Patent Application: US-95056710-A

Abstract:
the present invention provides methods of identifying candidate compounds for the treatment of type i diabetes and also provides methods for treating patients with type i diabetes and for limiting pancreatic beta cell apoptosis . the present invention also provides methods for diagnosing type i diabetes or a propensity to develop type i diabetes and methods for identifying diabetic patients to be treated with anti - apociii therapy .

Description:
within this application , unless otherwise stated , the techniques utilized may be found in any of several well - known references such as : molecular cloning : a laboratory manual ( sambrook , et al ., 1989 , cold spring harbor laboratory press ), gene expression technology ( methods in enzymology , vol . 185 , edited by d . goeddel , 1991 . academic press , san diego , calif . ), “ guide to protein purification ” in methods in enzymology ( m . p . deutshcer , ed ., ( 1990 ) academic press , inc . ); pcr protocols : a guide to methods and applications ( innis , et al . 1990 . academic press , san diego , calif . ), culture of animal cells : a manual of basic technique , 2 nd ed . ( r . i . freshney . 1987 . liss , inc . new york , n . y . ), gene transfer and expression protocols , pp . 109 - 128 , ed . e . j . murray , the humana press inc ., clifton , n . j . ), and the ambion 1998 catalog ( ambion , austin , tex .). in one aspect , the present invention provides methods of identifying candidate compounds for the treatment of type i diabetes comprising contacting pancreatic β cells with an amount effective of apolipoprotein ciii (“ apociii ”) effective to increase intracellular calcium concentration in the presence of one or more test compounds , and identifying those test compounds that inhibit apociii - induced increase in intracellular calcium concentration in the pancreatic β cells . as used herein , “ apociii ” refers to a protein comprising the amino acid sequence shown in seq id no : 2 ( human ) ( ncbi accession number caa25233 ), seq id no : 4 ( rat ) ( ncbi accession number aa40746 ), or seq id no : 6 ( macaque ) ( ncbi accession number caa48419 ), or functional equivalents thereof . the apociii may be substantially purified apociii , available , for example , from sigma chemical company ( st . louis , mo . ), wherein “ substantially purified ” means that it is removed from its normal in vivo cellular environment . alternatively , the apociii may be present in a mixture , such as blood serum from type i diabetic or partially or fully purified therefrom using standard techniques , such as those described below . in a preferred embodiment , substantially purified apociii is used . as discussed below , there are three known isoforms of human apociii that have the same amino acid sequence , but which differ in their glycosylation pattern . thus , in a preferred embodiment , glycosylated apociii is used , wherein the glycosylation is preferably sialylation . in an especially preferred embodiments , mono - sialylated or di - sialylated apociii is used . such glycosylated forms may be purchased , for example , from sigma chemical company , or may be partially or fully purified using standard techniques , such as those described below . as used herein , “ pancreatic β cells ” are any population of cells that contains pancreatic β islet cells . the cells can be obtained from any mammalian species , or may be present within the mammalian species when the assays are conducted in vivo . such pancreatic β islet cell populations include the pancreas , isolated pancreatic islets of langerhans (“ pancreatic islets ”), isolated pancreatic β islet cells , and insulin secreting cell lines . methods for pancreatic isolation are well known in the art , and methods for isolating pancreatic islets , can be found , for example , in cejvan et al ., diabetes 52 : 1176 - 1181 ( 2003 ); zambre et al ., biochem . pharmacol . 