Patent Application: US-52439505-A

Abstract:
dendritic cells that express the type ii c - type lectin dc - sign are located in the submucosa of tissues , where they mediate hiv - 1 entry . interestingly , the pathogen candidaalbicans , the major cause of hospital - acquired fungal infections , is found at similar sites . here it is demonstrated that dc - sign is able to bind candida albicans both in dc - sign transfected cell lines and in human monocyte derived dc . moreover , in immature dc , dc - sign is able to internalize candida in specific dc - sign enriched vesicles , distinct from those containing the mannose receptor , which is another candida receptor on dc . together , these results demonstrate that c . albicans has two major receptors on human monocyte derived dc , these receptors being dc - sign and mr . targeting of dc - sign offers novel opportunities to combat chronic forms of candidiasis .

Description:
c . albicans , both as a commensal as well as a true pathogen , is found on areas ( submucosa ) highly enriched in dc - sign positive dendritic cells . in addition to viruses ( hiv - 1 , siv , ebola ) ( geijtenbeek et al ., 2000b ; alvarez et al ., 2002 ; baribaud et al ., 2001 ) and parasites ( leishmania ) ( colmenares et al ., 2002 ), the c - type lectin dc - sign has now been found to also bind yeast ( c . albicans ). accordingly , c - type lectins on dendritic cells are major pathogen recognition receptors ( figdor et al ., 2002 ). distinct from the toll like receptors ( medzhitov and janeway , 2000 ), they mediate antigen uptake rather than activating dendritic cells . the binding is via a β - 1 , 2 - oligomannoside on c . albicans and other microorganisms . thus other microorganisms comprising β - 1 , 2 - oligomannoside on their cell surface will bind to dendritic cells . the binding of these microorganisms to dendritic cells can be blocked by a variety of compounds as discussed below . use of these compounds can prevent or alleviate infection by said microorganisms . c . albicans has two major receptors on human monocyte derived dc , these being dc - sign and the mannose receptor ( mr ). dc - sign is expressed at sites in the skin ( dermis ) and the mucosa ( geijtenbeek et al ., 2000b ) where c . albicans is known to enter the host . dc - sign positive dc might therefore , through these c - type lectin receptors , form the first encounter with these pathogens and initiate an immune response ( d &# 39 ; ostiani et al ., 2000 ; newman and holly , 2001 ). although not wishing to be bound by the proposed theory , it is believed , in accord with the publications by yamamoto et al . ( 1997 ), shibata et al . ( 1997 ), forsyth et al . ( 1998 ), marth and kelsall ( 1997 ) and szabo et al . ( 1995 ) as set forth by d &# 39 ; ostiani et al . ( 2000 ), that the type of immune response to a pathogen , e . g . candida , infection depends upon the receptor used to phagocytose the pathogen . it is believed that uptake via the mannose receptor generates a positive immune response ( see , e . g ., yamamoto et al . ( 1997 ) and shibata et al . ( 1997 )) whereas uptake via certain other receptors leads to suppression of the immune response to c . albicans or other pathogen ( see , e . g ., forsyth et al . ( 1998 ), marth and kelsall ( 1997 ) and szabo et al . ( 1995 )). it is here shown that pathogens such as candida do bind to dendritic cells via dc - sign and it is theorized that uptake via dc - sign on dendritic cells results in a suppression of the immune response . consequently it is proposed that uptake of candida or other pathogens via the mannose receptor leads to a positive immune response whereas uptake of the pathogens via dc - sign leads to a suppression of the immune response . it is proposed that inhibiting the uptake of pathogens via dc - sign will help prevent a suppression of the immune response and candida - related or other pathogen - related pathology . preventing binding of the pathogen to dc - sign while allowing binding of the pathogen to mannose receptors is proposed to be a useful method to promote a proper response to promote an immune response against the pathogen . some pathogens are destroyed through a th1 response while others rely on a th2 response to be effectively eliminated . again not wishing to be bound by the proposed theory , the finding reported herein that candida and other pathogens are able to bind to dendritic cells via dc - sign leads to the proposal that a favorable immune response will be aided by administration of antigens of the infecting pathogen to a patient . in this method an antigen , e . g ., a purified antigen such as one on the surface of a pathogen such as c . albicans , is administered to a person . the antigen can be taken up by dendritic cells via dc - sign , processed by the dendritic cells and presented to t cells thereby resulting in promotion of an immune response against the pathogen . if the antigen is one that does not naturally bind to dc - sign or even if it does , binding of the antigen to dendritic cells can be promoted by targeting the antigen to the dendritic cells , e . g ., by binding the antigen to an antibody which binds to dc - sign . methods of binding an antigen to an antibody are well known to those of skill in the art . see , e . g ., u . s . pat . no . 