Patent Application: US-90611707-A

Abstract:
the present invention provides the use of formulation with surfactant protein - d in the modulation of activity of human eosinophils derived from hypereosinophilic patients to an increased activation state and increased apoptosis . accordingly the utility of the invention can be extended in human subjects in resolution of eosinophilic inflammations in related diseases and disorders like neuromuscular and respiratory diseases with eosinophilia other than airway - hyperresponsiveness , allergy and asthma , hypereosinophilic leukemias , hypereosinophilc syndromes , skin diseases like eosinophilia - myalgia syndrome , eosinophilic fascitis , capillary leak syndromes , churg - strauss syndrome , toxic oil syndrome , parasitosis , etc ., where a large number of stimulated eosinophils accumulate and release a series of growth factors , cytokines , chemokines , bioactive lipid mediators , toxic oxygen metabolites .

Description:
the present invention provides the use of a formulation for inducing and increasing apoptosis in activated eosinophils comprising of complete or fragment ( s ) of native or recombinant human lung surfactant protein d , the said use comprising steps of : in an embodiment to the invention , isolation of human eosinophils was carried out from 50 ml peripheral blood of hypereosinophilic patients and healthy individuals by dextran sedimentation and density centrifugation , followed by negative immunomagnetic selection using antihuman cd16 microbeads . the standard protocol followed resulted in greater than 95 % pure and 99 % viable eosinophil population . in an embodiment to the invention , native human sp - d was found to be free from igg , igm , and ige contamination by elisa using anti - human igg , anti - human igm , and anti - human ige peroxidase conjugates , respectively . in another embodiment to the invention , a recombinant homotrimer containing eight gly - x - y collagen repeat sequences , an alpha - helical , coiled - coil neck region , and the carbohydrate recognition domain of sp - d was expressed as inclusion bodies in escherichia coli . this truncated sp - d , lacking the whole collagen domain and multimerising n - terminal region , retained the immunomodulatory properties displayed by full - length sp - d . cross - linking studies indicated that rhsp - d exists predominantly as a trimer in solution . its identity was confirmed by n - terminal sequencing , and was judged to be pure by sds - page and immunoblotting . in another embodiment to the invention , native human surfactant protein - d ( nhsp - d ) and rhsp - d were labeled with fitc for the binding studies . the presence of unlabelled rhsp - d competitively inhibited the binding of nhsp - d - fitc to the human eosinophils . in another embodiment to the invention , rhsp - d binding to human eosinophils was found to be calcium dependent and was inhibited in the presence of edta . in another embodiment to the invention , the pharmacologically active concentration of nhsp - d and rhsp - d was used ranging between 0 . 1 and 10 μg per ml of the protein . in another embodiment to the invention , there was an increase in apoptosis in eosinophils derived from hypereosinophilic patients in the presence of rhsp - d while eosinophils from healthy donors showed a decrease in apoptosis . in another embodiment to the invention , there was an increase in apoptosis in cytokine ( il - 5 ) primed eosinophils from healthy donors in the presence of rhsp - d to that of il - 5 primed eosinophils in absence of rhsp - d . five subjects ( 3 male and 2 female ) with mean age 28 years , range 22 - 31 , with elevated eosinophil counts and categorized as hypereosinophilic were included in this study . none of the subjects had received any medication for ≧ 24 h or steroids for ≧ 2 weeks before blood collection . the healthy donors ( 4 male and 2 female ) ( mean age 25 years , range 22 - 26 ) were defined on the basis of a lack of a clinical history of hypereosinophilia and had not been taking any medication 4 wk before the study . informed written consent was obtained from all the subjects . the project was approved by institutional human ethical committee and the guidelines were followed during sample collection . mixed granulocytes were obtained by dextran sedimentation and density centrifugation through histopaque - 1077 from the peripheral blood from hypereosinophilic patients and normals ( anticoagulated with 0 . 