57 : 1159 - 1164 ( 1999 ), and fagan et al ., surgery 124 : 254 - 259 ( 1998 ), and references cited therein . insulin secreting cell lines are available from the american tissue culture collection (“ atcc ”) ( rockville , md .). in a further embodiment where pancreatic β cells are used , they are obtained from ob / ob mice , which contain more than 95 % β cells in their islets , and are commercially available . as used herein , “ intracellular calcium concentration ” refers to cytoplasmic free ca 2 + concentration ([ ca 2 + ] i ) in the pancreatic β - cell . such concentrations can be measured by any method known in the art , such as the use of fluorescent calcium indicators , as disclosed herein . as used herein , “ increase intracellular calcium concentration ” refers to increasing the concentration during the course of the assay above that seen in the absence of test compounds . the method does not require a specific amount of increase in intracellular calcium concentration over baseline , so long as the compound ( s ) promotes an increase in intracellular calcium concentration above that seen in the absence of test compounds . in a preferred embodiment , the increase is a statistically significant increase as measured by standard statistical measurements . the contacting of the pancreatic β cells with the apociii may occur before , after , or simultaneously with contacting the cells with one or more test compounds . the contacting can be in vitro , in vivo , or ex vivo . the present invention further provides compounds identified by the above screening methods , and their use for treating subjects with type i diabetes . in another embodiment , the methods further comprise synthesizing the test compounds that inhibit apociii - induced increase in intracellular calcium concentration in the pancreatic β cells . when the test compounds comprise polypeptide sequences , such polypeptides may be chemically synthesized or recombinantly expressed . recombinant expression can be accomplished using standard methods in the art , as disclosed above . such expression vectors can comprise bacterial or viral expression vectors , and such host cells can be prokaryotic or eukaryotic . synthetic polypeptides , prepared using the well - known techniques of solid phase , liquid phase , or peptide condensation techniques , or any combination thereof , can include natural and unnatural amino acids . amino acids used for peptide synthesis may be standard boc ( nα - amino protected nα - t - butyloxycarbonyl ) amino acid resin with standard deprotecting , neutralization , coupling and wash protocols , or standard base - labile nα - amino protected 9 - fluorenylmethoxycarbonyl ( fmoc ) amino acids . both fmoc and boc nα - amino protected amino acids can be obtained from sigma , cambridge research biochemical , or other chemical companies familiar to those skilled in the art . in addition , the polypeptides can be synthesized with other nα - protecting groups that are familiar to those skilled in this art . solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided , such as by using automated synthesizers . when the test compounds comprise antibodies , such antibodies can be polyclonal or monoclonal . the antibodies can be humanized , fully human , or murine forms of the antibodies . such antibodies can be made by well - known methods , such as described in harlow and lane , antibodies ; a laboratory manual , cold spring harbor laboratory , cold spring harbor , n . y ., ( 1988 ). in one example , preimmune serum is collected prior to the first immunization with , for example , apociii . a substantially purified apociii , or antigenic fragments thereof , together with an appropriate adjuvant , are injected into an animal in an amount and at intervals sufficient to elicit an immune response . animals are bled at regular intervals , preferably weekly , to determine antibody titer . the animals may or may not receive booster injections following the initial immunization . at about 7 days after each booster immunization , or about weekly after a single immunization , the animals are bled , the serum collected , and aliquots are stored at about − 20 ° c . polyclonal antibodies against apociii can then be purified directly by passing serum collected from the animal through a column to which non - antigen - related proteins prepared from the same expression system without apociii bound . monoclonal antibodies can be produced by obtaining spleen cells from the animal . ( see kohler and milstein , nature 256 , 495 - 497 ( 1975 )). in one example , monoclonal antibodies ( mab ) of interest are prepared by immunizing inbred mice with apociii , or an antigenic fragment thereof . the mice are immunized by the ip or sc route in an amount and at intervals sufficient to elicit an immune response . the mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks . immunized mice are given one or more booster immunizations of by the intravenous ( iv ) route . lymphocytes , from antibody positive mice are obtained by removing spleens from immunized mice by standard procedures known in the art . hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner under conditions which will allow the formation of stable hybridomas . the antibody producing cells and fusion partner cells are fused in polyethylene glycol at concentrations from about 30 % to about 50 %. fused hybridoma cells are selected by growth in hypoxanthine , thymidine and aminopterin supplemented dulbecco &# 39 ; s modified eagles medium ( dmem ) by procedures known in the art . supernatant fluids are collected from growth positive wells and are screened for antibody production by an immunoassay such as solid phase immunoradioassay . hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of macpherson , soft agar techniques , in tissue culture methods and applications , kruse and paterson , eds ., academic press , 1973 . “ humanized antibody ” refers to antibodies derived from a non - human antibody , such as a mouse monoclonal antibody . alternatively , humanized antibodies can be derived from chimeric antibodies that retains or substantially retains the antigen - binding properties of the parental , non - human , antibody but which exhibits diminished immunogenicity as compared to the parental antibody when administered to humans . for example , chimeric antibodies can comprise human and murine antibody fragments , generally human constant and mouse variable regions . since humanized antibodies are far less immunogenic in humans than the non - human monoclonal antibodies , they are preferred for subsequent therapeutic antibody use . humanized antibodies can be prepared using a variety of methods known in the art , including but not limited to ( 1 ) grafting complementarity determining regions from a non - human monoclonal antibody onto a human framework and constant region (“ humanizing ”), and ( 2 ) transplanting the non - human monoclonal antibody variable domains , but “ cloaking ” them with a human - like surface by replacement of surface residues (“ veneering ”). these methods are disclosed , for example , in , e . g ., jones et al ., nature 321 : 522 - 525 ( 1986 ); morrison et al ., proc . natl . acad . sci ., u . s . a ., 81 : 6851 - 6855 ( 1984 ); morrison and oi , adv . immunol ., 44 : 65 - 92 ( 1988 ); verhoeyer et al ., science 239 : 1534 - 1536 ( 1988 ); padlan , molec . immun . 28 : 489 - 498 ( 1991 ); padlan , molec . immunol . 31 ( 3 ): 169 - 217 ( 1994 ); and kettleborough , c . a . et al ., protein eng . 4 ( 7 ): 773 - 83 ( 1991 ). the term antibody as used herein is intended to include antibody fragments thereof which are selectively reactive with apociii , or fragments thereof . antibodies can be fragmented using conventional techniques , and the fragments screened for utility in the same manner as described herein for whole antibodies . for example , f ( ab ′) 2 fragments can be generated by treating antibody with pepsin . the resulting f ( ab ′) 2 fragment can be treated to reduce disulfide bridges to produce fab ′ fragments . as used herein “ selectively reactive ” means that the antibodies recognize one or more epitope within apociii , but possess little or no detectable reactivity with control proteins , such as bovine serum albumin , under standard conditions such as those disclosed herein . when the test compounds comprise nucleic acid sequences , such nucleic acids may be chemically synthesized or recombinantly expressed as well . recombinant expression techniques are well known to those in the art ( see , for example , sambrook , et al ., 1989 , supra ). the nucleic acids may be dna or rna , and may be single stranded or double . similarly , such nucleic acids can be chemically or enzymatically synthesized by manual or automated reactions , using standard techniques in the art . if synthesized chemically or by in vitro enzymatic synthesis , the nucleic acid may be purified prior to introduction into the cell . for example , the nucleic acids can be purified from a mixture by extraction with a solvent or resin , precipitation , electrophoresis , chromatography , or a combination thereof . alternatively , the nucleic acids may be used with no or a minimum of purification to avoid losses due to sample processing . when the test compounds comprise compounds other then polypeptides , antibodies , or nucleic acids , such compounds can be made by any of