Patent Application: US-73393708-A

Abstract:
the present invention provides novel biomarkers for use in the diagnosis and prognosis of cancer and chronic inflammation and further of diseases which are mediated by chronic inflammation . the biomarkers are glycoproteins , the levels of which have been correlated by the inventors to correspond to particular disease conditions . the invention further extends to methods for monitoring the response to therapy of a treatment of a cancerous or chronic inflammatory condition .

Description:
as used herein , an “ antibody ” is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes . the recognized immunoglobulin genes include the kappa , lambda , alpha , gamma , delta , epsilon and mu constant region genes , as well as myriad immunoglobulin variable region genes . light chains are classified as either kappa or lambda . heavy chains are classified as gamma , mu , alpha , delta , or epsilon , which in turn define the immunoglobulin classes , igg , igm , iga , igd and ige , respectively . a typical immunoglobulin ( antibody ) structural unit comprises a tetramer . each tetramer is composed of two identical pairs of polypeptide chains , each pair having one “ light ” ( about 25 kd ) and one “ heavy ” chain ( about 50 - 70 kd ). the n - terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition . the terms variable light chain ( vl ) and variable heavy chain ( vh ) refer to these light and heavy chains respectively . antibodies exist as intact immunoglobulins or as a number of well - characterized fragments produced by digestion with various peptidases . thus , for example , pepsin digests an antibody below the disulfide linkages in the hinge region to produce f ( ab )′ 2 , a dimer of fab which itself is a light chain joined to vh - ch1 by a disulfide bond . the f ( ab )′ 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the ( fab ′) 2 dimer into a fab ′ monomer . the fab ′ monomer is essentially a fab with part of the hinge region ( see , fundamental immunology , w . e . paul , ed ., raven press , n . y . ( 1999 ), for a more detailed description of other antibody fragments ). while various antibody fragments are defined in terms of the digestion of an intact antibody , one of skill will appreciate that such fab ′ fragments may be synthesized de novo either chemically or by utilizing recombinant dna methodology . thus , the term antibody , as used herein , includes antibodies or fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant dna methodologies . antibodies include , e . g ., polyclonal and monoclonal antibodies , and multiple or single chain antibodies , including single chain fv ( sfv or scfv ) antibodies in which a variable heavy and a variable light chain are joined together ( directly or through a peptide linker ) to form a continuous polypeptide , as well as humanized or chimeric antibodies . antibodies , e . g ., antibodies specific for polypeptides bearing glycan markers of the invention , can be generated by methods well known in the art . such antibodies can include , but are not limited to , polyclonal , monoclonal , chimeric , humanized , single chain , fab fragments and fragments produced by a fab expression library . polypeptides do not require biological activity for antibody production . however , the polypeptide or oligopeptide is antigenic . peptides used to induce specific antibodies typically have an amino acid sequence of at least about 5 amino acids , and often at least 10 or 20 amino acids . short stretches of a polypeptide can optionally be fused with another protein , such as keyhole limpet hemocyanin , and antibodies produced against the fusion protein or polypeptide . numerous methods for producing polyclonal and monoclonal antibodies are known to those of skill in the art , and can be adapted to produce antibodies specific for polypeptides bearing markers of the invention . see , e . g ., coligan ( 1991 ) current protocols in immunology wiley / greene , ny ; and harlow and lane ( 1989 ) antibodies : a laboratory manual cold spring harbor press , ny ; stites et al . ( eds .) basic and clinical immunology ( 4th ed .) lange medical publications , los altos , calif ., and references cited therein ; goding ( 1986 ) monoclonal antibodies : principles and practice ( 2d ed .) academic press , new york , n . y . ; fundamental immunology , e . g ., 4th edition ( or later ), w . e . paul ( ed . ), raven press , n . y . ( 1998 ); and kohler and milstein ( 1975 ) nature 256 : 495 - 497 . other suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors . see , huse et al . ( 1989 ) science 246 : 1275 - 1281 ; and ward , et al . ( 1989 ) nature 341 : 544 - 546 . additional details on antibody production and engineering techniques can be found in u . s . pat . no . 