4 % ( w / v ) trisodium citrate ( ph 7 . 4 )) ( 25 ). eosinophils were subsequently isolated from the granulocyte pellet by negative immunomagnetic selection ( 26 ) by magnetic cell sorting of human leukocytes ( macs ) kit using anti - human cd16 microbeads , variomacs magnetic cell separator and ld depletion columns ( miltenyi biotec , ca , usa ). the protocol for eosinophil separation was followed as described by the manufacturer . the cd16 − eosinophils were separated from the cd16 + microbeads tagged neutrophils by passing the cell suspension through the magnetic column . this resulted in greater than 95 % pure eosinophil population . the eosinophil purity was assessed by hinkleman &# 39 ; s staining and may - grunwald giemsa staining . the viability of the purified eosinophils was determined by trypan blue dye exclusion and was consistently & gt ; 99 %. the eosinophils were washed and used further for the study . purified native human sp - d ( obtained commercially ) was judged to be pure by sds - page , western blot analysis , and amino acid composition . it was found to be free from igg , igm , and ige contamination by elisa using anti - human igg , anti - human igm , and anti - human ige peroxidase conjugates , respectively . a recombinant homotrimer containing eight gly - x - y collagen repeat sequences , an alpha - helical , coiled - coil neck region , and the carbohydrate recognition domain of rhsp - d was expressed as inclusion bodies in escherichia coli . the inclusion bodies were isolated , denatured in 6 m urea and slowly renatured overnight in decreasing amounts of urea . the soluble rhsp - d was purified on a maltose - agarose column and contaminating lps was removed using a polymixin - b column . cross - linking studies indicated that rhsp - d exists predominantly as a trimer in solution ( 24 ). its identity was confirmed by n - terminal sequencing , and was judged to be pure by sds - page and immunoblotting . for fitc labeling , the surfactant protein was dialyzed against fitc labeling buffer ( 0 . 05 m boric acid , 0 . 2 m sodium chloride , ph 9 . 2 ) at 4 ° c . with 2 - 3 changes over 2 days . 20 μl of 5 mg per ml fitc in dmso was then added for each mg of protein and incubated for 2 h at room temperature . the proteins were then dialyzed against buffer ( 0 . 1 m tris - hcl , ph 7 . 4 , 0 . 1 % w / v sodium azide , 0 . 2 m nacl ) and stored at 4 ° c . the flurochrome to protein ratio of 5 - 6 : 1 was considered optimum for flow cytometric analysis ( 28 ). to measure binding of fitc labeled rhsp - d or nhsp - d , purified eosinophils ( 5 × 10 5 cells per ml ) were washed and resuspended in the staining buffer ( pbs with 0 . 1 % sodium azide , 1 . 0 % bsa ) supplemented with 5 mm cacl 2 and 1 mm mgcl 2 or as indicated . 100 μl of this suspension was co - incubated with fitc labeled rhsp - d at indicated concentrations for 45 minutes at 4 ° c . cells were washed twice with washing buffer ( pbs with 1 % bsa ) by centrifugation at 4 ° c . at 200 × g and fixed with 4 % formaldehyde in pbs . for calcium - dependent studies , staining buffer containing 0 , 0 . 