the variety of methods in the art for conducting organic chemical synthesis . in another aspect , the present invention provides methods for treating patients with type i diabetes comprising administering to the patient an amount effective of an inhibitor of apociii to reduce apociii - induced increase in intracellular calcium concentration in pancreatic β cells . as used herein , an “ inhibitor ” of apociii includes compounds that reduce the transcription of apociii dna into rna , compounds that reduce translation of the apociii rna into protein , and compounds that reduce the function of apociii protein . such inhibiting can be complete inhibition or partial inhibition , such that the expression and / or activity of the apociii is reduced , resulting in a reduced ability to increase intracellular calcium concentration . such inhibitors are selected from the group consisting of antibodies that bind to apociii ; antisense oligonucleotides directed against the apociii protein , dna , or mrna ; small interfering rnas directed against the apociii protein , dna , or mrna , and any other chemical or biological compound that can interfere with apociii activity . in one embodiment , the inhibitor is identified using the compounds of the present invention . in another embodiment , the inhibitor is selected from the group consisting of ( a ) apociii - selective antibodies , ( b ) antisense nucleic acid constructs derived from the apociii mrna sequence ( seq id nos : 1 , 3 , and 5 ) ( ncbi accession numbers x00567 ( human ); j02596 ( rat ); and x68359 ( macaque ), respectively ), and ( c ) small interfering rna sequences derived from the apociii mrna sequence ( seq id nos : 1 , 3 , and 5 )) ( ncbi accession numbers x00567 ( human ); j02596 ( rat ); and x68359 ( macaque ), respectively ). methods for making antibodies against apociii or fragments thereof are disclosed above . antibodies against apociii are commercially available ( for example , from academy biomedical company ( texas , usa ) chemicon international ( california , usa ); united states biological ( massachusetts , usa ), novus biologicals ( colorado , usa rockland immunochemicals ( pennsylvania , usa ). methods for making antisense oligonucleotides and small interfering rna sequences against the apociii mrna seequence are well known to those of skill in the art , based on the apociii sequences disclosed herein . antisense oligonucleotides will be complementary to the mrna expressed from the apociii gene , in order to bind to the mrna to inhibit translation . in a preferred embodiment for using small interfering rnas , the rnas are double stranded rnas . methods for using such double stranded rnas are as described , for example in u . s . pat . no . 6 , 506 , 559 . for example , rna may be synthesized in vivo or in vitro . endogenous rna polymerase of the cell may mediate transcription in vivo , or cloned rna polymerase can be used for transcription in vivo or in vitro . for transcription from a transgene in vivo or an expression construct , a regulatory region ( e . g ., promoter , enhancer , silencer , splice donor and acceptor , polyadenylation ) may be used to transcribe the rna strand ( or strands ). the rna strands may or may not be polyadenylated ; the rna strands may or may not be capable of being translated into a polypeptide by a cell &# 39 ; s translational apparatus . rna may be chemically or enzymatically synthesized by manual or automated reactions . the rna may be synthesized by a cellular rna polymerase or a bacteriophage rna polymerase ( e . g ., t3 , t7 , sp6 ). if synthesized chemically or by in vitro enzymatic synthesis , the rna may be purified prior to introduction into the cell . for example , rna can be purified from a mixture by extraction with a solvent or resin , precipitation , electrophoresis , chromatography , or a combination thereof . alternatively , the rna may be used with no or a minimum of purification to avoid losses due to sample processing . the rna may be dried for storage or dissolved in an aqueous solution . the solution may contain buffers or salts to promote annealing , and / or stabilization of the duplex strands . in another aspect , the present invention also provides pharmaceutical compositions , comprising an inhibitor of apociii activity and a pharmaceutically acceptable carrier . in a preferred embodiment , the apociii inhibitor is selected from the group consisting of an antibody reactive with