5 , 482 , 856 , borrebaeck ( ed ) ( 1995 ) antibody engineering , 2nd edition freeman and company , ny ( borrebaeck ); mccafferty et al . ( 1996 ) antibody engineering , a practical approach irl at oxford press , oxford , england ( mccafferty ), paul ( 1995 ) antibody engineering protocols humana press , towata , n . j . ( paul ), ostberg et al . ( 1983 ) hybridoma 2 : 361 - 367 , ostberg , u . s . pat . no . 4 , 634 , 664 , and engelman et al . u . s . pat . no . 4 , 634 , 666 . specific monoclonal and polyclonal antibodies and antisera will usually bind with a kd of at least about 0 . 1 μm , preferably at least about 0 . 01 μm or better , and most typically and preferably , 0 . 001 μm or better . in practicing the present invention , many conventional techniques in molecular biology , microbiology , and recombinant dna technology are optionally used . these techniques are well known and are explained in , for example , berger and kimmel , guide to molecular cloning techniques , methods in enzymology volume 152 academic press , inc ., san diego , calif . ; sambrook et al ., molecular cloning — a laboratory manual ( 3rd ed . ), vol . 1 - 3 , cold spring harbor laboratory , cold spring harbor , n . y ., 2000 and current protocols in molecular biology , f . m . ausubel et al ., eds ., current protocols , a joint venture between greene publishing associates , inc . and john wiley & amp ; sons , inc ., ( supplemented through 2007 ). other useful references , e . g . for cell isolation and culture include freshney ( 1994 ) culture of animal cells , a manual of basic technique , third edition , wiley - liss , new york and the references cited therein ; payne et al . ( 1992 ) plant cell and tissue culture in liquid systems john wiley & amp ; sons , inc . new york , n . y . ; gamborg and phillips ( eds .) ( 1995 ) plant cell , tissue and organ culture ; fundamental methods springer lab manual , springer - verlag ( berlin heidelberg new york ) and atlas and parks ( eds .) the handbook of microbiological media ( 1993 ) crc press , boca raton , fla . methods of making nucleic acids ( e . g ., by in vitro amplification , purification from cells , or chemical synthesis ), methods for manipulating nucleic acids ( e . g ., site - directed mutagenesis , by restriction enzyme digestion , ligation , etc . ), and various vectors , cell lines and the like useful in manipulating and making nucleic acids are described in the above references . in addition , essentially any polynucleotide can be custom or standard ordered from any of a variety of commercial sources . in addition to other references noted herein , a variety of purification / protein purification methods are well known in the art , including , e . g ., those set forth in r . scopes , protein purification , springer - verlag , n . y . ( 1982 ); deutscher , methods in enzymology vol . 182 : guide to protein purification , academic press , inc . n . y . ( 1990 ); sandana ( 1997 ) bioseparation of proteins , academic press , inc . ; bollag et al . ( 1996 ) protein methods , 2nd edition wiley - liss , ny ; walker ( 1996 ) the protein protocols handbook humana press , nj ; harris and angal ( 1990 ) protein purification applications : a practical approach irl press at oxford , oxford , england ; harris and angal protein purification methods : a practical approach irl press at oxford , oxford , england ; scopes ( 1993 ) protein purification : principles and practice 3rd edition springer verlag , ny ; janson and ryden ( 1998 ) protein purification : principles , high resolution methods and applications , second edition wiley - vch , ny ; and walker ( 1998 ) protein protocols on cd - rom humana press , nj ; and the references cited therein . it is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims . accordingly , the following examples are offered to illustrate , but not to limit , the claimed invention . serum samples were collected at the university hospital zagreb . sera from a septic patient and a patient with acute pancreatitis were taken at the time of reporting to hospital and then three more times through the first eight days of hospitalization . patients claimed to report to hospital on the first day of feeling sick , so this was assumed to be the first day of disease . the blood from a healthy individual , matched by sex and age , was drawn on one occasion only . the possibility of any inflammatory condition in the control subject was additionally excluded by measuring c - reactive protein ( crp was lower than 0 . 