Patent Abstract:
the present invention is , among other things , concerned with suppressing neutrophil migration and the prevention of damage from neutrophil migration . a proteinaceous substance derivable from cryptococcus neoformans and capable of interfering with neutrophil migration in a mammalian host . also , functional equivalents of the proteinaceous substance and the use of this substance and / or equivalent in the preparation of a medicament or treatment of disese .

Detailed Description:
in a particular preferred embodiment the invention provides the use of mannoproteins , more preferable mannoprotein - 4 or a functional equivalent thereof in the preparation of a medicament for the treatment of a pathological condition or a disease involving neutrophil migration and in this sense as an anti - inflammatory agent . in inflammation , soluble factors called chemokines have been identified which attract various subtypes of leukocytes . the chemokine il8 is a potent chemoattractant for neutrophils ( 24 ). as expected , patients with bacterial meningitis have high levels of il8 as well as an increased number of neutrophils in their csf . we recently described that patients who suffer from a fungal meningitis caused by cryptococcus neoformans whose csf typically contains very little leukocytes , had high levels of il8 in their csf ( 25 ). therefore , in cryptococcal meningitis , an agent must be present that interferes with neutrophil migration toward il8 . cryptococcal polysaccharides have been shown to interfere with neutrophil movement ( 26 - 29 ). we and others in literature initially focused on the fungal capsular polysaccharide glucuronoxylomannan ( gxm ) as the cryptococcal agent responsible for inhibition of neutrophil migration . patients with cryptococcal infection have high titers of gxm in both serum and csf during infection ( 30 , 31 ). we demonstrated in vitro that gxm also interferes with neutrophil migration ( chemotaxis ) toward il - 8 if gxm is added directly to the leukocytes ( 25 ). we also demonstrated ( 32 ) that a low csf leukocyte cell count in patients with cryptococcal meningitis shows a significant inverse correlation to a high gxm titer in serum ( relative to the gxm titer in csf ). in a rabbit model for pneumococcal meningitis we have recently shown that intravenous administration of gxm not only delays entry of leukocytes ( polymorphonuclear cells , pmn ) into the csf and brain , but more importantly reduces tnfα levels in the csf and protects the brains of these rabbits against tissue damage in comparison to control animals ( 33 ). the exact mechanism how gxm interferes with neutrophil migration in cryptococcosis is not known . interestingly , in the second part of 1999 , we discovered that another class of cryptococcal proteins ( 34 ), the mannoproteins ( mp ) are even more powerful than gxm in reducing neutrophil migration , more in particular mannoprotein - 4 ( mp - 4 ). we compared gxm and three different mp &# 39 ; s ( mp - 1 , mp - 2 and mp - 4 ) for interference with different steps involved in transendothelial migration of pmn . 1 ., with the following results : 1 .] gxm ( 35 ) and mp , more in particular mp - 4 , inhibit migration of pmn towards il8 , fmlp , c5a and paf . 2 .] gxm ( 36 ) and mp , more in particular mp - 4 , were shown to cause shedding of l - selectin ( cd62l ; the specific targeting of neutrophils to venules in inflamed tissue involves cd62l binding to e - and p - selectin on endothelial cells ). 3 .] gxm and mp , more in particular mp - 4 , caused up - regulation of cd11b and cd18 ( the leukocyte function associated antigen - 1 , lfa - 1 ). 4 .] gxm and mp , more in particular mp - 4 , have intrinsic chemoattractive capacity . 5 .] gxm and mp , more in particular mp - 4 , cause ca - fluxes in leukocytes . 6 .] mp , more in particular mp - 4 , causes down - regulation of tnfa receptors p55 and p75 on leukocytes . 7 .] gxm and mp , more in particular mp - 4 , inhibit adhesion of neutrophils ( pmn ) to tnf stimulated endothelium . 8 .] gxm and mp , more in particular mp - 4 , inhibit upregulation of adhesion molecules e - selectin ( cd62e ), vcam , icam - 1 on tnf stimulated endothelium . 9 .] gxm and mp , more in particular mp - 4 , inhibit production of chemokines mcp - 1 , il - 8 , mip - 1a and rantes by tnf - stimulated endothelium . in all assays mp turned out to be much more active than gxm ; this holds true especially for mp - 4 which could be used at 20 - to 100 - fold lower concentrations than gxm while still causing higher absolute stimulation levels ( cd11b / 18 upregulation , chemotaxis , ca - flux ) or repression levels ( inhibition of chemotaxis levels , cd62l shedding ). furthermore , we know that mp can be easily purified ( 37 ) and is not toxic ; patients with cryptococcal meningitis can display relatively mild symptoms for a prolonged period of time despite the presence of mp , more in particular mp - 4 , in blood and csf . therefore mp is a potent therapeutic agent . off course functional equivalents of mp will be capable of interfering with neutrophil migration . since the protein and carbohydrate compositions of mp1 , mp2 and mp4 ( the most potent mannoprotein ) closely resemble each other , they are placed here together as “ mp ”. anti - inflammatory activity was detected in mp preparations , more