Patent Abstract:
phosphorylated glucomannans may be purified from naturally occurring sources and used as a supplement to swine feeds for the benefit of swine production .

Detailed Description:
according to one embodiment , the phosphorylated glucomannan is provided as an additive to swine feed that may be used at all stages of swine development . the phosphorylated glucomannan may , for example , be added and mixed into the feed as a concentrated raw product , a concentrated raw product with a non - covalently attached protein , raw product absorbed into a matrix , and / or a concentrated raw product with a non - covalently attached protein absorbed into a matrix . the phosphorylated glucomannan may be in the form of a dry powder that is capable of being added to or mixed with swine feed . dosing is by ratio or concentration that may vary according to the stage of swine development to provide a benefit to the swine by promoting the health of the swine and replacing , reducing or eliminating the use of subtherapeutic doses of antibiotics in swine nursery , grower , finisher and maintenance feeds . i ) a dry powder comprised of the phosphorylated glucomannan polysaccharides containing a subunit , repeated approximately 30 times , of 1 - 6 and 1 - 2 linkages between and within mannose and glucose residues at a ratio of 12 : 1 mannose : glucose mixed into swine feed at a concentration , ratio , or dose that provides the general benefits of good health and weight gain to the swine consuming the mixed feed . ii ) a dry powder comprised of phosphorylated glucomannan polysaccharides containing a subunit , repeated approximately 30 times , of 1 - 6 and 1 - 2 linkages between and within mannose and glucose residues at a ratio of 12 : 1 mannose : glucose and a non - covalently linked protein mixed into swine feed at a concentration , ratio , or dose that provides the general benefits of good health and weight gain to the swine consuming the mixed feed . iii ) a dry powder comprised of the phosphorylated glucomannan polysaccharides containing a subunit , repeated approximately 30 times , of 1 - 6 and 1 - 2 linkages between and within mannose and glucose residues at a ratio of 12 : 1 mannose : glucose and absorbed into a matrix and mixed into swine feed at a concentration , ratio , or dose that provides the general benefits of good health and weight gain to the swine consuming the mixed feed . iv ) a dry powder comprised of phosphorylated glucomannan polysaccharides containing a subunit , repeated approximately 30 times , of 1 - 6 and 1 - 2 linkages between and within mannose and glucose residues at a ratio of 12 : 1 mannose : glucose and a non - covalently linked protein and absorbed into a matrix and mixed into swine feed at a concentration , ratio , or dose that provides the general benefits of good health and weight gain to the swine consuming the mixed feed . the university of georgia college of agricultural & amp ; environmental sciences cooperative extension service , in bulletin 854 / revised may , 1995 , identifies the following tables that may be used to formulate swine diets . these tables may be used to formulate ideal swine feeds for various stages of swine growth . local variances in the content of various feed sources may be accounted for by laboratory food analysis to confirm the general guidelines presented below . 3 when adding large amounts of dried whey ( 10 % or more ), the added salt should be reduced (. 25 %) due to the high salt content of some whey products . 4 addition of 57 - 114 mg / lb of copper from copper sulphate has been shown to improve performance of young pigs ( up to 40 lbs ). 5 the maximum level of selenium allowed by fda is subject to change . check current feed regulations . 1 the nutrient requirement of lactating sows increases as feed intake decreases . the nutrient content should be increased 8 . 3 % for each pound below 12 . ( if sows will only consume 10 lb / day , the levels of required nutrients should be increased 16 . 6 %.) 2 the maximum level of selenium allowed by fda is subject to change . check current feed regulations . these feeds may be supplemented with minor amounts of a phosphorylated glucomannan , for example , as isolated from candida utilis , to achieve the instrumentalities described herein . other feed formulations may be provided by publicly available software , such as the user - friendly feed formulation program (“ uffda ”) based upon the book animal feed formulation — economics and computer applications , by g . m . pesti and b . r . miller , chapman and hall . the phosphorylated glucomannan mixed with this food to provide a dosage ranging from 1 to 5 mg of the phosphorylated glucomannan per kg of body weight in the swine . the preferred dosage is 3 mg per kg of body weight although higher doses may be used , such as doses of 20 mg / kg , the range from 1 mg to 5 mg per kg are generally minimal doses to achieve the desired effects . obtention of candida utlis polysaccharide with soy protein adsorbed on calcium phosphate the following laboratory - scale example teaches by way of example how to purify a phosphorylated glucomannan polysaccharide . the polysaccharide is characterized by a subunit that is repeated approximately 30 to 40 times , where the subunit contains 1 - 6 and 1 - 2 linkages between and within mannose and glucose residues at a ratio of 12 : 1 mannose : glucose , where also the phosphorylated