both r - boil - 1 and r - boil - 2 induce an infiltration of somatic cells into the milk in a dose related manner , while r - bogm - csf demonstrates no significant chemoattractant activity , fig1 . the minimal effective dose and maximal tolerable dose for r - boil - 1 and r - boil - 2 is 0 . 5 mg to 1000 mg and 0 . 5 mg to 30 mg respectively , while intramammary infusion of up to 5 mg of r - bogm - csf fails to elicit any significant influx of cells into the mammary gland . these doses are guidelines for quantitative induction of somatic cells , but significant beneficial qualitative changes can be induced with lower doses . the specific biological activity for r - boil - 1 as a chemoattractant is approximately 10 , 000 fold greater than r - boil - 2 , although the in vitro specific bioactivities are equivalent . active il - 2 inhibition or decreased biostability in milk might explain this discrepancy . the peak number of cells after r - boil - 1 infusion with a maximal tolerable dose is 4 - 5 fold greater than r - boil - 2 . the kinetics of pmn migration into the mammary gland is such that migration occurs 8 - 24 hours sooner for r - boil - 1 infusions or the boll - 2 infusion depending upon the dose . the dominant cell type after r - boil - 1 or r - boil - 2 infusion is pmns . in addition to quantitative changes in milk pmns after cytokine infusion , qualitative changes ( activation ) are also observed . r - boil - 1 induces an initial 150 × increase over the pre - treatment number of pmns at 16 hours , decreasing to 20 × at 40 hours , and back to normal levels by 112 hours , fig2 a . the inducible superoxide upon phorbol ester stimulation is significantly increased over the resident pmns , fig2 c . the inducible superoxide from resident milk pmns is significantly inhibited and is only 5 % of that from peripheral blood pmns from the same animal . phagocytosis is unaffected by intramammary r - soil - 1 administration , fig2 b , perhaps suggesting that the recruited pmns are only partially activated or primed . r - boil - 2 administration also elicit a quantitative increase in milk pmns , fig2 d , which diminishes after 72 hours , to control levels at the 2 mg dose . as with r - boil - 1 administration , inducible superoxide is significantly increased over the resident pmns , fig2 f , and remains throughout the 96 hours of sampling . phagocytosis is increased 2 - 3 fold over resident pmns within the 40 - 64 hours after time point and although the percent phagocytosis tapers off by 112 hrs , it is still above the pre - treatment level , fig2 e . the average number of ingested beads per phagocytic cell is also increased upon activation . the delay of peak phagocytic activity , as compared to somatic cell counts , suggests that activation and chemoattraction can be distinctly regulated . r - bogm - csf fails to elicit any significant quantitative increase in milk pmns , fig2 g , even with a 5 mg intramammary dose . however , the biological activity , as measured by inducible superoxide production is increased over the resident pmns , fig2 i . further , the percent of cells which are phagocytic , are enhanced , fig2 h . the increase in phagocytosis is maintained above pre - infusion levels for up to 112 hrs . in vivo measurement of daily milk production is also monitored for both intramammary infusions and intramuscular injection . cows are milked morning and evening and the milk weighed and recorded in total kilograms per day . the total dose of il - 2 administered per cow ranges from 2 to 96 mg . single or multiple intramammary infusions of il - 2 have variable effects on individual animal &# 39 ; s milk production based upon the total dose of r - boil - 2 administered per day , fig3 a ( intramammary ). production decreases as much as 30 - 40 % by the third infusion in the high dose animals ( 13 . 5 mg ), whereas only a 12 % decrease is observed at the 4 . 5 mg dose ( not statistically different from time 0 production or control animals ). no statistically significant effect on milk production has been observed if the total dose is less than 4 . 5 mg . this suggests that the secretary epithelium of the mammary gland can be induced to a physiological state of non - secretion by cytokines . this induction would be of great significance to assist in the morphological changes required during early lactation and serves to accelerate the protective mechanisms . similar effects on milk production are observed with intramuscular injection of r - boil - 2 at 2 . 