in particular the mp - 4 preparation . thus mp , more in particular mp - 4 , and functional equivalents thereof can be applied in the treatment of diseases or pathological conditions involving neutrophil migration . typically such diseases include infections and brain neurotrauma where neutrophils invade the tissue , diseases with damage due to reperfusion ischemia where neutrophils are attracted and cause secondary damage , intensive care patients with ards and other inflammatory diseases ( rheumatoid arthritis and inflammatory bowel disease ). mp , more in particular mp - 4 , will be especially useful in diseases involving il8 regulation . examples thereof are brain inflammatory disease such as meningitis — in particular bacterial meningitis —, neurotrauma ( brain , spinal cord ), lung disease ( ards and copd ), cardiovascular disease ( arteriosclerosis ), allergic skin reactions ( contact dermatitis , psoriasis ), auto - immune diseases or the protection of the brain against toxic medication administered systemically ( e . g . anti - cancer drugs ). other important applications will include the treatment of pathological conditions such as brain injury or ischemic events ( ischemia — reperfusion after cardiopulmonary bypass surgery or other forms of cardiac surgery ). in a further embodiment the invention provides a pharmaceutical composition comprising mp or functional equivalents thereof together with a suitable means for administration . typically such a composition will be given systematically . the dose of mp necessary to prevent neutrophil migration may vary , but will generally be between 250 mg / kg and 10 mg / kg , preferably between 1 mg / kg and 5 mg / kg . the composition may of course contain other drugs for the treatment of diseases involving neutrophil migration , such as anti - inflammatory drugs , corticosteroids or an antibiotic agent . the invention will be explained in more detail in the following experimental part . mannoproteins were purified from the supernatant of an a capsular mutant of c . neoformans , cap 67 ( e . s . jacobson , medical college of virginia ) to avoid contamination with gxm . cryptococcal cultures were grown for 5 days in rpmi supplemented with gentamycin to prevent bacterial contamination . supernatants were harvested by centrifugation and concentrated by ultrafiltration using a 3 . 5 kda cut - off filter . next , the retentate was applied to a concanavalin a column equilibrated in buffer a containing 10 mm tris ph 7 . 2 , 500 mm nacl , 1 mm mgcl 2 and 1 mm cacl 2 . the column was washed with buffer a and stepwise eluted with a - methyl - d - mannose pyranoside in pbs . mp - 1 and - 2 eluted at 200 mm amdm , mp - 4 eluted at 400 mm . mp - 1 and mp - 2 where further fractionated by anion exchange ( deae ) chromatography . purified mp is finally recovered by lyophilization . samples are analyzed by various methods to prove that none of the purified mannoproteins contains constituents other than those known to occur in mp ( 37 ): see table i . based on the purification schedule used to purify mp ( as described above ) we were able to draw the conclusion that mp , more in particular mp - 4 purified by our methods was never present in the previously used mp preparations ( 29 , 38 ), for the following reason . mp - 4 purified by our method reveals a polydispersed signal migrating between 20 and 28 kda on sds - page gels ( 37 ), whilst the culture filtrates ( 29 ) were concentrated using a 30 kda cut - off cassette . in all cases , confluent monolayers of human umbilical vein endothelial cells ( huvec ) were stimulated for 6 hours with 100 units / ml of tnfα after which the various assays were performed in the presence of polymyxin b . i . gxm / mp - 4 inhibit adhesion of neutrophils ( pmn ) to tnf stimulated endothelium in order to assess the effect of gxm / mp - 4 on pmn adhesion , we compared adhesion of fluorescence labeled neutrophils — either gxm / mp - 4 pretreated ( 1 hr at 37 ° c . ; ng - mg / ml range ) or untreated — to tnf - stimulated huvec in a static adhesion assay . after adhesion with pmn for 15 min . at 37 ° c . the monolayers were washed and the remaining adhesive pmn were fixed and counted in quadriple wells by means of fluorescence microscopy . ii gxm / mp4 inhibit upregulation of adhesion molecules e - selectin ( cd62e ), vcam , icam - 1 on tnf stimulated endothelium in order to assess the effect of gxm / mp - 4 on endothelial cells , we compared the expression of adhesion molecules on the surface of tnf - stimulated huvec cells — either gxm / mp - 4 pretreated ( 6 - 24 hrs at 37 ° c . ; ng - mg / ml range ) or untreated — by measuring the levels of rna encoding these molecules . for this , rna isolated using the trizol isolation method as described by the manufacturer was used as a template to produce cdna . specific cdna encoding adhesion molecules was amplified by the polymerase chain reaction ( pcr ) technique using gene - specific pcr primers . iii gxm / mp - 4 inhibit production of chemokines mcp - 1 , il - 8 , mip - 1α and rantes by tnf - stimulated endothelium in order to assess the effect of gxm / mp - 4 on endothelial cells , we compared the production of chemokines by tnf - stimulated huvec cells — either gxm / mp - 4 pretreated ( 12 - 48 hrs at 37 ° c . ; 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