glucomannan polysaccharide predominantly exists in the homo - trimeric form of an alpha helix . the polysaccharide may be obtained , for example , using the process described in ep1163911 , which is incorporated by reference , and describes the alternative use of soy or castor beans which are optionally omitted . the method of isolating phosphorylated glucomannan polysaccharides commences , for example , by soaking soybeans in water to provide soaked soybeans . these are ground to provide ground material and combined with candida utilis , water , and a first salt to provide an incubation mixture . the incubation mixture is incubated with stirring or agitation for extraction of the polysaccharide to provide a supernatant fluid . the supernatant is concentrated by filtration with a cutoff of about 20 kda . a second salt is added together with a low molecular weight ketone to form a precipitate . the precipitate is dried to yield an isolated polysaccharide product . in various aspects , the drying step is preferably performed at a temperature not more than 55 ° c . to avoid product degradation . the first salt is preferably a magnesium salt , such as mnso 4 . h 2 o . the incubation mixture may be provided with an amount of camphor that is miscible with the aqueous phase , and with heating to a temperature of from 30 ° c . to 40 ° c . concentration may be staged , for example , using an initial stage of filtering to remove cellular debris , ultrafiltration to the 20 kda cutoff to produce a concentrate of at least 1 / 10 the initial volume of the supernatant , and diafiltration of the concentrate against water in amount at least ten times the volume of the concentrate . the second salt is preferably a calcium salt , such as calcium chloride , where also the low molecular weight ketone is preferably acetone . the precipitate may be combined with an adsorption salt to stabilize the final product . suitable adsorption salts include , for example , calcium phosphate ( cahpo 4 . 2h 2 o ) and / or dihydrate calcium sulphate ( caso 4 . 2h 2 o ). the resulting isolated polysaccharide may be formulated by mixing with an animal feed carrier in a dosage formulation that is effective to reduce growth of non - beneficial microorganisms in the digestive tract of a predetermined animal . in one embodiment , starting materials include commercial pasteurized and spray - dried standard food grade candida utilis that is subjected to the preferred process described below : 1 . 1 weigh approximately 100 g of soy bean seeds . soak them for 24 hrs in water . 1 . 2 wash the seeds several times with water . 1 . 3 grind the seeds in a mortar or a mincer . 1 . 4 prepare an aqueous solution of 2 l containing 6 . 25 g / l of mnso 4 . h 2 o at a temperature of 37 ° c . add , stirring in a magnetic stirrer , 0 . 21 g / l of mno 2 , 0 . 6 g / l camphor , 62 . 5 g / l of desiccated c . utilis and 12 . 5 g / l of the seed milling . 1 . 5 incubate in orbital stirrer at 37 ° c . and 200 rpm 2 to 5 hours , until the concentration of the polysaccharide is between 3 to 4 g / l . 1 . 6 cool to a temperature less than 25 ° c ., allow to stand , separate the supernatant and filter through a hyflo ®/ standar super cell ® with a filter candle . 1 . 7 concentrate the filtrated supernatant by ultrafiltration with a cut off of 20 kda to a 1 / 10 of the original volume . 1 . 8 diafiltrate the concentrate against at least 10 times of its volume of water . 1 . 9 add , under stirring , calcium chloride to the concentrate / diafiltrate to a end concentration of 60 mm . let , under stirring , 30 minutes . 1 . 10 add , under stirring , calcium phosphate to a end concentration similar to three times the polysaccharide concentration . let , under stirring , 15 minutes . 1 . 11 add , under stirring , acetone to an end concentration of 40 % ( v / v ). 1 . 12 filter through nylon and separate the precipitate . 1 . 13 dry the precipitate in a vacuum oven at temperature not higher than 55 ° c . the above process is scalable to industrial level and implies an improvement respect to the prior art in the following points : a ) cobalt chloride is advantageously not needed . b ) filtration replaces centrifugation where filtration is a less expensive and more scalable process . c ) the former lyophilization may be replaced by adsorption on a salt , such as calcium phosphate , with precipitation . this renders a more stable product , due to the stabilizing action of the calcium phosphate . swine feed studies may be performed on a contract basis , for example , between a requesting agency and a testing agency . in one example , a study may be commissioned using two different test articles , namely : ( 1 ) glucomannan and ( 2 ) glucomannan plus a non - covalently linked protein . the test articles may be mixed in swine starter and grower feeds at varying concentrations for example : 1 mg / kg , 3 mg / kg and 20 mg / kg . a study of this type would show that swine fed either type of test article would perform better than the negative control having no antibiotic in feed , and as well as or better than positive control with antibiotic in the feed . possible parameters used for comparison of the test articles to negative and positive controls may include , for example , total weight gain , weekly weight gain , feed conversion , mortality , carcass weight , bacterial flora , blood chemistry , and peripheral blood cell populations . the two prebiotics including candida utilis phosphoglucomannan and candida utilis phosphoglucomannan - soybean proteins would be mixed prior to study initiation with a carrier , such as ( cahpo 4 . 