apociii or a fragment thereof , an antisense oligonucleotide against the apociii mrna sequence , and a small interfering rna sequence directed against the apociii mrna sequence . the inhibitors may be admixed with lactose , sucrose , starch powder , cellulose esters of alkanoic acids , stearic acid , talc , magnesium stearate , magnesium oxide , sodium and calcium salts of phosphoric and sulphuric acids , acacia , gelatin , sodium alginate , polyvinylpyrrolidine , and / or polyvinyl alcohol , and tableted or encapsulated for conventional administration . alternatively , the inhibitors may be dissolved in saline , water , polyethylene glycol , propylene glycol , carboxymethyl cellulose colloidal solutions , ethanol , corn oil , peanut oil , cottonseed oil , sesame oil , tragacanth gum , and / or various buffers . other adjuvants and modes of administration are well known in the pharmaceutical art . the carrier or diluent may include time delay material , such as glyceryl monostearate or glyceryl distearate alone or with a wax , or other materials well known in the art . the inhibitor may be made up in a solid form ( including granules , powders or suppositories ) or in a liquid form ( e . g ., solutions , suspensions , or emulsions ). inhibitor may be applied in a variety of solutions . suitable solutions for use in accordance with the invention are sterile , dissolve sufficient amounts of the antibody , and are not harmful for the proposed application . the inhibitor may be subjected to conventional pharmaceutical operations such as sterilization and / or may contain conventional adjuvants , such as preservatives , stabilizers , wetting agents , emulsifiers , buffers etc . as discussed below , our study shows that the sialylated forms of apociii were on average four - fold higher in sera from newly diagnosed t1d patients than in sera from healthy subjects . thus , in a further aspect , the present invention provides a method for diagnosing type i diabetes comprising ( b ) determining an amount of sialylated apociii in the blood serum sample ; and ( c ) comparing the amount with an amount of sialylated apociii in a blood serum sample from a non - diabetic patient ; and ( d ) diagnosing those subjects with an elevated amount of sialylated apociii in the blood serum sample relative to the amount of sialylated apociii in a blood serum sample from a non - diabetic patient as having type i diabetes . as disclosed herein , the inventor has discovered that the sialylated forms of apociii predominate in type i diabetic patients , and that analyzing the levels of sialylated apociii in blood serum relative to control will provide a better read out for diagnosis of type i diabetes than analyzing apociii levels as a whole . in a further embodiment , the level of mono - sialylated apociii in the subject relative to control is analyzed ; in another embodiment , the level of di - sialylated apociii in the subject relative to control is analyzed ; and in a further embodiment , both measurements are made . the method does not require a specific amount of increase in sialylated apociii in the blood serum sample over control , although it is preferred that the increase is a statistically significant increase as measured by standard statistical measurements . the diagnostic methods of the invention can be used in combination with any other diagnostic methods known in the art , in order to increase the accuracy of the assays . media the basal medium used both for isolation of cells and for experiments was a hepes buffer ( ph 7 . 4 ), containing ( in mm ): 125 nacl , 5 . 9 kcl , 1 . 3 cacl 2 , 1 . 2 mgcl 2 , 25 hepes . bovine serum albumin was added to the medium at a concentration of 1 mg / ml . for cell culture , rpmi 1640 medium was supplemented with 100 μg / ml streptomycin , 100 iu penicillin and 10 % fetal calf -, normal human - or diabetic serum . preparation of cells adult mice from a local colony ( 3 ) were starved overnight . pancreatic islets were isolated by a collagenase technique and cell suspensions were prepared as previously described ( 4 , 5 ). cells were seeded onto glass coverslips and cultured at 37 ° c . in a humidified atmosphere of 5 % co 2 in air . preparation and purification of sera : sera from t1d patients and control subjects were collected , identically sterile - processed and stored frozen at − 20 ° c . until used . the sera were heat - inactivated by incubation at 56 ° c . for 30 min . thereafter β - cells were incubated overnight in rpmi 1640 culture medium with 10 % of the sera and changes in [ ca 2 + ] i were recorded , subsequent to depolarization with 25 mm kcl . the five t1d sera that induced an enhanced [ ca 2 + ] i response were centrifuged and the supernatant was passed through a 0 . 