in order to determine the immunoactivity of the lysozyme dimer , an assay in human peripheral blood lymphocytes with facs analysis has been employed . mitogenic stimulation in human peripheral lymphocytes is a well established method to test reactivity of the most important cells of the immune system . to test influences of therapeutical substances on the activation and proliferation of lymphocytes from healthy blood donors , the mitogen is added in a suboptimal dose and the lymphocyte response is finally measured quantitatively with immunorelevant parameters . the results are compared with the control value measured without medication . to stimulate lymphocytes , cona in a concentration of 20 μg / ml of medium was used . initial cell concentration was 10 6 cells / ml . the short - time cultures in a co 2 incubator was carried out during one ( il - 2 receptors on lymphocytes and hla - dr ) or two days ( all other tests ). the following parameters have been measured as criteria for cellular activation : the following observations regarding cell products were made in the culture supernatant : neopterin -- produced by t - lymphocytes during the immune response and in stimulated cultures -- in the present experiments was not markedly elevated by the lysozyme dimer above the control value in cona stimulated cells without the tested dimer . with increased concentration of the tested dimer , the neopterin values were slightly higher . similarly , β - 2 - microglobulin values in all concentrations of the lysozyme dimers fluctuated around the control value . il - 2 receptors -- being shed from the lymphocyte surface during culture and giving information about total il - 2 receptor turnover -- were on a comparable level with the il - 2 receptors on the lymphocyte surface ( reported below ) and showed a clear suppression at the highest concentration of the tested dimer . interleukin - 6 showed a clear tendency to dose - dependent higher values in higher concentrations . this molecule is very important in hematopoiesis , cell differentiation and immune reaction . in the test , the first three values had to be extrapolated , since the highest standard was only 2000 pg / ml . the results relating to the remaining molecules are shown in table 1 below . unlike the interleukin - 6 , the tnf - α was unexpectedly table 1______________________________________influence of lysozyme dimer on the human peripheral blood lymphocytes______________________________________ hla - dr / no sample cd3 il - 2 rec ki - 67 / cd8 ki - 67 / cd4______________________________________control 3 . 3 8 . 9 6 . 0 2 . 71 . 1 mg / ml 2 . 2 2 . 6 4 . 8 5 . 3 2 . 0 . 3 mg / ml 1 . 9 6 . 3 4 . 1 3 . 0 3 . 0 . 1 mg / ml 3 . 0 6 . 1 6 . 5 ( 6 . 9 ) 4 . 33 μg / ml 3 . 0 5 . 9 6 . 3 3 . 0 5 . 10 μg / ml 3 . 4 6 . 0 7 . 7 3 . 7 6 . 3 . 3 μg / ml 2 . 4 5 . 0 7 . 5 3 . 7 7 . 1 μg / ml 2 . 5 4 . 9 7 . 0 4 . 1 8 . 0 . 3 μg / ml 3 . 3 5 . 8 7 . 5 3 . 9 9 . 0 . 1 μg / ml 2 . 8 4 . 0 7 . 9 3 . 8 10 . 33 ng / ml 3 . 1 4 . 6 8 . 0 4 . 1______________________________________no sample tnf thym .- kinase ifn - α______________________________________ control 205 . 11 9242 4 . 971 . 1 mg / ml 38 . 07 923 8 . 81 2 . 0 . 3 mg / ml 17 . 19 10914 9 . 44 3 . 0 . 1 mg / ml 13 . 75 7254 17 . 96 4 . 33 μg / ml 36 . 11 9525 3 . 67 5 . 10 μg / ml 22 . 22 5492 7 . 54 6 . 3 . 3 μg / ml 54 . 91 5198 6 . 26 7 . 1 μg / ml 18 . 47 5840 33 . 91 8 . 0 . 3 μg / ml 14 . 47 6839 10 . 07 9 . 0 . 1 μg / ml 94 . 16 3672 4 . 97 10 . 33 ng / ml 172 . 46 7312 10 . 07______________________________________ present in culture supernatants in dramatically reduced concentrations , except the last two dilution steps , which proved to be ineffective in suppression of tnf concentration . thymidine kinase is measured in the lymphocyte cytoplasma after freezing and thawing of the cell pellet . the thymidine kinase is upregulated in dividing cells making it a good marker for cell proliferation . table 1 data show a depression by the highest tested lysozyme dimer concentrations ; in the other dilution steps , no clear tendency can be seen . interferon - α , in turn , under the same conditions shows values above the control value of cona alone at higher concentrations . a visible increase occurs from the second value to the third dilution . hla - dr / cd3 , being a histocompatibility marker , is expressed on activated t - lymphocytes during