2h 2 o ) and / or dihydrate calcium sulphate ( caso 4 . 2h 2 o ). the negative control would be considered to have 0 mg test article / kg diet . as an example , the test articles would be titrated into the negative control feed at levels to approximate 1 , 3 , and 20 mg of active test article / kg body weight . bmd 60 1 would be added to the negative control diet at one pound per ton diet and this would be considered the positive control ration . thus , there would be 8 treatment groups with 26 swine / group . each treatment group would be divided into 2 pens of 13 swine , designated replicate a and replicate b . the eight rations would be fed ad libitum to 2 pens of 13 swine each for the duration of the study . body weight , feed consumption and feed efficiency would be measured weekly and feed efficiency corrected for any mortality . 1 bmd 60 contains bacitracin at 60 mg / lbs . bmd 60 produced by carl s . akey , inc . po box 5002 , lewisburg , ohio 45338 . the study described below may be replicated in relevant time intervals for swine in any one of nursery , feeder or maintenance stages , or a combination of these stages . the study below is commissioned for the feeder stage . blood will be collected weekly from 3 predetermined swine from replicate a of each treatment group and submitted for cbc / chemistries . additionally , six ( 6 ) swine per treatment group ( 3 per pen ) will be sacrificed on day 90 for cbc & amp ; chemistries . at the conclusion of the study , gut samples will be taken from three of swine from each treatment group ( replicate b ) and sent off to determine levels of salmonella spp . and campylobacter spp . present . it is common practice in the swine industry to include antibiotics and other growth promoters in the feed of the swine ; this is the most cost efficient method . the duration of the study is designed to mimic a standard growth phase commonly found in the feeder swine industry . these products are designed to replace antibiotics at sub therapeutic levels in feeder swine . the number of animals in the protocol is considered to be the minimum necessary to evaluate the effects of the test articles in comparison to sub therapeutic doses of antibiotics in feeder swine . the current dose levels for the two test articles will be 0 , ˜ 1 mg active test article / kg body weight , ˜ 3 mg active test article / kg body weight , and ˜ 20 mg active test article / kg body weight . these doses are considered to be safe doses for the two prebiotics including the aforementioned candida utilis phosphoglucomannan and candida utilis phosphoglucomannan - soy bean proteins . test article 1 ( candida utilis phosphoglucomannan adsorbed in calcium phosphate ). candida utilis phosphoglucomannan 10 - 13 % ( w / w ), dihydrated calcium phosphate 87 - 90 % ( w / w ). this compound may be prepared , for example , by industrial farmaceutica cantabria and provided to a test agency prior to study initiation . test article 2 ( candida utilis phosphoglucomannan - soy bean proteins adsorbed in calcium phosphate — calcium sulphate ). candida utilis phosphoglucomannan — soy bean proteins 5 - 10 % ( w / w ), dihydrated calcium phosphate dihydrated calcium sulphate 90 - 95 % ( w / w ). this compound may be prepared by industrial farmaceutica cantabria and provided to the test location prior to study initiation . unless otherwise noted , the identity , strength , purity , composition , stability and method of synthesis , fabrication and / or derivation of each batch of the test and control articles is documented by the test agency before its use in the study . this documentation is maintained by the test agency . an archival sample from each lot of test article is taken and stored in the archives of the test agency , pending shipment to the requesting agency . given the desired dose ( approximate ) levels of the test articles of 1 , 3 , and 20 mg active test article / kg body weight , the average ratio of grams feed intake / day / kg body weight is taken from the nrc ( 1994 ) for the swine of age 1 to 3 weeks and 3 to 9 weeks . this value is used to determine the mg total product / kg feed to mix for each treatment group and time period . the following table 6 outlines the values that may be used for each treatment : table 6 shows the dosing levels for each test article . table 6 lists the amount of test article to add based on the required dose level of the active article consumed per kilogram of feed consumed . the swine are fed suitable amounts of food for their age and size to meet the study dosing requirements . due to the nature of the test articles there is currently no accurate methodology to quantitate the amount of test article or its activity in the test diets other than an empirical study , for example , as described herein . prior to the receipt of the swine the facility is cleaned and sanitized removing all organic matter . each pen is set up so as to isolate it from all other pens ; this is done in order to prevent possible cross contamination among pens . each pen is uniformly provided with suitable equipment for the raising of swine . swine are housed in an environmentally controlled room at the test agency for the duration of the study . during the acclimation phase ( days − 6 to − 1 ) all swine are fed ad libitum the negative control diet , containing no test article or antibiotic . each pen is initially fed a suitable amount of the designated ration . the feed intake is observed daily and feed is weighed and added as necessary in order to insure the swine are maintained on ad libitum feeding . at approximately day − 6 of the experimental phase all swine are ear tagged with a unique number identification in the right ear . at day 0 of the experimental phase all swine are individually weighed . swine selection and randomization procedures is conducted by test agency personnel ( other than the investigator or co - investigator ) using microsoft ® excel 2002 ( 10 . 