45 mm sterile filter . samples were loaded on sep - pak c 18 ( waters , ma ) preconditioned with 0 . 1 % tfa . after a wash with 0 . 1 % tfa , proteins were eluted with 60 % acetonitrile in 0 . 1 % tfa and thereafter lyophilized . batches of one milligram of the lyophilized sample were dissolved in 500 μl 0 . 1 % tfa , centrifuged and injected into a rp - hplc with a vydac c 18 ( 0 . 46 × 25 cm ) column ( grace vydac , hesperia , ca ). the separation was made using a linear gradient of 20 - 60 % acetonitrile in 0 . 1 % tfa for 40 min at 1 ml / min . fractions of 1 ml were collected and lyophilized . purification of isoforms of apolipoprotein ciii ( apociii ) apociii was purified from human serum by adsorption to a lipid emulsion and delipidation , followed by chromatography of the lipid - associated proteins under denaturing conditions in guanidinium chloride and urea , respectively , as previously described ( 6 ). the apociii isoforms were dialyzed against ammonium bicarbonate and lyophilized before use . measurements of [ ca 2 + ] i cells , attached to coverslips , were pretreated with the different compounds as described in the results and thereafter incubated in basal medium with 2 μm fura - 2am ( molecular probes , eugene , oreg .) for 30 min . the coverslips were mounted as the bottom of an open chamber and cells were perfused with medium . fluorescence signals were recorded with a spex fluorolog - 2 system connected to an inverted zeiss axiovert epifluorescence microscope . the excitation and emission wavelengths were 340 / 380 and 510 nm , respectively . the results are presented as 340 / 380 excitation ratios , directly representative of [ ca 2 + ] i ( 7 ). patch clamp whole - cell ca 2 + currents were recorded by using the perforated - patch variant of the whole - cell patch - clamp recording technique to eliminate the loss of soluble cytoplasmic components . electrodes were filled with ( in mm ): 76 cs 2 so 4 , 1 mgcl 2 , 10 kcl , 10 nacl , and 5 hepes ( ph 7 . 35 ), as well as amphotericin b ( 0 . 24 mg / ml ) to permeabilize the cell membrane and allow low - resistance electrical access without breaking the patch . pancreatic β - cells were incubated in rpmi 1640 medium with apociii ( 10 : g / ml ) or vehicle overnight . the cells were bathed in a solution containing ( in mm ): 138 nacl , 10 tetraethylammonium chloride , 10 cacl 2 , 5 . 6 kcl , 1 . 2 mgcl 2 , 5 hepes and 3 d - glucose ( ph 7 . 4 ). whole - cell currents induced by voltage pulses ( from a holding potential of − 70 mv to several clamping potentials from − 60 to 50 mv in 10 mv increments , 100 ms , 0 . 5 hz ) were filtered at 1 khz and recorded . all recordings were made with an axopatch 200 amplifier ( axon instruments , foster city , calif .) at room temperature ( about 22 ° c .). acquisition and analysis of data were done using the software program pclamp6 ( axon instruments , foster city , calif .). protein characterization primary sequence was obtained in abi 494c and clc sequencers . protein molecular weights were determined by electrospray mass spectrometry ( autospec hybrid tandem mass spectrometer , micromass ). for recording of positive - ion conventional - es spectra , samples ( 16 μmol / ml ) were introduced into the es interface by infusion or loop injection at a flow rate of 3 ml / min . to determine the position of the glycosylation , the native protein was digested with trypsin 1 : 10 w / w ( promega , madison , wis .). the resulting fragments were separated by hplc using a vydac c 8 ( 2 . 1 × 150 mm ) and a gradient of 0 - 50 % b in 50 min ( buffer a , 5 % acetonitrile / 0 . 1 % tfa ; b , 80 % acetonitrile / 0 . 