5 mg / dl ). the enrolled patients were individuals who fulfilled clinical criteria of sepsis or acute pancreatitis and who had signed the informed consent to participate . this study conformed to the ethical guidelines of the 1975 declaration of helsinki and was approved by the institutional review boards of the university hospital center zagreb and the university of zagreb faculty of pharmacy and biochemistry . the n - glycans from 10 μl of sera were analyzed as described previously ( royle et al manuscript submitted ). briefly , the proteins from sera were immobilized in a block of sds - polyacrylamide gel and n - glycans were released by digestion with recombinant n - glycosidase f ( pngase f , roche diagnostics ). after extraction , glycans were fluorescently labeled with 2 - aminobenzamide ( ludgertag 2 - ab labeling kit ludger ltd ., abingdon , uk ). released glycans were then subjected to normal - phase high performance liquid chromatography ( np - hplc ) on a tsk amide - 80 250 × 4 . 6 mm column ( anachem , luton , uk ) at 30 ° c . with 50 mm formic acid adjusted to ph 4 . 4 with ammonia solution as solvent a and acetonitrile as solvent b . 180 and 120 min runs were on a 2695 alliance separations module ( waters , milford , mass .). hplcs were equipped with a waters temperature control module and a waters 2475 fluorescence detector set with excitation and emission wavelengths of 330 and 420 nm . the system was calibrated using an external standard of hydrolyzed and 2 - ab - labeled glucose oligomers from which the retention times for the individual glycans were converted to glucose units ( gu ) ( royle , l ., radcliffe , c . m ., et al . 2006 ). glycans were analyzed on the basis of their elution positions and measured in glucose units then compared to reference values in the “ glycobase ” database ( available at : http :// glycobase . ucd . ie / cgi - bin / glycobase . cgi ) for preliminary structure assignment ( royle et al submitted ). glycans were separated according to the number of sialic acids by weak anion exchange hplc . the analysis was performed using a vydac 301vhp575 7 . 5 × 50 - mm column ( anachem ltd ., luton , bedfordshire , uk ) ( royle , l ., radcliffe , c . m ., et al . 2006 ). compounds were retained on the column according to their charge density , the higher charged compounds being retained the longest . separated fractions were collected and subjected to np - hplc . glycans , both from total glycan pool and wax separated fractions , were sequenced by exoglycosidase digestions followed by np - hplc ( royle , l ., radcliffe , c . m ., et al . 2006 ). the exoglycosidase digestions of 2 - ab labeled glycans were carried out with the following enzymes obtained from prozyme , san leandro , calif ., usa : abs , arthrobacter ureafaciens sialidase ( glyko sialidase a ,) specific for α2 - 3 , 6 , 8 sialic acids ; nan1 , streptococcus . pneumoniae neuraminidase —( sialidase s ) releases α2 - 3 , 8 linked sialic acid ; btg , bovine testes β - galactosidase specific for β1 - 3 , 4 & amp ; 6 linked galactose ; spg , s . pneumoniae β - galactosidase , specific for β1 - 4 - linked galactose ; bkf , bovine kidney α - fucosidase digests α1 - 2 , 6 & gt ;& gt ; 3 , 4 - fucose ; guh , β - n - acetyl - glucosaminidase digests n - acetylglucosamine but not the bisect ; jbm , jack bean α - mannosidase , amf , almond meal α - fucosidase - removes α1 - 314 - fucose ; xmf , xanthomonus sp . α1 - 2 - fucosidase ( new england biolabs ( hitchin , herts , uk ). samples were incubated overnight at 37 ° c . in 50 mm sodium acetate buffer , ph 5 . 5 . except jbm digestion which were in 100 mm sodium acetate , 2 mm zn2 +, ph 5 . 0 . desialylated glycan samples ( 1 μl of an aqueous solution ) were cleaned with a nafion 117 membrane ( börnsen , k . o ., mohr , m . d ., widmer , h . m . 1995 ) before analysis with 2 , 5 - dihydroxybenzoic acid ( dhb ) on a waters - micromass ( manchester , uk ) tofspec 2e reflectron - tof mass spectrometry operated in reflectron mode with delayed extraction ( harvey , d . j . 1993 ) nano - electrospray mass spectrometry was performed with a waters - micromass quadrupole - time - of - flight ( q - tof ) ultima global instrument . samples of non - 2ab labelled glycans in 1 : 1 ( v : v ) methanol : water containing 0 . 