4524 . 4219 ) sp - 2 . random numbers are generated using the “ rand ” function of excel and are captured using the “ copy / paste special / values ” commands . the “ rank ” function in excel is used to assign swine to groups within blocks by random number . in addition , single factor anova data analysis ( α = 0 . 05 ) in excel is used to assess the outcomes of randomizations for homogeneity of variance ( f statistic & lt ; f critical value ) between groups . anova is conducted for body weight between pens . each pen has an additional 3 swine ( n total swine = 13 per pen ) included at day 0 to provide 3 swine per pen ( 6 per treatment ) on day 3 for sacrifice and blood collection . selection of the 3 swine for the day 3 sacrifice is by a random number assignment . all thirteen swine in each pen receive a random number generated in excel . the three swine with the highest random numbers within each pen are selected for the day 3 collection . ten ( 10 ) swine remain in each pen to complete the study . three ( 3 ) swine from the replicate a pen of each treatment group are selected for blood draw each week of the experimental phase . within replicate a , swine are selected for blood collection based on their random numbers . the swine with the lowest 3 random numbers are drawn on weeks 1 , 4 , and 7 , the next 3 lowest on weeks 2 , and 5 , and the next 3 lowest on weeks 3 and 6 . the tenth swine within the replicate is considered an extra swine . unused test article mixtures and containers are returned to the requesting agency . collection equipment used in the study are autoclave and disposed of in the biohazard / sharps solid waste stream at the test agency . any commercial breeder capable of supplying a group of animals having a uniform breeding standard . at this stage , the feeder swine begin acclimation to study conditions at about 5 to 7 days prior to the initiation of the trial . during acclimation , all swine are checked for viability twice daily . prior to assignment to study , all swine are examined to ascertain suitability for study by a staff veterinarian . at approximately day − 7 of the study clinical observations are made by a staff veterinarian for each swine . any swine that is found abnormal is rejected from the study . at approximately day − 6 of the study all swine are individually ear tagged with a unique numerical identification in the right ear . at approximately day 30 the swine are given an additional ear tag in the left ear , this is the same number as was placed in the right ear at day approximately − 6 . currently acceptable practices of good animal husbandry are followed , e . g ., as shown in the guide for the care and use of laboratory animals ; national academy press , 1996 . the test agency , for example , may be fully accredited to perform contract studies by the association for assessment and accreditation of laboratory animal care international ( aaalac ). suitable standards are imposed , for example , to provide space requirements for the growth of swine at the feeder stage . swine are monitored by the technical staff for any conditions requiring possible veterinary care . if any such conditions are identified , a staff veterinarian is notified for an examination and evaluation . during the study the swine are uniformly provided with ambient lighting that is know to be suitable for the production of swine . during the study the swine are uniformly provided with ambient temperature that is known to be suitable for the production of swine . humidity is monitored in accordance with standard procedure at the test agency , but is not controlled . the swine are housed in groups of 10 in individual floor pens in an environmentally controlled room for the duration of the study . swine are allowed ad libitum feeding . from days − 6 to 0 all swine are given the negative control diet ( containing no test articles or antibiotics ). from day 0 forward each pen is given its respective diet ad libitum . clean , fresh water from an on - site deep well is available ad libitum during the study . an approximate mixture of 50 / 50 ( v / v ) of fresh straw and straw that has been previously used for feeder swine bedding is used in this trial . the purpose of the litter contamination is to increase the pathogen burden in the test swine to better reflect the normal farm husbandry condition . it is also desirable to have a pressure of infection to determine the efficacy of the test article . nutritional certification of batches of feed provided by the manufacturer ( via manufacturer &# 39 ; s bag label ) is included in the raw data . there are no known contaminants in the food which are expected to interfere with the objectives of this study . a copy the test agency &# 39 ; s