6 , 548 , 275 and references cited therein . the antigen - anti - dc - sign antibody complex will bind to dc - sign and be taken up by and processed by the dendritic cells . it is proposed that binding to dc - sign on dendritic cells of just an antigen , rather than the complete pathogen , results in a positive immune response against the pathogen rather than the suppression of the immune response as is proposed to result when the complete pathogen binds to dc - sign and is taken up by dendritic cells . compounds that can be used in the compositions to inhibit binding of microorganisms in accordance with this disclosure include inhibitors for the c - type lectins known per se , including but not limited to those described in wo 93 / 01820 as mentioned above . in general , these are compounds that can bind or adhere to , preferably in a reversible manner , or that can serve as a ligand for , the c - type lectins , in particular the c - type lectin dc - sign or natural variants or equivalents thereof . examples are mannose carbohydrates such as mannan and d - mannose ; fucose carbohydrates such as l - fucose ; plant lectins such as concanavalin a ; antibiotics such as pradimicin a ; sugars such as n - acetyl - d - glucosamine and galactose ; as well as suitable peptidomimetic compounds and small drug molecules , which can for instance be identified using phage display techniques . furthermore , proteins such as gp120 and analogs or fragments thereof or similar proteins with binding capacity to c - type lectins on dendritic cells may be used , as well as isolated icam - receptors and analogs thereof , including parts or fragments thereof . such parts or fragments should then preferably still be such that they can bind to the c - type lectins on the surface of dendritic cells or macrophages . however , the use of carbohydrates is usually less desired from a therapeutic point of view , as they can be rapidly metabolized in vivo . also , the use of plant lectins such as concanavalin a and pradimicin antibiotics can have disadvantages in a therapeutic setting , in particular when treating patients with autoimmune disorders and / or hiv infections . preferably , one or more physiologically tolerable and / or pharmaceutically acceptable compounds are used , such as defined in wo 93 / 01820 . for instance , the use of gp120 or derivatives thereof may cause undesired side effects , in particular on the nervous system ( vide wo 93 / 01820 ). therefore , according to particularly useful embodiments of the present disclosure , preferably an antibody directed against a c - type lectin as present / expressed on the surface of a dendritic cell , or a part , fragment or epitope thereof , is used . as used herein , the term antibodies includes inter alia polyclonal , monoclonal , chimeric and single chain antibodies , as well as fragments ( e . g ., fab , f ( ab ′) 2 , f ( ab ′), fv , fd ) and an fab expression library . furthermore , “ humanized ” antibodies may be used , for instance as described in wo 98 / 49306 . such antibodies against the c - type lectins can be obtained as described hereinbelow or in any other manner known per se , such as those described in wo 95 / 32734 , wo 96 / 23882 , wo 98 / 02456 , wo 98 / 41633 and / or wo 98 / 49306 . for instance , polyclonal antibodies can be obtained by immunizing a suitable host such as a goat , rabbit , sheep , rat , pig or mouse with a c - type lectin or an immunogenic portion , fragment or fusion thereof , optionally with the use of an immunogenic carrier ( such as bovine serum albumin or keyhole limpet hemocyanin ) and / or an adjuvant such as freund &# 39 ; s , saponin , iscom &# 39 ; s , aluminum hydroxide or a similar mineral gel , or keyhole limpet hemocyanin or a similar surface active substance . after an immune response against the c - type lectin has been raised ( usually within 1 - 7 days ), the antibodies can be isolated from blood or serum taken from the immunized animal in a manner known