5 mm ammonium phosphate were infused through proxeon ( proxeon biosystems , odense , denmark ) nanospray capillaries as detailed in harvey , d . j ., 2005 . the major n - linked glycans structures from the serum glycoproteins of patients with sepsis or acute pancreatitis were identified and quantified during the first eight days of the disease , and compared to glycans present on normal serum glycoproteins . n - linked glycans were released from sequentially taken sera of a septic patient and a patient with acute pancreatitis as well as from a healthy individual . np - hplc profiles of patients through the first eight days of disease , as well as the glycan profile from the healthy individual are shown in fig1 . structures of the glycans in each peak were identified by combinations of exoglycosidase digestions , chromatographic ( np - hplc and wax - hplc ) and mass spectrometric techniques . the np - hplc and wax - hplc in combination with exoglycosidase digestions enabled identification and quantification of the intact ( sialylated ) compounds , including those which coelute in the undigested profile . quantifications of structures in chromatographic data are expressed as % of all integrated peaks . mass spectrometric techniques , which were performed on desialylated and unlabelled glycans , complemented the chromatographic data and provided details such as the branching pattern of the triantennary glycans ( see legend to fig2 for details ). glycans in the major peaks from the hplc profiles , identified by combination of all techniques mentioned , are listed in table 1 together with the relative percentages . these structures are consistent with those already reported in healthy individuals ( royle et al submitted ). major glycan structures present in the untreated glycan pool released from sera . ( f = found , but in either very small amount or can not be differentiated ( exact area value ) from other peak ). table 2 lists the structures of the glycans that were identified by ms techniques and fig3 shows the maldi profiles of the desialylated glycans taken on day 8 of disease compared with that from the control patient . measured masses were within 0 . 1 mass units for the esi spectra and 0 . 3 mass units for maldi - tof . symbol representation of glycan structure is : glcnac , black square ; galactose , white diamond ; fucose , diamond with a dot inside ; sialic acid , black star ; βlinkage , solid line ; alinkage , dotted line ; 1 - 6 linkage , \; 1 - 4 linkage , −; 1 - 3 linkage , /; 1 - 2 linkage , |. ( a . average mass ; b . position of fucose on 3 - antenna based on known structure of α1 - acid glycoprotein .) the analysis of the glycan profiles from patients with acute pancreatitis and sepsis identified several deviations from the healthy profile , as well as changes that occurred during the course of the disease . the most obvious and persistent changes during the first eight days of both conditions were changes in peaks numbered 20 to 26 ( fig1 ), identified as being trisialylated and tetrasialylated structures with and without sialyl lewis x . changes in trisialylated structures in glycan profiles were persistent in both diseases throughout the studied period . np - hplc analysis of trisialylated glycans isolated by wax - hplc ( fig4 ) showed that the amount of the trisialylated glycan a3g3s3 , decreased relative to the amount of the fucosylated a3fg3s3 ( sialyl lewis x ) during the period studied , particularly in sepsis . the non fucosylated form decreased in sepsis from 5 . 4 to 3 . 4 % in the total glycan pool and in pancreatitis it decreases from 6 . 5 to 5 . 5 %, whilst in the healthy control this fraction represented 8 % of total glycan pool . the fucosylated sialyl lews x form increased in sepsis from 6 . 6 to 6 . 8 %, while in pancretitis this increase was more expressed at 6 . 0 to 9 . 7 % of the total glycan pool ,. compared to 6 % in the control ( see table 1 ). hplc and exoglycosidase sequencing showed that the a3fg3s3 structure digested with almond meal α - fucosidase ( amf ) which removes fucose α1 - 3 or α1 - 4 linked to glcnac and that the galactose on this glcnac was removed by β1 - 4 galactosidase indicating that this was a lewis x rather than a lewis a structure . the outer - arm fucosylation was also found by negative ion ms / ms analysis ( fig2 e ) on desialylated monofucosylated triantennary glycans . the location of the none core fucose was shown to be on a glcnac residue by the absence of a c 1 ion corresponding to gal - fuc in the ms / ms analysis . negative ion ms / ms showed the presence of both core and outer arm fucosylated structures . the relative percentage of the core to antenna - mono - fucosylated triantennary glycans , as measured by the ratio of the 2 , 4 a r ions in the negative ion ms / ms spectrum was 34 % antenna and 66 % core of the control sample , whereas in the sepsis day 1 and day 8 samples , the amount of