1 , 1 or 5 mm calcium chloride or buffer with 10 mm edta was used . flow cytometry was performed on at least 10 , 000 cells with a facscan ™ flow cytometer ( becton dickinson ). the specific mean fluorescence ( smf ) was determined by subtracting the non - specific mean fluorescence ( nmf ) of the cells . the relative smf ( rsmf ), which is the ratio of smf emitted by bound surfactant protein to that of the nmf of the corresponding control , was calculated . the results were expressed as rsmf or % rsmf or as their mean fluorescence intensity ( mfi ) or % mfi . the effect of rhsp - d on the viability of eosinophils of hypereosinophilic patients and healthy donors was determined . for this , 100 μl of cells ( 6 × 10 5 cells / ml ) were suspended in medium ( rpmi - 1640 containing 10 % fcs , 50 μm 2 - mercaptoethanol , 1 mm sodium pyruvate and gentamicin ( 50 μg / ml )) and cultured with rhsp - d ( 0 . 1 - 10 μg / ml ), il - 5 ( 25 ng / ml ) or the culture medium alone and incubated for 48 h . the cells were then evaluated for any changes in viability or apoptotic cell numbers by hypotonic propidium iodide staining . to study rhsp - d induced changes in the viability of in vitro primed cells , 100 μl of human eosinophils isolated from healthy donors ( 6 × 10 5 cells / ml ) were incubated with il - 5 ( 25 ng / ml ) for 1 h [ il - 5 primed eosinophils ] and cultured with rhsp - d ( 0 . 1 - 10 μg / ml ). the cells as such [ control ] or il - 5 alone ( il - 5 primed eosinophils ) [ il - 5 ] or with rhsp - d ( 10 μg / ml ) [ il - 5 + rhsp - d ] were incubated for 48 and 60 h in the culture medium . the evaluation of apoptosis was done by hypotonic propidium iodide staining of dna , cd95 expression ( apoptotic cell marker ) and giemsa staining for analysis of cell morphology ( 29 , 30 ). hypotonic propidium iodide ( pi ) solution was used to differentiate and identify the proportion of apoptotic eosinophils displaying a hypodiploid dna peak to that of viable cells using modification of previous protocols ( 29 ). the method is demonstrated to be as sensitive as performing dna agarose gel electrophoresis with the added advantage , that quantification of the degree of dna fragmentation can be readily performed ( 29 ). for this , the washed eosinophils were gently resuspended in 200 μg of hypotonic propidium iodide ( pi ) solution ( 50 μg / ml in 0 . 1 % sodium citrate plus 0 . 1 % triton x - 100 ). the red pi fluorescence of the eosinophil nuclei was analyzed on at least 2 , 000 cells from each sample . for demarcating the two populations of normal and apoptotic eosinophils in each experiment , il - 5 incubated eosinophils were included , as il - 5 is known to maintain eosinophil survival and shows distinct peak for normal eosinophils . the number of cells belonging to the m region divided by the total cell count was expressed as the percentage cell apoptosis (% cell apoptosis ). eosinophil expression of the apoptosis marker , cd95 or fas - receptor ( apo - 1 ) is demonstrated to increase as cells undergo apoptosis ( 30 ). the cell surface staining of cd95 on eosinophils was performed with the fitc labeled mouse anti - human cd95 igg1 mabs ( serotec ltd ., oxford , uk ). incubation with the mab was performed for 30 min at 4 ° c . the cells were then washed twice and analyzed by flow cytometry . the mouse igg1 was used as an isotype control for the study . apoptosis was confirmed by morphological analysis of cells spun onto cytospin slides , fixed with methanol and stained with may - grünwald - giemsa . the eosinophils were enumerated in randomly - selected fields and a minimum of 200 total cells were counted . the present invention is useful in providing the use of formulation with sp - d in resolution of eosinophilic inflammations by inducing and / or increasing apoptosis towards the beneficial effects in human subjects suffering eosinophil related diseases and disorders . these include neuromuscular and respiratory diseases with eosinophilia , hypereosinophilic leukemias , hypereosinophilc syndromes ( rare hematological diseases ), skin diseases like eosinophilia - myalgia syndrome , eosinophilic fascitis , capillary leak syndromes ( il - 2 ), churg - strauss syndrome , toxic oil syndrome , parasitosis , etc . the invention is illustrated by the following examples which are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention . this example describes the preparation of human eosinophils from the peripheral blood . mixed granulocytes were obtained by dextran sedimentation and density centrifugation through histopaque - 1077 from the human peripheral blood of hypereosinophilc patients and healthy donors ( anticoagulated with 0 . 