1 % tfa ). the fragments separated were applied to mass analysis . quantification of apociii sera were collected and prepared as described above . the relative amounts of apociii in t1d serum and control serum , respectively were evaluated by comparisons of the peak area corresponding to apociii in the second rp - hplc . flow cytometric analysis of cell death rinm5f cells were cultured for 36 h in the presence of 10 % control serum , control serum and 40 μg / ml apociii or t1d serum with or without 100 or 200 μg / ml anti - apociii . the whole cell population was collected and stained with egfp - conjugated annexin v and propidium iodide ( pi ) ( bd pharmingen ) and analyzed on a facscan using cellquest acquisition software ( becton dickinson , immunocytometry system ). facs gating , based on forward and side scatter , was used to exclude cellular debris and autofluorescence and typically 10 000 cells were selected for analysis . statistical analysis statistical significance was evaluated by student &# 39 ; s t - test and p values & lt ; 0 . 05 were considered significant . data are expressed as means ± sem . apociii plays a key role in the regulation of the metabolism of triglyceride - rich lipoprotein ( trl ) ( 8 ). it controls the catabolism of trl by inhibiting the activity of lipoproteinlipase ( lpl ) ( 9 , 10 ), thereby inducing hypertriglyceridemia . apociii also inhibits the binding of remnant lipoproteins to catabolic receptors like the ldl receptor related protein ( lrp ) ( 11 ). when the apociii gene was disrupted in knock - out mice , there was a 70 % reduction in triglyceride levels ( 12 ). overexpression of human apociii in transgenic mice results in hypertriglyceridemia ( 13 ). apociii is a 79 - residue , 8 . 8 kda polypeptide ( 14 ) with three known isoforms that differ in the extent of glycosylation , ciii 0 ( no sialic acid ), ciii 1 ( one sialic acid residue ), ciii 2 ( two sialic acid residues ), contributing approximately 10 %, 55 % and 35 %, respectively , of total plasma apociii ( 15 ). mutagenesis of the glycosylation site and expression in stable cell lines suggest that intracellular glycosylation is not required for the transport and secretion functions ( 16 ). lack of glycosylation does not affect the affinity of apociii for vldl and hdl ( 16 ). insulin is involved in the regulation of the apociii gene and induces a dose - dependent down - regulation of the transcriptional activity . overexpression of the apociii gene could contribute to the hypertriglyceridemia seen in t1d patients ( 17 ). however , mice transgenic for the human apociii gene are neither insulin - resistant nor hyperinsulinemic ( 18 ). the concentration of apociii has previously been found to be higher in diabetic patients than in normal subjects ( 19 - 27 ). in insulin deficient rats there was no significant change in apociii in one study ( 28 ), while others have reported an increase in the proportions of the sialylated apociii ( 29 , 30 ). we have tested sera from seven newly diagnosed t1d patients ( table 1 ). mouse pancreatic β - cells were cultured overnight with 10 % sera from patients or normal subjects . sera from five of the patients induced a significantly higher increase in [ ca 2 + ] i , when cells were depolarized with 25 mm kcl , leading to an opening of voltage - gated l - type ca 2 + - channels , than sera from healthy blood donors ( fig1 ). positive sera were pooled , concentrated and fractionated by reversed phase ( rp )- hplc . when fractions were tested on isolated mouse pancreatic β - cells , one fraction ( no . 3 , fig2 a ) eluting between 52 - 60 % acetonitrile , induced a more pronounced increase in [ ca 2 + ] i when cells were depolarized with high concentrations of k + . after further purification of the component ( s ) in this fraction by repeated rp - hplc runs ( fig2 b , d ), all fractions obtained were tested for effects on [ ca 2 + ] i by incubation with mouse β - cells overnight . the results from this second purification ( fig2 b ) showed a higher activity in fraction 2 ( fig2 c ). the protein that induced an increase in [ ca 2 + ] i indicated by the bar in fig2 d was determined . sequence information was obtained both by c - terminal and n - terminal degradations . the sequences were identical to those of human apociii for 20 n - terminal and 5 c - terminal residues . we analyzed the apociii purified from t1d sera by mass spectrometry for subcomponent identification . the major components had apparent masses of 9423 and 9714 da ( fig2 e ), corresponding to the mono - and di - glycosylated forms of apociii ( theoretical , calculated values are : ciii 0 8764 da , ciii 1 9420 da , ciii 2 9712 da ). to determine the positions of glycosylation , the protein was digested with trypsin and the fragments were separated by rp - hplc . when the separated fragments were analyzed by mass spectrometry , seven of the eight fragments showed masses identical to the theoretical