mhhhhhhssglvprgsgmketaaakferqhmdspdlgtddddkamadigslmsvk mgkkyknivllkglevindyhfrmvksllsndlklnlkmreeydkiqiadlmeek frgdaglgklikifediptledlaetlkkeklkvkgpalsrkrkkevdatspaps tsstvktegaeatpgaqkrkkstkekagpkgskvseeqtqppspagagmstamgr spspktslsappntsstenpktvakcqvtprrnvl ) or c - terminal ( ifi16 c - term , residues 525 - 729 , ( seq id no . : 3 mhhhhhhssglvprgsgmketaaakferqhmdspdlgtddddkamadigsmvlna tesfvyepkeqkkmfhatvatenevfrvkvfnidlkekftpkkiiaianyvcrng flevypftlvadvnadrnmeipkglirsasvtpkinqlcsqtkgsfvngvfevhk knvrgeftyyeiqdntgkmevvvhgrlttinceegdklkltcfelapksgntgel rsvihshikviktrknkkdilnpdssmetspdfff ) fragments of ifi16 were amplified by pcr and cloned in frame in the pet30a vector . expression and affinity purification were performed following standard procedures . the purity of the proteins was assessed by coomassie blue staining following 10 % sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( sds - page ). as negative controls for elisa and immunoblotting , the polypeptide encoded by the pet30a empty vector ( seq id no . : 4 ; control peptide , mhhhhhhssglvprgsgmketaaakferqhmdspdlgtddddkamad igsefelrrqacgrtrappppplrsgc ) was expressed and purified with the same protocol . 250 ng of recombinant ifi16 ( seq id no . : 1 ), ifi16 - n - term ( seq id no . : 2 ), ifi16 c - term ( seq id no . : 3 ) or control peptide ( seq id no . : 4 ) were separated using sds - page under reducing conditions and transferred to nitrocellulose membranes . the membranes were blocked in 3 % top block ( fluka , st . louis , usa ) and then incubated with 1 : 100 dilution of the patients sera . after washing , the membranes were incubated with horse radish peroxidase ( hrp )- conjugated rabbit anti - human igg ( dakocytomation , glostrup , denmark ). the immunocomplexes were detected by enhanced chemiluminescence ( ecl , ge healthcare ) and signals acquired with versadoc3000 ( bio - rad laboratories inc ., hercules , usa ). after stripping , the membranes were reprobed with anti - ifi16 c - terminal rabbit polyclonal antibody ( 13 ) or anti - ifi16 n - terminal mouse monoclonal antibody ( santa cruz biotechnology , santa cruz , usa ) to confirm the presence of the recombinant proteins . immunohistochemical analysis for ifi16 expression was performed on sections from paraffin - embedded tissues placed on silane - coated glass slides as previously described ( 6 ). polystyrene microwell plates for enzyme - linked immunoabsorbent assay ( elisa ) ( nunc - immuno maxi - sorp , nunc , roskilde , denmark ) were coated for 16 h at 4 ° c . with 2 μg / ml of either human recombinant ifi16 ( seq id no . : 1 ) or control peptide solubilized in 0 . 2 ml of phosphate - buffered saline ( pbs ). after blocking with coating buffer containing 3 % bovine serum albumin ( bsa ) in pbs , patients sera , diluted 1 : 100 , were added in duplicate and incubated for 1 h at 37 ° c . after washing , hrp - conjugated rabbit anti - human igg ( dilution 1 : 4800 ) ( dakocytomation ) was added and incubated for 1 h at 37 ° c . after substrate addition , absorbance was measured at 490 nm using a bio - rad microplate reader ( bio - rad ). the results were corrected by subtracting the background reactivity of the reference mixture . statistical analysis was performed by spss statistical software ( spss inc ., chicago , usa ) using the one - way anova with bonferroni multiple comparisons tests . either fisher &# 39 ; s exact test or chi - square test was used for measuring association . the independent effect of significant variables was assessed using forward conditional logistic regression . positivity cut - off values were calculated as the 95 th percentiles in the control population . with substantial deviation from normality , data was natural log transformed before parametric analysis was performed . anti - ifi16 autoantibody levels by elisa and skin ifi16 immunoreactivity were elevated in ssc and sle as recently reported in the normal epidermis from healthy controls , ifi16 expression was restricted to the basal layer ( fig1 a ). notably , as shown in the representative sections in fig1 , ifi16 expression was greatly increased and found ubiquitously expressed in all layers of the epidermis in the lesional skin from both ssc ( fig1 b ) and sle ( fig1 c ) patients . furthermore , the dermal inflammatory infiltrate showed ifi16 positive staining , indicating that it is expressed to a high level in lymphocytes , fibroblasts and endothelial cells . to verify whether the increased expression of ifi16 in the affected skin was related to the presence of anti - ifi16 autoantibodies , their presence and levels in serum samples from 100 sle patients and 82 ssc patients were assessed using elisa with recombinant ifi16 protein . other autoimmune diseases , including sjs , ra and cu as well as healthy individuals as normal controls were also examined . in addition , to further assess the clinical specificity of anti - ifi16 autoantibodies , 40 sera from patients with chronic hcv infection were also subjected to anti - ifi16 elisa . absorbance values higher than the 95 th percentile of the control population ( 0 . 360 ) were considered to be positive in this study . as depicted in fig2 a , patients with ssc , sle and sjs exhibited significantly ( one - way anova p & lt ; 0 . 0001 ) higher anti - ifi16 antibody levels of the igg isotype than normal controls did ( ssc p & lt ; 0 . 0005 , sle p & lt ; 0 . 002 and sjs p & lt ; 0 . 