5 μg / kg body weight / day for 5 days , fig3 b ( intramuscular ). to specifically study the presence of cytokines after a single intramammary infusion into dry cows , 5 quarters from four different cows , are infused with 1 mg r - boil - 2 at dry - off . since a dry gland does not have its contents milked out every 12 hours , the retention of biological activity of r - boil - 2 infused into the dry gland at 72 hours or greater is examined . secretions from dry cow quarters treated with 1 mg of r - boil - 2 are collected at 72 , 144 and 240 hours after dry cow treatment . as with the milk , a 1 : 50 dilution of dry secretion samples is made and these solutions assayed by the bioassay using bt - 2 and compared to r - boil - 2 diluted in media . as shown in fig4 no detectable r - boil - 2 bioactivity ( less than 0 . 024 ηg / ml ) is demonstrated in treated glands as early as 72 hours after infusion . therefore , cytokines exert a beneficial long - term change in the protective mechanisms of the gland which significantly exceeds the time limit of detection for biological activity in the gland . the potential benefits of exogenous cytokine administration to the host , as it relates to the differential induction of quantitative and qualitative changes in milk pmns , is further explored in applied preventative and therapeutic experiments . r - boil - l , r - boil - 2 and r - bogm - csf are all capable of protecting the host from subsequent s . aureus challenge , table 1 . the protective effect of r - boil - 1 is most efficacious if administered 24 hours prior to s . aureus challenge . this maximal protection correlates with the peak quantitative biological response for this cytokine . the protective effect of r - boil - 2 is most efficacious if administered 48 hours prior to s . aureus challenge . this also correlates with the peak quantitative biological response of this cytokine and also marks the qualitative increase in phagocytosis and inducible superoxide , fig2 d & amp ; 2e . unlike r - boil - 1 and r - boil - 2 , r - bogm - csf elicit only minor quantitative changes in milk pmns . however , good protection from s . aureus is observed when r - bogm - csf is administered both 24 and 48 hours prior to challenge . both these time periods are shown to increase phagocytic ability of the cells in the milk of treated glands , fig2 h . table 1__________________________________________________________________________prevention of s . aureus mastitis by recombinant bovine cytokinestreatment . sup . 1 glands . sup . 2 dose . sup . 3 administration . sup . 4 % infected . sup . 5 % control . sup . 6__________________________________________________________________________control 1 * 10 -- -- 100 0il - 1 10 1 μg 0 hrs 13 87 10 &# 34 ; - 24 hrs 0 100 10 &# 34 ; - 48 hrs 22 78il - 2 10 1 mg 0 hrs 77 23 10 &# 34 ; - 24 hrs 11 89 10 &# 34 ; - 48 hrs 37 63control 2 * 10 -- -- 82 0gm - csf 10 1 mg - 24 hrs 20 76 10 &# 34 ; - 48 hrs 20 76__________________________________________________________________________ . sup . 1 single intramammary infusion administered after the pm milking . a total of 100 mammary glands from 22 different lactating holstein dairy cattle are treated . . sup . 2 all glands are free of bacteria on 5 successive samples after the am milking prior to the initial infusion . . sup . 3 the total dose in mass of protein is indicated . the biological activity of rboil - 1 , rboil - 2 and rbogm - csf are 32 , 000 units / μg , 22 , 000 units / μg and 38 , 000 units / μg respectively . . sup . 4 infusions of cytokines were administerd simultaneously with s . aureus 0 , 24 or 48 hours proir to challenge with 30 - 200 cfus of s . . sup . 5 individual quarters are considered cured if the milk samples remai free of s . aureus . for 14 days after intramammary challenge with of s . aureus . . sup . 