the immune reaction . the results obtained show a certain percentage of activated t - cells in the control culture ; with various concentrations of lysozyme dimer in the cultures , the values vary around the control value . il - 2 receptors on lymphocytes : interleukin - 2 is a cytokine produced by t - helper lymphocytes after activation through il - 1 . il - 2 is autocrine and paracrine . t - helper lymphocytes not only produce il - 2 but are also stimulated to proliferation by this molecule . the receptors for il - 2 at the surface of t - helper lymphocytes are upregulated upon activation . table 1 shows that at highest concentration of lysozyme dimer there is a marked suppression of the il - 2 receptors on lymphocytes , while the remaining values do not differ more than within the biological bandwidth . table 1 also contains data related to ki - 67 / cd8 and ki - 67 / cd4 . ki - 67 is a proliferation molecule appearing in cells undergoing mitosis . ki - 67 is an important parameter to assess stimulated cells and in tumor diagnosis . in the reported results , a slight inhibition of cell proliferation is seen in ki - 67 suppressor ( cd8 ) cells with the highest two doses of lysozyme dimer . in helper ( cd4 ) lymphocytes with the highest dose , there appears to be substantial increase in the percentage of positive cells . with lower doses , the percentage of cells expressing ki - 67 / cd4 is slightly higher than the control value . the immunological parameters listed above and chosen by their potential importance in the immune response were analyzed with the method based on measuring the influence of a tested substance on suboptimally concanavalin a ( cona ) stimulated human peripheral lymphocytes . the method being well established , sensitive and allowing to evaluate many different parameters . at some 20 concentrations of lysozyme dimers there are marked differences of the test results as compared to the values of lymphocytes stimulated with cona alone , while as regards for example tnf and ifn - a , the observed effects are generally within the range of all tested dilutions . laboratory tests have been performed to determine the effect of lysozyme dimer on the phagocytic activity of milk and blood cells in vitro . it was earlier determined that in the standard in - vitro test , lysozyme dimer does not inhibit proliferation of the microorganisms isolated from the infected mammary glands of cows . as the clinical use of intraudderly and simultaneously intravenously administered lysozyme dimer effectively eliminates the infection of the mammary glands in cows , it was clear that the main antibacterial mechanism in mammary glands of cows is phagocytosis . accordingly , blood and milk of both healthy and infected cows were used in in - vitro tests aiming to determine the effect of lysozyme dimer on phagocytosis . for comparison , the experiments were conducted with the same concentration of substance tested and the same incubation time using the cells isolated from healthy and infected cows or even from an infected and a healthy section of the mammary gland of the same cow , in order to eliminate individual response differences . in the tests performed , both the purified dimeric form and a mixture of dimer and small fractions of trimers and higher oligomers of lysozyme were added to the blood or milk of the healthy and infected cows in concentrations of 25 -- 0 . 25 pg / ml and the mixture was incubated at 37 ° c . for 0 . 5 - 24 hours . the percentage of phagocytizing cells ( phagocytosis index according to the method of wisniewski et al ., zycie wet . 1994 69 : 168 ) and the percentage of granulocytes nitroblue tetrazolium test - positive ( according to the method of park , b ., lancet , 1968 , 22 : 532 ) were determined for each sample . the lysozyme dimer enhances phagocytic activity of leukocytes in the in - vitro tests . the effects are dependent on the dose and on the incubation time . higher concentrations of lysozyme dimer are necessary to activate milk leukocytes than blood leukocytes . excessively high concentrations of the dimer slightly reduce phagocytic activity in - vitro . selected results -- mainly showing that as long as the post - polymerization reaction mixture does not contain any cytotoxic monomeric form of the dimer , comparable results are obtained for highly purified and less pure dimerized lysozyme -- are shown in the following tables 2 and 3 in which a term &# 34 ; lysozyme dimer +&# 34 ; is used to indicate a composition containing a small fraction of trimers and higher oligomers of lysozyme as mentioned earlier in the specification . table 2______________________________________effect of lysozyme dimer on phagocytic activity of milk leukocytes from healthy cows ( concentration of the dimer 20 μg / ml , incubation time 3 hours ) healthy cow milk leukocytesindicator preparation cow no . 