most recent water analysis is included with the raw data . there are no known contaminants which are expected to interfere with the objectives of this study . each swine is weighed once a weekly , this information is recorded in the study records . feed is weighed out prior to feeding . all feed added to a pen is weighed and recorded in the study records . once weekly the feeders is weighed and weights recorded in order to determine feed disappearance . once daily the minimum , maximum and current temperature and humidity are recorded in the study records . mortality is recorded daily for each pen in the study book . the body weight is recorded for each mortality and recorded in the study book . the feeder swine are euthanized by an intravenous overdose of sodium pentobarbital ( 390 mg / ml )/ sodium phenylion ( 50 mg / ml ) at 0 . 22 ml / kg , followed by cervical dislocation ( e . g ., as src sop pr . 04 . 01 ). blood is collected for determination of cbc with differential and chemistries on study days 3 , 7 , and weekly thereafter . the samples are collected by test agency personnel and sent to a suitable analytical company , such as antech diagnostics for analysis . for the day 3 draw the swine are sacrificed and blood is collected via a direct heart draw . from days 7 on the blood is collected from the brachial artery . for the cbc approximately 1 ml of whole blood is drawn using a drop for the blood smear and the rest drawn into an edta microtainer for storage and reuse . the differential for the cbc is automated . the analytical chemistry requires approximately 0 . 50 ml serum from each swine . on day 90 of the study 3 swine from each pen are sacrificed for gut collection . a sample of the small intestine is collected from each swine , from the ileum - cecal junction to the meckel &# 39 ; s diverticulum . sub samples from this portion of the small intestine is taken and sent to antech diagnostics laboratories to determine salmonella spp . and campylobacter spp . counts . this data is recorded in the study records . on day 90 of the study all swine are sacrificed and the carcass is weighed . this data is recorded in the study records on day 90 of the study all swine are sacrificed and a staff veterinarian performs a gross necropsy and gross pathology . these results are included in the study report . all data documenting experimental details and study procedures and observations are recorded and maintained as raw data . at the completion of the study , all reports and study specific original raw data , and copies of certain study related facility data are reported . an exact copy of the report and raw data is maintained in the test agency &# 39 ; s archives for a period of at least 1 year after submission of the signed final report . all plasma samples are shipped to the test requestor . the test requestor is responsible for retaining samples of the test article . anova statistical analysis is performed on study data including body weight gain , feed consumption , feed efficiency corrected for mortality , and carcass meat yield . alpha is set at 0 . 05 . those skilled in the art will appreciate that the foregoing description teaches by way of example , and not by limitation . accordingly , what is shown and described should be construed in a manner that is consistent with the scope and spirit of the invention . the following documents are incorporated by reference to the same extent as though fully replicated herein : 1 choct m c ( 2001 ) alternatives to in - feed antibiotics in monogastric animal industry . asa technical bulletin vol . an30 - 2001 p . 1 - 6 2 mathew a ( 2002 ) seeking alternatives to growth promoting antibiotics . depart . of animal science , the uni . of tennessee , knoxville tenn ., usa 3 turner j l , pas , dritz s s , and minton j e (?) review : alternatives to conventional antimicrobials in swine diets . the professional animal scientist 17 . p . 217 - 226 4 mitchener b ( 1999 ) eu moves toward a total ban of antibiotics in animal feed . wall street journal , jul . 28 , 1999 5 newman k ( 1994 ). mannan - oligosaccharides : natural polymers with significant impact on the gastrointestinal microflora and the immune system . in : lyons t p and j , k a ( ed .) biotechnology in the feed industry . nottingham university press , nicholasville , ky ., p . 167 - 180 6 waldroup p w , oviedo - rondon e o , fritss ca ( 2003 ) comparison of bio - mos and antibiotic feeding programs in broiler diets containing copper sulfate . international journal of poultry science 2 p 28 - 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( 2002 ) immunoferon , a glycoconjugate of natural origin , regulates the liver response to inflammation and inhibits tnf - α production by an hpa axis - dependent mechanism . internation immunopathology vol . 275 p 24 brieva a , guerrero a , alonso - lebrero j l , pivel j p ( 2001 ). immunoferon , a glycoconjugate of natural origin , inhibits lps - induced tnf - α production and inflammatory responses . international immunopharmacology vol 1 p 1979 - 1987 25 johnson r w , ( 1997 ). inhibition of growth by pro - inflammatory cytokines : an intergrated view . j anim sci vol 75 p 1244 - 1255 26 podzorski r p , gray g r , and nelson r d ( 1990 ) different effects of native candida albicans mannan and mannan - derived oligosaccharides on antigen - stimulated lymphoproliferation in vitro . the journal of immunology vol . 144 p 707 - 716 .