values . the mass difference was localized to the c - terminal fragment , previously shown to be glycosylated ( 31 ). the absence of a non - glycosylated c - terminal fragment indicated that the isolated apociii forms were glycosylated . the relative amounts of apociii in t1d and control sera were evaluated by comparisons of the peak area corresponding to apociii in the second rp - hplc ( fig3 a ). in t1d sera the levels of the sialylated isoforms of apociii ( apocill i and apociii 2 ) were four - fold higher than in non - diabetic sera . the non - sialylated isoform ( apociii 0 ) could not be detected . the concentration of apociii has been reported to be between 6 - 14 mg / dl in control subjects and 9 - 27 mg / dl in diabetics ( 19 , 20 , 24 - 27 ). these variations may to a certain extent reflect the fact that various methods have been used for the determinations . in our experiments we have used 10 % t1d serum in the culture medium instead of 10 % fetal calf serum normally used , and therefore we chose to use concentrations in the range 10 - 50 μg / ml . we have tested concentrations from 1 - 50 μg / ml and with 1 , 3 and 6 μg / ml we did not see any effects , but with the concentrations 10 - 50 μg / ml we had responses . commercially available apociii ( sigma ), which constitutes a mixture of apociii 1 and apociii 2 , was tested at a concentration of 10 μg / ml and was shown to stimulate ca 2 + influx as the product isolated from t1d sera ( fig3 b ). co - incubation of β - cells with 100 μg / ml of a polyclonal antibody against human apociii ( academy biomedical company , houston , tex .) blocked the activity of both the commercial apociii and the t1d serum ( fig3 b , c ). the polyclonal antibody had no activity by itself ( data not shown ). when testing the three isoforms of apociii by incubation of β - cells at a concentration of 10 μg / ml , both the glycosylated ( ciii 1 and ciii 2 ) and the un - glycosylated isoform caused significantly higher increase in [ ca 2 + ] i than cells that had been incubated with only the vehicle , 0 . 1 % trifluoroacetic acid ( tfa ) ( fig3 d ). to study the effect of possible binding of apociii to serum lipoproteins in the culture medium , cells were incubated in basal buffer containing no serum and 10 μg / ml apociii 1 for 2 and 6 h . there was a significantly elevated increase in [ ca 2 + ] i upon depolarization in all the experiments where the cells had been exposed to apociii 1 for 6 h , but only in one out of three experiments where the incubation time was only 2 h ( data not shown ). there was a higher percentage of dead cells in the cell population exposed to t1d serum . this effect was prevented by the addition of anti - apociii ( fig3 e ). furthermore , the addition of pure apociii to culture medium with control serum resulted in an increased cell death . to elucidate the molecular mechanism underlying the stimulatory effect of apociii on [ ca 2 + ] i the activity of voltage - gated ca 2 + - channels was analysed in β - cells incubated with 10 μg / ml apociii . apociii - treated cells displayed larger ca 2 + - channel currents than control cells during depolarizations in the range − 10 to 10 mv , from a holding potential of − 70 mv ( fig4 a , b ). these data demonstrate that apociii modulated the activity of the voltage - gated l - type ca 2 + - channel and that the effect occurred in the range of physiological depolarizations . so far immunoblot experiments have not revealed a direct interaction of apociii with the ca 2 + - channel ( data not shown ). future experiment will clarify to what extent this reflects imperfectness in the immunoprecipitation protocol or the actual true situation . our study shows that the sialylated forms of apociii were on average four - fold higher in sera from newly diagnosed t1d patients than in sera from healthy subjects . apociii induced both an increase in [ ca 2 + ] i and β - cell death . the molecular mechanism underlying the stimulatory effect of apociii on [ ca 2 + ] i reflected an activation of the voltage - gated l - type ca 2 + - channel . addition of an antibody against apociii blocked the effects of both t1d serum and apociii on [ ca 2 + ] i as well as on β - cell death . this suggests that the ca 2 + dependent cytotoxic effect of t1d serum on the pancreatic β - cell is mediated by apociii . 1 . efendic , s ., kindmark , h . & amp ; berggren , p . o . 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