477 cow no . 463______________________________________ % phagocytosis control 77 . 8 80 . 0 lysozyme dimer + 100 100 lysozyme dimer 92 . 6 100 phagocytosis control 2 . 7 4 . 1 index lysozyme dimer + 4 . 4 6 . 4 lysozyme dimer 4 . 8 8 . 4 % reduction control 3 . 4 2 . 5 nbt lysozyme dimer + 5 . 7 3 . 8 lysozyme dimer 3 . 4 6 . 8______________________________________ table 3______________________________________effect of lysozyme dimer on the phagocytic activity of milk leukocytes from infected cows ( concentration of the dimer 20 μg / ml , incubation time 30 min .) infected cow ( no . 490 ) milk leukocytes infected */ indicator preparation section healthy section______________________________________ % phagocytosis control 98 58 lysozyme dimer + 98 90 lysozyme dimer 100 100 phagocytosis control 10 . 9 2 . 7 index lysozyme dimer + 8 . 7 14 . 9 lysozyme dimer 10 . 9 8 . 9 % reduction control 4 . 2 4 . 2 nbt lysozyme dimer + 5 . 9 8 . 4 lysozyme dimer 5 . 5 7 . 0______________________________________ */ = on set of inflammation ; cow not treated it is clear that the degree of purification of lysozyme dimer has no marked effect on the observed phagocytic activity of milk leukocytes . it may appear further that the effector cells may be granulocytes . in the further examples , results of in - vivo studies are reported . for the clinical tests , the formulation of 2 mg of lysozyme dimer in 10 ml of pbs solution was used . this preparation is referred to as klp - 602 . when administered intravenously , klp - 602 was shown to stimulate phagocytic activity of blood granulocytes in healthy and sick calves and in healthy foals as well as in milk cows following intraudder application . the effect is manifested through an increased number of neutrophils and increased ability to absorb staphylococcus and to reduce nbt . this phenomenon occurs primarily during the first 12 - 24 hours following the injection of the preparation . the effect of klp - 602 upon phagocytic activity in the udder depended upon the dose and the form of the drug and the response of the individual animal . lysozyme dimer was used in the therapy of infectious diseases of cattle , pigs , horses and dogs . in different doses and at different time intervals , the preparation was administered intravenously , intramuscularly , subcutaneously , intraudderly and intrauterinely . the medication was administered to 346 cows , 274 calves , 110 sows and male pigs , 294 piglets , 709 sucking piglets , 35 foals and 107 dogs . alternative treatment was applied as a control . due to the nature of the test animals , none was left without therapeutical treatment for morality reasons . nevertheless , the conclusions could be drawn in comparison to the clinical picture of the treated diseases known from veterinary literature . klp - 602 was most effective in the treatment of diseases affecting pigs . 100 % or nearly 100 % of animals recovered from the following diseases : post - partum agalactia ( mma syndrome ), dysentery , pyometritis , influenza and colibacillosis . application of the medicament in cases of edema disease and bronchopneumonia gave slightly less prominent but still significantly better results than those obtained by alternative therapies . prompt recession of diarrhoea ( usually during the first 24 hours ) and fever as well as reappearance of milk secretion of particular importance in cases of post - partum udder and uterine inflammations ( saving the life of the piglets ) could be observed . the effectiveness of klp - 602 therapy in comparison to alternative treatments is shown in table 4 below . when treated with klp - 602 , 100 % of foals suffering from enteritis and 83 . 