per se , which optionally may involve a step of screening for an antibody with desired properties ( i . e . specificity ) using known immunoassay techniques , for which reference is again made to , for example , wo 96 / 23882 . monoclonal antibodies may be produced using continuous cell lines in culture , including hybridoma and similar techniques , again essentially as described in the above cited references . fragments such as f ( ab ′) 2 and fab may be obtained by digestion of an antibody with pepsin or another protease and reducing disulfide - linkages and treatment with papain and a reducing agent , respectively . fab - expression libraries may for instance be obtained by the method of huse et al . ( 1989 ). preferably , a monoclonal antibody against the c - type lectin ( s ) on dendritic or macrophage cells is used , more specifically against dc - sign or an antigenic part thereof . hereinbelow , the invention will be illustrated by means of monoclonals herein referred to as azn - d1 , azn - d2 , and azn - d3 although similar monoclonals with comparable specificity for c - type lectins may also be used . hybridomas that produce the monoclonals azn - d1 and azn - d2 were deposited on apr . 8 , 1999 with the european collection of cell cultures under provisional ecacc accession numbers 99040818 and 99040819 , respectively . a hybridoma that produces azn - d3 was deposited on jul . 18 , 2003 with the european collect of cell cultures under ecacc accession number 03071801 . antibodies against the c - type lectins on dendritic cells , specifically against dc - sign , have been described by wo 00 / 63251 . for a further description of the methods and techniques known per se in which the antibodies of the invention can be used , reference is made to general textbooks , such as sites et al ., basic and clinical immunology 8th ed ., prentice - hall ( 1994 ) and roitt et al ., immunology , 2nd ed ., churchill livingstone ( 1994 ); both incorporated herein by reference . particular reference is made to the general uses of antibodies and techniques involved therein as mentioned in sections 2 . 7 to 2 . 17 of the general reference work by janeway - travers , immunobiology , the immune system in health and disease , third edition , which is incorporated herein by reference . a composition of the invention may contain one or more of the above - mentioned active compounds . for instance , an anti - c - type lectin antibody can be formulated with mannose or fucose carbohydrates , lectins and / or antibiotics such as pradimicin a , whereby a synergistic effect may be obtained . the present compositions may also contain or be used in combination with known co - inhibitory compounds , such as anti - lf3a ; as well as other active principles known per se , depending upon the condition to be treated . for instance , the present compositions may be formulated or used in combination with immunosuppressants or immunomodulants . compositions in accordance with this disclosure can further be formulated using known carriers and / or adjuvants to provide a pharmaceutical form known per se , such as a tablet , capsule , powder , freeze dried preparation , solution for injection , etc ., preferably in a unit dosage form . such pharmaceutical forms , their use and administration ( single or multi dosage form ), as well as carriers , excipients , adjuvants and / or formulants for use therein , are generally known in the art and are for instance described in wo 93 / 01820 , wo 95 / 32734 , wo 96 / 23882 , wo 98 / 02456 , wo 98 / 41633 and / or wo 98 / 49306 , all incorporated herein by reference . furthermore , the formulation can be in the form of a liposome , as described in wo 93 / 01820 . pharmaceutical formulations of antibodies , their administration and their use are generally described in wo 95 / 32734 , wo 96 / 23882 , wo 98 / 02456 , wo 98 / 41633 and / or wo 98 / 49306 . the present compositions may farther be packaged , for instance in vials , bottles , sachets , blisters , etc . ; optionally with relevant patient information leaflets and / or instructions for use . the compound or composition is in particular administered in such an amount that the interaction between dc - sign on dendritic cells and yeast or pathogen cells are altered or modified , more in particular in such an amount that the adhesion of dendritic cells to yeast or pathogen cells is reduced . alternatively , the compound or composition in administered in such an amount that the interaction between dc - sign on dendritic cells and an antigen , e . g ., a purified antigen , is enhanced . in all the above methods and embodiments , the