antenna - fucosylated glycans rose to 52 % and 63 % for the two days respectively . in pancreatitis , the relative amount of antenna - fucosylated to core fucosylated triantennary glycan rose to 78 % on day 2 and 85 % on day 8 this shows a clear increase in the lewis x structure relative to the core fucosylated structure with time in both diseases . from the hplc profiles shown in fig1 it is evident that the tetrasialylated structures were elevated compared to the control profile and that the amount of these structures in the whole glycan pool changed during the course of both diseases . changes observed were additionally analyzed by np - hplc analysis of the tetrasialylated fraction obtained from wax hplc which revealed an increase in outer - arm fucosylated structures over non - fucosylated ones ( fig5 ). negative ion ms / ms of the desialylated monofucosylated tetra - antennary showed that 54 % of the fucose was outer arm in the control sample compared to sepsis samples at day 1 ( 75 %) and day 8 ( 86 %) and 92 % in both of the pancreatitis samples . this also suggests a move to the lewis x structure on tetra - antennary structures during the course of disease . the presence of outer arm fucosylation on di - sialylated biantennary structures was difficult to measure quantitatively due to it low abundance . however negative ion ms / ms ( ratio of the 2 , 4 a r ions ) analysis of the disialylated samples detected a trace of the outer - arm - fucosylated biantennary glycan present in the control and sepsis day 1 sample ( about 2 % of the fucosylated biantennary structures ) which rose to about 7 % on sepsis day 8 . the amount of antenna - fucosylated biantennary glycans in the pancreatitis samples appeared to be somewhat higher , reaching approximately 10 % on days 2 and 8 . the fragmentation pattern showed that the fucose was mainly substituted in the 6 - antenna ( shifts in the d and [ d - 18 ] − ions . changes in fucose levels through the course of disease were also seen when we analyzed all fucose - containing glycans after exoglycosidase digestions ( abs + spg ) or ( abs + spg + amf ) of whole glycan pools . both conditions showed increase in the outer arm fucosylation ( sepsis ˜ 50 % difference between day 1 and day 8 , pancreatitis ˜ 30 % increase ), while core fucosylation decreased in both conditions (˜ 15 %). np - hplc profile of neutral fractions ( prepared by wax hplc ) showed peaks identified as being oligomannose structures ( peaks man5 , man6 , man7 , man8 and man9 ) by digestion with jack bean mannosidase ( jbm ). the relative amounts of these mannose structures changed between the first and eighth day of diseases and also in comparison with the control serum ( fig6 ). the amount of fa2b in the peak which co - elutes with man5 was subtracted before comparisions between the mannose peaks were calculated for fig6 b . the degree of branching was measured after treating the glycan pool with a combination of a sialidase from arthrobacter ureafaciens ( abs ), β - galactosidase from s . pneumoniae ( spg ) and α - fucosidase from bovine kidney ( bkf ), which reduced the glycans into the core antenna structures ( glcnac only attached to the trimannosyl chitobiose core mono -, bi -, tri - and tetraantennary structures ). as shown in fig7 a , the degree of branching in pancreatitis and sepsis was different from the control serum and also changed during the course of the diseases . the control and sepsis samples also contained low levels of glycans with an n - acetyl - lactosamine extension as detected by negative ion ms / ms . the spectra contained a very abundant f - type ion at m / z 789 confirming the antenna structure as hex 2 hexnac 2 . the high relative abundance of this ion is consistent with n - acetyl - lactosamine substitution on the 6 - branch of the 6 - antenna ( reference davids paper ). both sepsis day 1 and day 8 had an additional n - acetyl - lactosamine - extended glycan with an additional fucose . the spectrum from day 1 was weak but that from day 8 showed 80 % of the fucosylated structures had outer - arm - fucose substitution in one of the antennae , and an f ion at m / z 935 ( m / z 789 + 146 ) showed that at least some of this fucose was located on the n - acetyl - lactosamine - extended antenna . this compound was not detected in the early pancreatitis sample but was present on day 8 . the results demonstrate that changes of serum glycans occur very early in acute inflammation . the proportions of different glycans changed daily ; some of them continuously in the same direction , while others varied during the course of acute pancreatitis and sepsis . the most prominent changes that consistently followed disease progression were observed for tri - and tetrasialylated structures as well as for oligomannose structures . these structures were also found to be altered in the first day of both pancreatitis and sepsis ( when compared to the control serum ). in sepsis , the proportion of the trisialylated triantennary a3g3s3 in total glycan pool constantly decreased while in pancreatitis the proportion of the sialyl lewis x structure a3fg3s3 constantly increased . the acute - phase protein α1 - acid glycoprotein , which is elevated early in the acute - phase response , has been recognized as a principal carrier of this lewis antigen ( brinkman - van der linden , e . c ., de haan , p . f ., et al . 1998 ). haptoglobin and α1 - antichymotripsyn were also found to contribute to the elevation of sialyl - lewis x , but to lesser extent . earlier studies also reported elevation of the expression of sialyl - lewis x on α1 - acid glycoprotein induced by inflammation , independent of the increase in the concentration of α1 - acid glycoprotein ( higai , k ., aoki , y ., et al . 2005 ). a similar observation was made in malignant diseases ( croce , m . v ., salice , v . c ., et al . 2005 ). it has been postulated that this increase might have beneficial effects by protecting the organism from overreaction that can occur during inflammation and which could be fatal ( bone , r . c . 1996 ). since the enzyme responsible for the addition of fucose to a3g3s3 is α1 - 3 fucosyltransferase , the levels of this enzyme are crucial . a study on α1 - acid glycoprotein suggested that inflammatory cytokines regulate the expression of α1 - 3 fucosyltransferase vi responsible for α1 - 3 fucosylation in liver tissue ( de graaf , t . w ., van der stelt , m . e ., et al . 1993 , higai , k ., aoki , y ., et al . 2005 ) as well as the expression of α2 - 3 sialyltransferase required for sialyl - lewis x formation ( since only structures containing α2 - 3 - linked neu5ac can be fucosylated ). work on the prognostic value of α1 - acid glycoprotein glycosylation in septic shock ( brinkman - van der linden , e . c ., van ommen , e . c ., et al . 1996 ), indicated that a modest elevation in biantennary glycans in combination with a strong increase in sialyl - lewis x was associated with higher mortality than a high transient increase in biantennary with gradually increasing sialyl - lewis x expression . this clearly demonstrates that the manner of changes in glycan structures can be associated with the severity of a disease . the amounts of oligomannose structures were found to constantly decrease with the progression of acute pancreatitis , while in sepsis they varied slightly throughout the days . however , in both diseases these structures were markedly increased on day 1 ( compared to control ) and then decreased on day 8 ( man6 and man9 fell to below the control level ). these types of glycan structures can be found on the c3 component of complement ( hirani , s ., lambris , j . d ., et al . 1986 ) which is also one of the positive acute - phase proteins . the complement pathway is derived from many small plasma proteins that form the biochemical cascade of the immune system . it is designed to destroy infectious microbes and damaged host material ( ritchie , g . e ., moffatt , b . e ., et al . 2002 ). in contrast to most of the components synthesized mainly in the liver that have complex biantennary structures , c3 contains only oligomannose types ( hase , s ., kikuchi , n ., et al . 1985 , hirani , s ., lambris , j . d ., et al . 1986 ) with predominantly man8 and man9 on the a chain and man6 on the β chain . increases in biantennary glycans have been reported in patients with acute and chronic inflammatory conditions as well as in cancer ( higai , k ., aoki , y ., et al . 2005 ). these compounds were also elevated , especially in the later stage of the acute response . in acute pancreatitis , biantennary glycans with bisecting glcnac were markedly elevated compared to the control . tetraantennary structures were elevated in both diseases , although this elevation was more prominent in pancreatitis . enzymes responsible for synthesising antennae on n - glycans are n - acetylglucosaminyl transferases ( gnt ). gnt iii is the enzyme responsible for adding β - glcnac to the 4 - position of the mannose in the core of n - glycans forming a bisecting β1 → 4 - glcnac structure . gnt iv is responsible for forming triantennary structures by adding β1 → 4glcnac to the 3 - antenna of the tri - mannosyl - chitobiose core while tetraantennary glycans are produced by subsequent actions by this enzyme and gnt v that adds β - glcnac to the 6 - position of the mannose of the 6 - antenna ( brockhausen , i ., hull , e ., et al . 