3 % of foals suffering from bronchopneumonia have recovered faster than the control group with known preparations . klp - 602 was tested in the treatment of some diseases in dogs , such as folliculitis ( 100 % effective ), infection of the upper and lower respiratory tracts and infection of gastrointestinal tracts manifesting itself through diarrhoea . prevalent in this group was parvovirosis , which is known to be practically an incurable disease . nevertheless , a recovery rate of about 75 % was observed in parvovirosis cases . in the tests performed with the animals affected with naturally occuring diseases , as described above , several important observations were made : 1 . the therapy proved to be effective and very simple in treatment of diseases which have a multi - factor etiology and pathogenesis ( such diseases are prevalent in animal populations and are hard to handle especially in the breeding farms where epidemic spreading of diseases is very easy ). such findings prove the modulating effect of the lysozyme dimer on the natural defensive mechanisms . table 4__________________________________________________________________________animals treated with klp - 602 animals treated with other prepartionsno . of duration recovered not recovered no . of preparation recoverycases ( days ) number % number % cases used duration % __________________________________________________________________________i . colibacillosis of piglets434 1 - 2 421 97 13 3 400 . sup . x antibiotics 3 - 5 75 - 80 . sup . x glucose , vit . bii . dysentery of pigs29 1 - 2 29 100 -- -- 219 antibiotics 5 - 9 75 stolmed , ridowetiii . post - partum agalactia30 1 - 2 30 100 -- -- 40 . sup . x antibiotics 3 - 5 80 . sup . x cortisone , sulfamideiv . influenza of piglets38 1 - 5 38 100 -- -- 200 . sup . x antibiotics 3 - 9 60 - 80 . sup . x ( latent formulations ) v . morbus oedematosus82 1 - 3 78 95 4 5 120 . sup . x antibiotics 3 - 6 60 . sup . x penicillin , tetra - cycline vit . b , fluids + glucose__________________________________________________________________________ . sup . x approximate data 2 . the therapy proved to be effective in diseases which involve in their natural course the presence of endotoxic shock or other disorders such as high and long - lasting fever , affecting the status of an animal for long period of time after recovery and thus extremly detrimental for the animals such as horses ( foals )-- bred for races and other sports , as well as those bred mainly for the food industry , in which case the production costs are the critical issue . quick recoveries will allow significant reduction of costs for the treatment of such diseases . 3 . results observed in vivo confirm suppressed levels of tnf in various naturally occuring infections treated with lysozyme dimers and thus support the invention as claimed . the effect of lysozyme dimer on the activity of antibiotics against various different microorganisms was studied in order to select the bacteria for further tests , to determine the doses of antibiotics most effective for the selected microorganisms and to find the range of the lysozyme dimer concentrations within which the expected effects will be observed . in the experiments , two lots of lyophilized purified lysozyme dimer were used , one produced in 1991 and another one produced in 1992 . in the experiments , antibiotics available on the polish market , as supplied by polfa , a polish manufacturer of the drugs , such as penicillin , neomycin , erythromycin , cephalosporine ( sefril ), were used . the antibiotics to be tested were suspended in a buffered nacl - solution ( pbs - biomed ) or in a bovine serum . a lysozyme dimer was then added to the suspension in such a manner that in the tested samples the concentration of the dimer was always 5 μg / ml . the concentrations of the antibiotics being tested were different in each test due to varying sensibility of the microorganisms used . the effect of the antibiotic alone or in combination with a lysozyme dimer was tested in a pbs or bovine serum suspension in vitro on escherichia coli , salmonella enteritidis , staphylococcus aureus and streptococcus uberis isolated from sick animals . laboratory strains of sarcina lutea 9341 atcc and staphylococcus aureus 209 p were also used in the experiments . due to the preliminary character of the tests , each antibiotic was tested against 1 , 2 or 3 different kinds of bacteria as follows : a 0 . 