4 % w / v trisodium citrate , ph 7 . 4 ) ( 25 ). eosinophils were subsequently isolated from the granulocyte pellet by negative immunomagnetic selection by magnetic cell sorting of human leukocytes ( macs ) kit using anti - human cd16 microbeads , variomacs magnetic cell separator and ld depletion columns ( miltenyi biotec , ca , usa ) ( 26 ). the cd16 − eosinophils were separated from cd16 + microbead tagged neutrophils by passing the cell suspension through the magnetic column that yielded & gt ; 95 % pure eosinophil population . the eosinophil purity was assessed by hinkleman &# 39 ; s staining and may - grunwald giemsa staining . the viability of purified eosinophils was determined by trypan blue dye exclusion and was consistently & gt ; 99 %. this example describes the preparation and purification of native human sp - d from the lung lavage obtained from alveolar proteinosis patients and preparation of a recombinant homotrimer containing eight gly - x - y collagen repeat sequences , an alpha - helical , coiled - coil neck region , and the carbohydrate recognition domain of rhsp - d , which is expressed under bacteriophage t7 promoter as inclusion bodies in escherichia coli ( 24 ). this example describes the isolation of rhsp - d from the inclusion bodies , which is further denatured in 6 m urea and slowly renatured overnight by dialyzing against buffer containing decreasing amounts of urea . the soluble rhsp - d is purified on a maltose - agarose column and contaminating lps removed using a polymyxin - b column . the identity of the protein is confirmed by n - terminal sequencing and is judged to be pure by sds - page and immunoblotting . estimating the binding of native and recombinant human sp - d to human eosinophils this example describes the binding of different concentrations of purified human rhsp - d ( 0 . 1 - 10 μg / ml ) in phosphate buffered saline with 0 . 1 % w / v sodium azide , 1 % w / v bsa , 5 mm cacl 2 and 1 mm mgcl 2 , incubated with eosinophils prepared from peripheral blood of hypereosinophilic patients and healthy donors at 5 × 10 5 cells / ml counts . prior to this the native and recombinant surfactant proteins are labeled with fitc as per standard methods . the eosinophils are incubated with fitc labeled surfactant proteins at various concentrations for 45 minutes at 4 ° c . the binding of rhsp - d to eosinophils derived from hypereosinophilic patients and healthy individuals was compared . the competitive experiment of native and recombinant surfactant protein was carried out . fitc - sp - d ( 10 μg / ml ) was allowed to bind eosinophils in presence of unlabelled rhsp - d ( 10 , 20 , 50 , 100 and 300 μg / ml ) in the binding buffer . flowcytometric analysis was performed on at least 10 , 000 cells using facscan ™ ( becton dickinson ) to check the binding of sp - d to human eosinophils . the specific mean fluorescence ( smf ) is determined by subtracting the non - specific mean fluorescence ( nmf ) of the cells from the observed mean fluorescence intensity ( mfi ). the relative smf ( rsmf ), which is the ratio of smf emitted by bound surfactant protein to that of the nmf of the corresponding control , was calculated . the results expressed either as rsmf , or % rsmf or as their mean fluorescence intensity ( mfi ), or % mfi . the binding of rhsp - d and nhsp - d to human eosinophils was dose dependent as shown in fig1 a . native sp - d ( 10 μg / ml ) showed a dose dependent binding to eosinophils that was inhibitable to 50 % by unlabelled rhsp - d at 224 . 