6 the % control = ( 1 - {% infected after therapy divided by infected in control quarters )} × 100 . * quarters in control 1 are challenged with 250 cfus of s . aureus .. quarters in control 2 are challenged with 30 cfus of s . aureus . infected mammary glands are also effectively treated with intramammary infusion of r - boil - 1 and r - boil - 2 , see table 2 . both cytokines have a dose response therapeutic efficacy . r - boil - 1 and r - boil - 2 are most efficacious if doses are administered over 3 consecutive milkings , rather than in a single bolus . the percentage of quarters clearing the infection for greater than 1 milking ( responding ), as compared to the percentage of quarters which actually cure their infections ( cure ) is a measure of the failure of the host cells to kill the bacteria . the relapse rate is the greatest for r - boil - 2 ( combined relapse rate of 40 % for the 10 mg dose x &# 39 ; s 3 ). the high relapse rate for r - boil - 1 suggests that although many host cells may indeed ingest s . aureus , these cells are inefficient at killing the bacteria . the relapse rate for r - boil - 1 ( 54 %) is comparable to the na ( sodium ) cephapirin treatment ( 57 %), while the relapse rate for r - boil - 1 ( 54 %) is more closely approximated by the cefa - lak ® treatment ( 16 %), albeit the overall cure rates are not as efficacious as cefa - lak ®. this suggests that activation of phagocytic cells in the milk is an essential component of effective therapy for infectious agents . furthermore , if activation of the host cells is effective , as indicated by the r - boil - 2 and r - bogm - csf , then a combination therapy with r - boil - 1 or antibiotics is useful . the biological activity of pmns and number of pmns during mastitis infections are both variable , both within the animal and in each individual infected gland . clearly , activation of phagocytosis is necessary to efficiently eliminate bacteria but is insufficient without proper activation of the bactericidal components of the phagocytic cell . such a dissociation of activation of phagocytosis without activation of bactericidal activity could in fact contribute to a source of reinfection as stated earlier . this biological activity can be precisely modulated by local in vivo administration of recombinant homologous cytokines to a targeted organ . table 2__________________________________________________________________________efficacy of recombinant bovine cytokines as mastitis therapeuticstreatment . sup . 1 dose . sup . 2 interval % response . sup . 3 % cures . sup . 4 % relapse . sup . 5__________________________________________________________________________il - 1 ( single ) 10 μg -- 11 11 0 200 μg -- 20 0 100 600 μg -- 10 0 100il - 1 ( multiple ) 10 μg 24 hrs 77 8 90 10 μg 48 hrs 39 0 100 200 μg 24 hrs 79 7 91 200 μg 48 hrs 64 29 54il - 2 ( single ) 2 μg -- 0 0 -- 10 μg -- 20 0 100 30 μg -- 10 10 0il - 2 ( multiple ) 2 mg 24 hrs 55 9 84 10 mg 48 hrs 33 8 76 10 mg 24 hrs 58 25 57 10 mg 48 hrs 33 25 23na cephapirin 200 mg 12 hrs 86 42 51cefa - lak ® 200 mg 12 hrs 92 77 16__________________________________________________________________________ . sup . 1 s . aureus infections are established 2 - 3 weeks prior to treatment . treatments are administered after the am or pm milking by 1 - 3 successive intramammary infusions . a total of 120 mammary glands from 28 different lactating holstein dairy cattle are used . recombinant bovine interleukin1 ( il - 1 ) and interleukin2 ( il - 2 ) are obtained from immunex corp . seattle , wa . na cephapirin is prepared is pbs ; and cefalak ® ( bristolmeyers ) is used according to package # insert instructions . . sup . 2 cytokines are infused intramammary through the teat canal after th pm milking at the indicated intervals . the total dose is delivered in a total volume of 10 ml of sterile pbs . the biological activity of rboil - 1 and rboil - 2 is 32 , 000 units / μg and 22 , 000 units / μg , respectively . a total dose per infusion 200 mg of na cephapirin and cefalak ® is administered after consecutive pm and am milkings , according to described procedures . . sup . 3 the % responding = quarters which transiently (& gt ; 1 milking ) are free of s . aureus divided by the total number of quarters treated . . sup . 4 the % cured quarters = # of quarters which clear their infection and remain free of s . aureus divided by the total number of quarters treated . infections are considered to have relapsed if there are & gt ; 1 cfus from three consecutive am milkings , or & gt ; 20 cfus for two consecutive afte therapy at anytime during the 14 day monitoring period . . sup . 