compounds / compositions used will be administered in a therapeutically effective amount , for which term reference is generally made to wo 93 / 01820 , wo 95 / 32734 and / or wo 96 / 23882 . the administration can be a single dose , but is preferably part of a multidose administration regimen carried out over one or more days , weeks or months . all terms used herein have the normal meaning in the art , for which reference can be made to inter alia the definitions given in wo 93 / 01820 , wo 95 / 32734 , wo 96 / 23882 , wo 98 / 02456 , wo 98 / 41633 and / or wo 98 / 49306 , analogously applied . furthermore , although the invention is described herein with respect to the specific 44 kda c - type lectin receptor dc - sign , it is not excluded that other , generally similar c - type lectins , including natural variants of dc - sign , may also be present on dendritic cells and / or may be involved in dendritic cell / yeast ( or other pathogen ) cell interaction . such variants will usually have a high degree of amino acid homology ( more than 80 % to more than 90 %) with , and / or be functionally equivalent to dc - sign . also , any such receptor will generally display properties similar to those as described herein ; in particular that inhibition of this receptor , either by carbohydrate inhibitors or specific antibodies , will lead to an alteration of dendritic cell / yeast cell interaction . the following examples are offered by way of illustration and are not intended to limit the invention in any manner . standard techniques well known in the art or the techniques specifically described below are utilized . fluorescein isothiocyanate ( fitc ) was from fluka . mannan , d - mannose , and l - fucose were from sigma chemical co . ( st . louis , mo .). il - 4 and granulocyte macrophage colony stimulating factor ( gm - csf ) used for culturing monocyte - derived dendritic cells were from schering - plough ( international , kenilworth , usa ). the following abs were used : azn - d1 , azn - d2 , azn - d3 ( igg1 , anti - dc - sign ( geijtenbeek et al ., 2000a )); azn - l50 ( igg1 , anti - alcam ) ( nelissen et al ., 2000 ); nki - l19 ( igg1 anti - β 2 integrins ); mab clone 19 . 2 against mannose receptor was from bd biosciences pharmingen . β - 1 , 2 - oligomannoside was isolated as described ( shibata et al ., 1996 ; shibata et al ., 1985 ). c . albicans , strain uc820 , a clinical isolate that has been well described ( forsyth and mathews , 1996 ), was maintained on agar slants at 4 ° c . previous experiments showed that strain uc820 can develop hyphae and pseudohyphae in vitro and in vivo to the same extent as a panel of virulent control c . albicans . c . albicans uc820 was inoculated into 100 ml of sabouraud broth and was cultured for 24 hours at 37 ° c . after 3 washes with pyrogen - free saline by centrifugation at 1500 × g , the number of yeast cells was counted in a hemocytometer ; occasional strings of 2 yeast were counted as 1 cfu of c . albicans . the suspension was diluted to the appropriate concentration with pyrogen - free saline . microscopy confirmed that the suspension consisted of blastoconidia . when necessary , the blastoconidia were heat killed either at 56 ° c . for 1 hour or at 100 ° c . for 30 minutes . immature dendritic cells ( imdc ) were generated from human peripheral blood monocytes as described previously ( vissers et al ., 2001 ). briefly , monocytes were isolated by adherence to plastic and cultured in the presence of il - 4 ( 500 u / ml ) and gm - csf ( 800 u / ml ) for 6 - 7 days . k562 transfectants either expressing dc - sign or alcam were generated by transfection of k562 cells with 10 μg of plasmid by electroporation as described previously ( geijtenbeek et al ., 2000b ; nelissen et al ., 2000 ). positive cells were sorted several times to obtain stable transfectants with similar expression levels of dc - sign . labeling of candida cells was performed as follows : viable or heat killed ( 60 minutes at 56 ° c . or 30 minutes at 100 ° c .) yeast cells were resuspended to 2 × 10 8 / ml in 0 . 1 mg / ml fitc in 0 . 05 m carbonate - bicarbonate buffer ( ph 9 . 5 ). after incubation for 15 minutes at room temperature in the dark , fitc - labeled candida cells were washed twice in pbs containing 1 % bsa ( pba buffer ) and analyzed by flow cytometry . dc or k562 were stained in pba with primary abs and fitc - conjugated secondary abs and were analyzed by flow cytometry using the facscalibur ( bd biosciences , mountain view , calif .). isotype - specific controls were included . dc or k562 transfected cells were stained with anti - cd45 - apc prior to exposure to fitc labeled live or heat inactivated candida . dc or k562 cells expressing dc - sign or alcam ( control ) were or were not preincubated for 10 minutes at room temperature with mannan ( 300 μg / ml ), mannose ( 100 mm ), facose ( 100 mm ), egta ( 5 mm ), or a mixture of azn - d1 and azn - d3 ( 20 μg / ml ), in 20 mm tris ph 8 . 