001 respectively ). anti - ifi16 titers above the 95 th percentile of the controls were observed in 21 % of ssc , 26 % of sle and 50 % of sjs respectively . by contrast , anti - ifi16 prevalence was 4 % in ra , 5 % in cu and 10 % in hcv patients respectively . thus , igg autoantibody levels were increased in sle , sjs and ssc but not in other autoimmune diseases , including ra and cu or in patients with increased polyclonal reactivity such as hcv - positive patients . since the presence of anti - ifi16 autoantibodies had already been reported in both sjs and sle , where a significant serological heterogeneity is well known to occur ( 14 ), but not in ssc , a decision was taken to gain more insight into the actual role of anti - ifi16 autoantibodies in ssc pathogenesis . univariate analysis showed that anti - ifi16 autoreactivity in ssc patients was not associated with either disease duration or disease severity as measured by medsger stage , haq disability index , organ involvement and positivity to other autoantibodies ( data not shown ). by contrast , a strict association between anti - ifi16 reactivity and the cutaneous form of the disease was found , with patients in the limited cutaneous scleroderma ( lc - ssc ) having higher anti - ifi16 igg titers than patients with the diffuse form ( dc - ssc ) ( p = 0 . 017 ). indeed , as shown in fig2 b , anti - ifi16 titers above the 95 th percentile of the controls were observed in 28 % of patients with lc - ssc but only in 4 % of dc - ssc ( 95 % confidence interval for the difference 5 - 37 %). in line with previous reports , the presence or titers of anti - ifi16 antibodies did not correlate with clinical manifestations or disease activity in both sle and sjs patients ( data not shown ). in our samples the cutaneous form of ssc was significantly associated with both anti - centromere ( χ 2 = 18 . 771 p & lt ; 0 . 0005 ) and anti - topoisomerase ( χ 2 = 32 . 689 p & lt ; 0 . 0005 ) autoantibodies . logistic regression showed that all the three serological markers were independent predictors of the cutaneous form of scleroderma , and their combination was able to explain 62 % of the associated variability . this model was able to correctly predict 89 % of the clinical presentation forms of scleroderma . moreover , anti - ifi16 reactivity displayed lower sensitivity ( 28 %) and higher specificity ( 96 %) than those found with either anti - topoisomerase i ( 95 % and 67 % respectively ) or anti - centromere ( 65 % and 92 % respectively ). the combined use of anti - ifi16 and anti - centromere markers gave rise to the highest sensitivity and specificity score ( 79 % and 92 % respectively ). interestingly , in the ssc subgroup negative for both anti - centromere and anti - topoisomerase i reactivity , all patients with anti - ifi16 positivity displayed the limited cutaneous form of ssc ( specificity 100 % and positive predictive value 100 %). the presence of anti - ifi16 antibodies was also evaluated by immunoblotting analysis using recombinant ifi16 protein either full length or deleted fragments as schematically represented in fig3 a . western blots were performed on 25 ssc sera , 17 from patients with anti - ifi16 titers above and 8 with titers below the 95 th percentile of the controls . as illustrated in the representative immunoblotting in fig3 , low - titer sera did not exhibit reactivity with ifi16 , thus confirming the specificity of the elisa technique . by contrast , only 10 of the 17 ifi16 high - titer sera were positive , very likely because immunoblot detects antibodies directed against linear epitopes while elisa either linear and conformational epitopes . correlation between anti - ifi16 autoantibody titers and the intensity of immunoreactive bands was not depicted . to further characterize the antigenic specificity of the ifi16 positive sera , the 10 sera recognizing linear epitopes were analyzed for their reactivity against the n - terminal ( ifi16 n - term ) and c - terminal ( ifi16 c - term ) fragments of ifi16 respectively . as shown in the representative immunoblot in fig3 c , 4 sera displayed reactivity against the n - terminal fragment , 3 against the c - terminal fragment , 2 recognized both fragments and 1 displayed no reaction . all together these data suggest a polyclonal immune response against ifi16 in patients with ssc . the presence of anti - ifi16 autoantibodies and their levels in serum samples from 163 patients affected by multiple sclerosis ( ms ) and 64 healthy individuals as normal controls were examined by elisa with recombinant ifi16 protein . as shown in fig4 , patients with ms exhibited significantly ( unpaired t - test p & lt ; 0 . 05 ) higher anti - ifi16 antibody levels of the igg isotype than normal controls did . naturally , while the principle of the invention remains the same , the details of construction and the embodiments may widely vary with respect to what has been described and illustrated purely by way of example , without departing from the scope of the present invention as defined in the appended claims . 1 . landolfo s , gariglio m , gribaudo g , lembo d . the ifi 200 genes : an emerging family of ifn - inducible genes . biochimie 1998 ; 80 ( 8 - 9 ): 721 - 8 . 2 . ludlow l e , johnstone r w , clarke c j . the hin - 200 family : more than interferon - inducible genes ? exp cell res 2005 ; 308 ( 1 ): 1 - 17 . 3 . rozzo s j , allard j d , choubey d , vyse t j , izui s , peltz g , et al . evidence for an interferon - inducible gene , ifi202 , in the susceptibility to systemic lupus . immunity 2001 ; 15 ( 3 ): 435 - 43 . 4 . seelig h p , ehrfeld h , renz m . interferon - gamma - inducible protein p16 . a new target of antinuclear antibodies in patients with systemic lupus erythematosus . arthritis rheum 1994 ; 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