05 ml portion of an 18 hours broth culture of the bacteria strain to be tested was added to a 14 ml sample of enriched agar ( biomed ). the mixture was stirred and poured out on a plate of 10 mm diameter . after cooling and solidification of the agar , sterile cylinders were placed thereon , and the cylinders were filled with the antibiotic solutions to be tested . a 10 mg sample of the antibiotic to be tested was initially dissolved in pbs ; next , the required volume of this solution was added to the predetermined volume of pbs or bovine serum so as to obtain a concentration of the antibiotic as close as possible to mic ( minimal inhibitory concentration ); solutions of 3 different concentrations were always prepared . 10 mg of lyophilized lysozyme dimer was initially dissolved in 10 ml of pbs . further dilutions were prepared with either pbs or with bovine serum and added to the antibiotic solutions prepared earlier as described above . the following combinations were tested : the concentration of antibiotic was the same in each cylinder ; the concentration of the lysozyme dimer was 5 μg / ml . after the cylinders were filled with the solutions , the plates were kept at room temperature for 2 hours ; then , the cultures were incubated at 37 ° c . the plates were removed from the heater after 18 hours of incubation , and the diameter of the bacteria growth inhibition zone ( lack of colonies ) was measured around the cylinders . no difference was found in the size of bacteria growth inhibition zones around the cylinders filled with the antibiotic suspensions in pbs without and with addition of lysozyme dimer in a concentration of 5 μg / ml . the size of the inhibition zone , however , decreased around the cylinders filled with the antibiotic suspension in bovine serum , but increased -- around those filled with the antibiotic + lysozyme dimer suspensions in bovine serum . the zones were bigger than those seen around cylinders filled with pbs suspensions as well as bigger than those around cylinders filled with serum suspensions of antibiotics alone , free of lysozyme dimer . the phenomenon occured in the tests with penicillin used against sarcina lutea . ampicillin showed a synergism in combination with lysozyme dimer in inhibiting in vitro growth of escherichia coli , salmonella enteritidis and staphylococcus epidermidis . it was noted that there is an increase of activity of erythromycin in the presence of lysozyme dimer against staphylococcus aureus 209 p and streptococcus uberis as well as of sefril -- against staphylococcus aureus 209 p . escherichia coli and salmonella enteritidis were resistant to this antibiotic . the increase of antibacterial activity of the tested antibiotics was observed only when the bovine serum was used as the solvent . as a rule , the increase averaged 50 %, but in some instances , the presence of lysozyme dimer resulted in a 100 % increase in the activity of the antibiotics . lysozyme dimer ( without any conservant ) in concentrations of 5 μg / ml exhibits in vitro a synergism with some antibiotics used in mic ( minimal inhibitory concentration ) in the presence of bovine serum in inhibiting bacteria growth . the results obtained until now make it clear that further studies will be essential . ito et al have recently reported that tnf - α can antagonize the anti - hiv activity of azt ( ito , m . et al . : &# 34 ; tumor necrosis factor antagonizes inhibitory effect of azidothymidine on human immunodeficiency virus ( hiv ) replication in vitro ; biochem . biophys . res . commun . 1990 , 166 ; 1095 - 1101 ). aids patients in an advanced stage suffer from many occasional infections . some infective agents may induce elevation of tnf - α , il - 6 and other cytokines that may be either immunosuppressive or may promote hiv replication . accordingly , treatment with azt alone is not sufficiently effective . in order to increase the anti - hiv activity of azt , it is therefore proposed to combine the azt treatment with the administration of lysozyme dimer to inhibit the synthesis of tnf -- the factor antagonizing an anti - hiv activity of azt .
dimerized lysozyme is used prophylactically or as a treatment for diseases associated with tumor necrosis factor . administration of the dimerized lysozyme by mouth , injection , an impregnated tampon , an antiseptive dressing , a gel or an ointment results in lowered tumor necrosis factor concentrations . therapy proved to be effective and very simple in the treatment of diseases which have a multi - factor etiology and pathogenesis .