5 μg / ml concentration ( fig1 c ). eosinophils from healthy donors showed a significantly higher binding to rhsp - d ( 10 μg / ml ) as compared to eosinophils derived from hypereosinophilic patients ( fig1 b ). to evaluate the effect of calcium ion concentration on rhsp - d binding to human eosinophils , binding assays in the presence or absence of 5 mm calcium chloride or in the presence of 10 mm edta were carried out . the binding was estimated by flowcytometry as defined in the above example . rhsp - d ( 10 μg / ml ) showed a significant increase in binding to eosinophils from hypereosinophilic patients and healthy donors in presence of 5 mm calcium and was inhibited in the presence of 10 mm edta ( fig2 ). the effect of rhsp - d on the viability of eosinophils of hypereosinophilic patients and healthy donors was determined . for this , 100 μl of cells ( 6 × 10 5 cells / ml ) were suspended in medium ( rpmi - 1640 containing 10 % fcs , 50 μm 2 - mercaptoethanol , 1 mm sodium pyruvate and gentamicin ( 50 μg / ml )) and cultured with rhsp - d ( 10 μg / ml ), il - 5 ( 25 ng / ml ) or the culture medium alone and incubated for 48 h . the cells were then evaluated for any changes in viability or apoptotic cell numbers by hypotonic propidium iodide staining . to study rhsp - d induced changes in the viability of in vitro primed cells , 100 μl of human eosinophils isolated from healthy donors ( 6 × 10 5 cells / ml ) were incubated with il - 5 ( 25 ng / ml ) for 1 h [ il - 5 primed eosinophils ] and cultured with rhsp - d ( 10 μg / ml ). the cells as such [ control ] or il - 5 alone ( il - 5 primed eosinophils ) [ il - 5 ] or with rhsp - d ( 10 μg / ml ) [ il - 5 + rhsp - d ] were incubated for 48 and 60 h in the culture medium . the evaluation of apoptosis was done by hypotonic propidium iodide staining of dna , cd95 expression ( apoptotic cell marker ) and giemsa staining for analysis of cell morphology . fig3 depicts representative histogram of rhsp - d incubated eosinophils of a hypereosinophilic patient showing a significant increase in % apoptotic cells in comparison to the control ( cells cultured in medium alone ) and il - 5 cultured cells , measured by hypotonic pi staining of the cells . eosinophils from hypereosinophilic patients showed an increase in the apoptosis in the presence of rhsp - d (% increase in apoptotic cell number relative to control , median , 27 . 5 ± 5 . 33 , n = 4 ) while , eosinophils from healthy donors showed a decrease in apoptosis in presence of rhsp - d (% decrease in apoptotic cell number relative to control , median , 13 . 2 ± 4 . 2 , n = 5 ) ( 48 h ) ( p = 0 . 015 , mann whitney test ), suggesting significantly different responses from the two eosinophil types . incubation of rhsp - d ( 10 μg / ml ) with il - 5 primed eosinophils from healthy donors resulted in a significant increase in apoptosis in these cells in comparison to the eosinophils incubated with il - 5 ( 25 ng / ml ) alone observed at 48 h ( data not shown ) and maximally at 60 h ( fig4 ). rhsp - d treated il - 5 primed eosinophils [ il - 5 + rhsp - d ] of the healthy donors showed higher apoptosis (% apoptotic cells , 70 . 57 ± 10 . 19 , n = 3 ) in comparison to il - 5 primed eosinophils in absence of rhsp - d [ il - 5 ] (% apoptotic cells , 53 . 2 ± 13 . 68 , n = 3 ) observed by hypotonic propidium iodide staining ( fig4 a ). rhsp - d treated il - 5 primed eosinophils [ il - 5 + rhsp - d ] ( 60 h ) also showed a significant increase in cd95 expression in comparison to eosinophils incubated with il - 5 alone [ il - 5 ] ( fig4 b ). fig4 c inset shows microscopic analysis of giemsa stained eosinophils . eosinophils with a typical bilobed nucleus were considered as cells with normal morphology and were counted as viable ( fig4 c inset i ). eosinophils with condensed nuclei were considered as cells with apoptotic morphology and were counted as apoptotic ( fig4 c inset ii ). the microscopic analysis of rhsp - d ( 10 μg / ml ) treated il - 5 primed eosinophils [ il - 5 + rhsp - d ] showed a significant decrease in cells with normal morphology in comparison to il - 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