1989 ). increased activities of these enzymes have been reported in many human malignancies ( dennis , j . w . and laferte , s . 1989 , guo , j . m ., zhang , x . y ., et al . 2001 , jin , x . l ., liu , h . b ., et al . 2004 , takamatsu , s ., oguri , s ., et al . 1999 , yao , m ., zhou , d . p ., et al . 1998 ). the results suggest an increase in the activity of these enzymes in the acute - phase response , especially in pancreatitis . an isoform of the triantennary glycans with a branched 6 - antenna represents a significant proportion of the triantennary structures in both sepsis and pancreatitis , while in normal serum the amount of this structure is almost negligible ( table 2 ). comparison of tri - and tetra - antennary structures reveals that the ratio of α1 - 3 - fucosylated forms to core fucosylated or non - fucosylated forms was increased in both pancreatitis and sepsis . this alteration in core fucosylation is different from the findings in different human cancers ( block , t . m ., comunale , m . a ., et al . 2005 , ito , y ., miyauchi , a ., et al . 2003 ), as well as in pancreatic cancer ( barrabes , s ., pages - pons , l ., et al . 2007 ), where the increase in core fucosylation was observed and even suggested as a new diagnostic and prognostic marker . these changes in fucose levels can be a part of the regulatory processes during inflammation since it was suggested that it participates in immune modulation ( bone , r . c . 1996 ) ( listinsky , j . j ., listinsky , c . m ., et al . 2001 ). in general , the results show that changes of serum glycans can be observed very early in acute inflammation and that the proportions of different structures change daily . since glycan profiles in healthy serum are more or less constant , these changes apparently mirror the disease . the observed differences between sepsis and acute pancreatitis are probably due to the fact that , in these two conditions , the acute - phase response is triggered by different stimuli and is , thus , associated with different patterns of production of specific cytokines . this complexity is not surprising knowing how complex and diverse the acute - phase response is and how important roles glycans play in many different processes . all documents referred to in this specification are herein incorporated by reference . various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention . although the invention has been described in connection with specific preferred embodiments , it should be understood that the invention as claimed should not be unduly limited to such specific embodiments . indeed , various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention . 2 - ab — 2 - aminobenzamide ; abs — sialidase from arthrobacter ureafaciens ; amf — almond meal α - fucosidase ; bkf — α - fucosidase from bovine kidney ; btg — β - galactosidase from bovine testes ; crp — c - reactive protein ; dhb — dihydroxybenzoic acid ; esi — electrospray ionization ; gnt — n - acetylglucosaminyl transferase ; gu — glucose unit ; guh — β - n - acetyl - hexosaminidase ; hplc — high - performance liquid chromatography ; jbm — jack bean α - mannosidase ; maldi — matrix - assisted laser desorption / ionization ; ms — mass spectrometry ; nan1 — neuraminidase from streptococcus pneumoniae ; np — normal - phase ; btg — β - galactosidase from bovine testes ; spg — β - galactosidase from s . pneumoniae ; bkf — α - fucosidase from bovine kidney ; guh — β - n - acetyl - hexosaminidase ; jbm — jack bean α - mannosidase ; amf — almond meal α - fucosidase ; pngase f — n - glycosidase f , qtof — quadripole time - of - flight ; spg — β - galactosidase from s . pneumoniae ; wax — weak anion exchange ; xmf — α - fucosidase from xanthomonus sp . ax , number of antenna ( glcnac ) on trimannosyl core ; f at the start of the abbreviation indicates a core fucose α1 - 6 linked to the inner glcnac ; mx , number ( x ) of mannose on the core glcnac ; gx , number ( x ) of β31 - 4 linked galactose on antenna ; g1 [ 3 ] and g1 [ 6 ] indicates that the galactose is on the antenna of the α1 - 3 or α1 - 6 mannose ; f ( x ), number ( x ) of fucose linked α1 - 3 to antenna glcnac ; lac ( x ), number ( x ) of lactosamine ( galβ1 - 4glcnac ) extensions ; sx , number ( x ) of sialic acids linked to galactose ; the numbers 3 or 6 in parentheses after s indicate whether the sialic acid is in an α2 - 3 , α2 - 6 linkage . block , t . m ., et al . 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