0 , containing 150 mm nacl , 1 mm cacl 2 , 2 mm mgcl 2 , and 1 % bsa ( tsa buffer ) or , when egta was used , in pbs . subsequently , fitc labeled candida were resuspended to the appropriate concentrations either in tsa or pbs and added in various cell / c . albicans ratios . after incubation , cell - candida conjugates were analyzed by flow cytometry , and the relative difference in mean fluorescence intensity of the double labeled events compared with that of control cells was calculated . cells were labeled with anti - cd45apc to discriminate cells binding fitc - labeled yeast particles from yeast aggregates . the fluorescent beads adhesion assay was performed as described earlier ( geijtenbeek et al ., 1999 ; nelissen et al ., 2000 ). briefly , carboxylate - modified transfluorspheres ( 488 / 645 nm , 1 . 0 μm ; molecular probes , eugene , oreg .) were coated with icam - 3 - fc or alcam - fc , and adhesion was determined by measuring the percentage of cells that have bound fluorescent beads by flow cytometry . in inhibition studies , the bead adhesion assay was performed in the presence of 0 . 3 mg / ml mannan , 5 mm egta , or 20 μg / ml antibodies against dc - sign or alcam . immature dc ( 5 × 10 5 ) were incubated with unopsonized live fitc - labeled candida cells ( 2 . 5 × 10 6 ) in a total volume of 500 μl at 37 ° c . in a water bath with orbital shaking at 150 rpm for various periods of time . at the end of the incubation period , the dc binding candida are separated from unbound candida by a ficoll gradient . the samples were then adhered on poly - l - lysine coated glass coverslips , fixed in 1 % paraformaldehyde in pbs for 15 minutes at room temperature , and permeabilized in cold methanol for 5 minutes on ice . after a blocking step in pbs / 3 % bsa for 60 minutes at room temperature , cells were labeled with mab ( 10 μg / ml in pbs / 3 % bsa ) for 60 minutes at room temperature and subsequently incubated with cy5 - conjugated goat - anti - mouse f ( ab ′) 2 fragments for 30 minutes at room temperature . finally , the anti - bleach reagent mowiol was added , and samples were analyzed using a mrc1024 confocal microscope ( bio - rad ). candida albicans is a ligand of dendritic cell specific icam - 3 grabbing non - integrin ( dc - sign ) the erythroleukemic cell line k562 transfectants stably expressing dc - sign ( k - sign ) ( geijtenbeek et al ., 2000a ) were used to investigate the potential of c . albicans to bind dc - sign in the absence of any other known c . albicans , receptors . binding to icam - 3 fluorescent beads was used as a positive control for dc - sign function . k562 cells transfected with the homotypic activated leukocyte cell adhesion molecule alcam ( nelissen et al ., 2000 ) ( k - alcam ), which is expressed by dc but does not bind any of the known ligands of dc - sign , were used as negative control . the monoclonal antibodies azn - d1 and azn - l50 were used to detect dc - sign and alcam , respectively . the k562 transfectants stably express dc - sign and alcam , ( fig1 a - 1 through 1 a - 3 ) with expression levels similar to that observed previously ( geijtenbeek et al ., 2000a ; nelissen et al ., 2000 ). transfectant cells were labeled with cd45apc to discriminate cells binding fitc labeled yeast from yeast aggregates . results from a representative experiment are shown in fig1 b . dc - sign clearly mediated adhesion to both c . albicans and icam - 3 - fc - coated beads . k - alcam cells , which are able to bind only alcam - fc beads , were used as negative control . c . albicans and icam - 3 specific adhesion was determined in the presence of blocking anti - dc - sign ( azn - d1 ) mab ( 20 μg / ml ). binding of c . albicans by k - sign is significantly inhibited by blocking antibodies against dc - sign ; in addition , the calcium chelator egta ( present at 5 mm ) abrogates binding ( fig1 c ). this ca 2 + dependence confirms that binding to c . albicans is mediated by the c - type lectin domain of dc - sign . depending on the concentration of c . albicans ( fig1 d ) and on the incubation time ( fig1 e ), binding of k - sign to the yeast cells increases significantly . additional studies were performed to determine the effect on binding of c . albicans to k562 transfectants . fig1 f shows results for k562 cells transfected with k - alcam , k - sign , k - sign - v351 ( k562 transfected with dc - sign with a mutation of valine to glycine at amino acid 351 ), k - signe324 ( k562 transfected with dc - sign with a mutation of glutamic acid to glutamine at amino acid 324 ), and d - l - sign ( k562 transfected with l - sign ( see wo 02 / 50119 or genbank accession no . af290887 for sequence of l - sign that is a protein related to dc - sign )). binding of c . albicans to each of these cells was performed in the presence of either : no blocker , azn - d1 + azn - d3 antibodies , l15 ( an antibody isotypic for azn - d1 and azn - d3 ), mannose , azn - d1 + azn - d3 + mannose , fucose , mannan , or egta . the results indicate binding of candida to k562 cells transfected with wild - type dc - sign . binding was not seen to either mutated form of dc - sign , to l - sign or to alcam . incubation in the presence of azn - d1 + azn - d3 greatly inhibited this binding . egta also prevented the binding . the presence of either mannose or fucose resulted in some inhibition of binding of c . albicans to the k562 cells transfected with wild - type dc - sign . a control experiment using an antibody isotypic to azn - d1 and to azn - d3 showed only a minor effect . further studies were performed as above but using other strains or species of yeast and also comparing binding of the conidiae versus binding of the hyphal form of the organism . fig6 shows the binding to k - sign cells of conidiae and hyphae of c . albicans strains v13 - 12 , v15 - 31 and v18 - 17 and c . dubliniensis strains v18 - 13 , 4247 and 3588 . these are clinical strains isolated from patients . it is seen that the conidiae or single - celled form of the yeast bind to dc - sign better than do the hyphae . the binding of each form to k - sign cells is inhibited by anti - dc - sign antibody and by egta ( fig6 ). fig7 is a different representation of the effect of anti - dc - sign antibody on binding of each of live conidiae and hyphae to immature dendritic cells for each of c . albicans and to c . dubliniensis . in each case the binding in buffer alone in the absence of antibody was set as 100 %. this shows that anti - dc - sign has a small but real inhibition of binding of the two species for both conidiae and hyphal forms . much of the binding to the immature dendritic cells is to mannose receptors , which binding is not affected by the anti - dc - sign antibody . fig8 a - f show the binding of various species of candida as well as aspergillus fumigatus to k - sign cells . the amount of binding varies between the species , but in all cases , except possibly c . tropicalis and c . parapsilosis which each had very low binding , the binding is inhibited by anti - dc - sign and by edta . this was true for both the conidiae and hyphae ( no data for c . glabrata for hyphae ). dc - sign binds live and heat inactivated yeast forms of candida albicans in various experiments , heat inactivated c . albicans were used instead of live cells . in order to exclude the possibility that the binding of dc - sign to heat - inactivated c . albicans was due to artifacts derived from the heat treatment , k - sign were allowed to interact with both live and heat - inactivated c . albicans yeast . as shown in fig2 , the percentage of binding did not increase significantly upon heat inactivation , when compared with binding to live yeast cells . in addition , the blocking of binding by using ab against dc - sign is also not profoundly altered by heat treatment . however , in similar experiments using c . dubliniensis it was found that heat killing the yeast changed the binding affinity , the heat killing resulting in a dramatic decrease in the binding of the single celled yeast form from about 47 % to about 10 %. dc are specialized in binding and uptake of antigen . ( sallusto et al ., 1995 ), and recently it has been published that the interaction between dc and candida is mediated by the mannose - fucose receptor ( d &# 39 ; ostiani et al ., 2000 ; newman and holly , 2001 ). however , considering the present findings on k - sign and the observation that c . albicans can be found in areas of the body ( sub - mucosa ) highly enriched in dc - sign positive cells , the contribution of dc - sign on immature dc in binding candida was further investigated by the present work . in fig3 a it is shown that human monocyte - derived immature dc are able to bind c . albicans , and that this interaction increases with time . for this experiment 50 × 10 3 dendritic cells were incubated with heat inactivated candida ( 500 × 10 3 ). this is in agreement with published results that dc are able to internalize c . albicans within 10 - 20 minutes of incubation at 37 ° c ., reaching a maximum after 60 minutes ( d &# 39 ; ostiani et al ., 2000 ; newman and holly , 2001 ). in an attempt to establish the contribution of both the mannose receptor and dc - sign , immature dc were incubated with specific inhibitors before interacting with c . albicans . immature monocyte derived dendritic cells ( 50 × 10 3 ), which were cd45apc labeled , were incubated with heat - inactivated candida ( 500 × 10 3 ) in the absence or presence of azn - d1 and azn - d3 , anti - dc - sign mab ( 20 μg / ml each ), mannose ( 100 mm ), and egta ( 5 mm ). the results ( an average of five independent experiments ) are shown in fig3 b . the binding to c . albicans in the absence of inhibitors was set as 100 %. * indicates p & lt ; 0 . 01 and ** indicates p & lt ; 0 . 001 percentage of binding in the presence of blocking agent vs . percentage of binding in the absence of blocking agent . the results show that antibodies against dc - sign block binding , though partially ( about 20 %) and to a minor extent with respect to the blocking exerted by mannose on the mr ( about 60 - 70 %). the combination of both anti - dc - sign abs and mannose increases blocking up to about 80 - 85 %. the observation that egta is the most effective blocking agent corroborates the finding that indeed c - type lectins are primarily involved in the binding of c . albicans on immature dc . the bar labeled “ isotype ” in fig3 b is a control incubation including an irrelevant isotypic antibody , the results indicating that inhibition of binding by anti - dc - sign is due to the specificity of the azn - d1 and azn - d3 antibodies rather than to the isotype of the anti - dc - sign antibodies . experiments were performed to measure the amount of each of dc - sign and mannose receptor on the surface of immature dendritic cells . the cells were incubated with antibody ( azn - d1 ) against dc - sign or with antibody against the mannose receptor . both antibodies stain the same number of cells with approximately the same intensity . these results are shown in fig3 c - 1 and 3 c - 2 . the results show that both receptors ( dc - sign and the mannose receptor ) are present in equal quantities on the surface of dendritic cells . additional studies were performed showing the effect on binding of increasing the ratio of candida to immature dendritic cells and the ability of various agents to inhibit this binding . the results are shown in fig3 d . immature dendritic cells and c . albicans were mixed at ratios of 1 : 2 , 1 : 5 and 1 : 10 . it is seen that binding increases as the ratio of candida to imdc increases . it is further seen that the antibodies azn - d1 and azn - d3 can only partially inhibit the binding of candida to imdc . mannose and fucose each have greater inhibitory effects on binding than do the anti - dc - sign antibodies ; the combination of antibodies , mannose and fucose has the greatest inhibitory effect . mannan did not block the binding , whereas egta largely eliminated binding . in addition to dc - sign and the mr , dc are known to express high levels of the β 2 - integrin mac1 , which has already been implicated in the binding of lymphocytes to c . albicans ( forsyth et al ., 1998 ). nevertheless , no blocking of c . albicans binding was detected when anti - β 2 - integrin antibody ( nki - l19 ) was used , suggesting that mac - 1 is not likely involved in c . albicans binding on human immature monocyte derived dc . moreover , the use of laminarin to interfere with the interaction between the dc specific c - type lectin dectin - 1 and c . albicans did not show any block either . these observations strongly suggest that on immature dc the binding of c . albicans is due to a greater extent to mr and also to a lesser extent to dc - sign . it was recently shown that dc - sign is an antigen uptake receptor , facilitating phagocytosis within minutes ( engering et al ., 2002 ). in order to determine whether dc - sign contributes also to the internalization of c . albicans by immature dc , experiments were performed allowing dc to interact with fitc - labeled candida for 60 minutes at 37 ° c . to allow phagocytosis . subsequently , the dc were fixed , permeabilized and fluorescently labeled with specific abs against various receptors . confocal microscopy images ( fig4 a - e ) of immature dc show that binding of fitc labeled c . albicans ( green ) co - localizes ( merged ) with cy5 labeled dc - sign ( blue ). labeling of β 2 integrins ( nki - l19 ), alcam ( nki - l50 ) and mannose receptor was used as controls . fig4 a and 4b represent labeling of the β 2 - integrin mac - 1 and alcam , respectively ; labeling of these receptors was used as negative control , since they were shown not to be involved in the binding of candida by dc . as expected , none of these adhesion receptors was detectable in vesicles containing candida . in contrast , fig4 c clearly shows colocalization of dc - sign with some of the internalized candida , indicating the involvement of this lectin in binding and uptake of this pathogen . the fact that the majority of vesicles do not seem to contain dc - sign is explained by the primary role of mr in the phagocytosis of c . albicans . in fact , as shown in fig4 d , a great colocalization between mr and ingested candida was observed . in order to determine whether dc - sign was able to internalize candida also in presence of inhibitors of mr , dc were allowed to phagocytose candida in the presence of mannose . as shown in fig4 e , vesicles containing dc - sign colocalizing with fitc - labeled candida could still be clearly observed despite the presence of mannose . the presence of egta almost completely blocked phagocytosis . binding of c . albicans to immature dendritic cells was performed in the presence of numerous possible inhibitors of binding . the results are shown in fig5 . binding was performed in the presence of buffer ( tsa ), anti - dc - sign antibodies azn - d1 + azn - d3 , mannose , fucose , mannose + fucose , azn - d1 + azn - d3 + mannose , azn - d1 + azn - d3 + fucose , azn - d1 + azn - d3 + mannose + fucose , nki - l19 ( an igg1 anti - β 2 - integrin )+ mannose + fucose , nki - l19 , mannan , laminarin ( a β - 1 , 3 - linked oligomannoside ), antibody to dcir ( a c - type lectin , see bates et al . ( 1999 )), β - 1 , 2 - oligomannoside , and egta . as seen above , the azn - d1 and azn - d3 inhibited the imdc / candida binding to only a small extent . also , l19 , mannan , laminarin and anti - dcir had only minor effects upon the binding . mannose and fucose inhibited binding to a large degree as did egta . additionally , it was found that β - 1 , 2 - oligomannoside greatly inhibited binding , contrasting with the lack of inhibition by laminarin . this indicates that binding between dc - sign and candida occurs via the β - 1 , 2 - oligomannoside on c . albicans . additional experiments will test the ability of an anti - candida monoclonal antibody that binds to β - 1 , 2 - oligomannoside to block the binding of c . albicans to dc - sign bearing cells . furthermore , a peptidomimetic ( fhenwps ( seq id no : 1 )) for this carbohydrate will be tested . jouault et al . ( 2001 ) have described that vaccination with this peptide gave anti - candida antibodies . dendritic cells are not only equipped with a highly specialized antigen presentation machinery , but they also possess a unique mechanism that enables them to migrate into tissues and to reach lymphoid compartments when activated to present antigen . due to the low number of dc present in the peripheral blood , dc were generated from human monocytes after culture in the presence of gm - csf and il - 4 . in addition to the known alterations in chemokine receptors , in performing experiments it was noticed that there were major phenotypical switches in adhesion molecules upon dendritic cell development and maturation . interestingly , it was observed that lfa - 1 as well as the other β 2 - integrins p150 . 95 and mac - 1 lose their ability to bind to icam - 1 , although the expression levels of these receptors are increased . moreover , lfa - 1 becomes unable to bind to icam - 3 ; in fact , the binding to this molecule on dc is completely dc - sign dependent . to explain these differences in adhesive properties , the cell surface distribution of these receptors was investigated . employing high resolution microscopy techniques , including electron microscopy and near - field scanning optical microscopy , clearly distinct patterns of distribution of lfa - 1 in comparison with dc - sign on these cell types were observed . the distribution in defined microdomains on the cell surface directly correlates with ligand binding . it will be appreciated that the methods and compositions of the instant invention can be incorporated in the form of a variety of embodiments , only a few of which are disclosed herein . it will be apparent to the artisan that other embodiments exist and do not depart from the spirit of the invention . thus , the described embodiments are illustrative and should not be construed as restrictive . aderem a and underhill d m ( 1999 ). annu . rev . immunol . 17 : 593 - 623 . colmenares m , et al . 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