5 the % relapse = [ 1 - {% responding divided by % cured }] × 100 . phagocytic activity of cells from dry cow secretions after cytokine treatment somatic cells are isolated from mammary gland lavages on days 3 and 6 after treatment with cefa - dri ® plu6 il - 2 or cefa - dri ® alone . the cells are incubated with fluorescent latex micropheres and analyzed on the flow cytometer for their ability to phagocytize the beads . fig5 shows that phagocytic activity increases on day 6 as compared to day 3 in the infected quarters for both treatment groups and yet no biologically active il - 2 is detected after 3 days , fig4 . in contrast , phagocytic activity decreases in the uninfected quarters on day 6 as compared to day 3 for both treatment groups . this data suggests that cells from glands with an established infection at the time of dry - off maintain a higher level of phagocytic activity than cells from glands that are clear of infection at the time of dry - off . also , treatment with il - 2 seems to enhance the phagocytic activity of cells of the dry gland . two groups of 17 mammary quarters from 10 different holstein dairy cows are assigned to receive routine dry cow therapy cefa - dri ® or dry cow therapy plus 1 mg of recombinant bovine interleukin - 2 ( il - 2 ). antibiotic treatment of nocardia infections has been shown to be ineffective . cefa - dri ® treated quarters result in 72 % of the glands becoming infected with nocardia after challenge with broth dipping , table 3 . however , only 53 % of the glands become infected if the dry cow therapy includes recombinant il - 2 . table 3__________________________________________________________________________prevention of nocardia infections by r - boil - 2 in dry cowspercent quarters infected . sup . 1treatment . sup . 2 # glands il - 2 dose . sup . 3 dry - off d / o + 10 days . sup . 4 d / o + 28 days__________________________________________________________________________cefa - dri ® 17 -- 0 24 72cefa - dri ® + rboil - 2 17 1 mg 0 6 51__________________________________________________________________________ . sup . 1 individual quarters are dipped in broth of 10 . sup . 8 cfu / ml of nocardia at dryingoff . all teats are then dipped in iodidine solution and treated with respective drycow therapy . . sup . 2 single intramammary infusion is administered after the last pm milking . a total of 34 mammary glands from 10 different holstein dairy cattle entering the dry period are treated . . sup . 3 the total dose in mass of protein is indicated . the biological activity of rboil - 2 is 22 , 000 units μg . infusions of rboil - 2 are administered simultaneously with cefadri ®. . sup . 4 all quarters were stripped at indicated times and their dry secretions plated for the presence of nocardia . prevention of new infection of staphylococcus aureus early in mammary gland involution two groups of 12 s . aureus infected mammary quarters from 6 different holstein dairy cows are assigned to receive routine dry cow therapy cefa - dri ® or dry - cow therapy plus 1 mg of recombinant bovine interleukin - 2 ( il - 2 ). 8 - 10 days after treatment at drying - off , dry secretion samples are taken and tested for the presence of residual antibiotics or bacteria . all quarters are then re - challenged with 50 - 250 cfu of s . aureus and the animals allowed to calf . upon the initiation of lactation , samples are taken on three successive milkings and tested for the presence of s . aureus . all quarters treated with cefa - dri ® alone are found to be infected . however , 40 % of the cefa - dri ® plus il - 2 treated quarters are free of infection at initiation of lactation , table 4 . table 4__________________________________________________________________________prevention of s . aureus infections by r - boil - 2 after chalenge during dryperiodpercent quarters infected . sup . 1treatment . sup . 2 # glands il - 2 dose . sup . 3 dry - off d / o + 10 days . sup . 4 calving + 5 days . sup . 5__________________________________________________________________________cefa - dri ® 10 -- 100 0 100cefa - dri ® + rboil - 2 10 1 mg 100 0 60__________________________________________________________________________ . sup . 1 all quarters are all infected with staphylococcus aureus ( new boul strain 305 ) for a minimum of 2 weeks prior to dryingoff . . sup . 2 single intramammary infusion is administered after the last pm milking for cefadri ®. the rboil - 2 treated animals were infused with rboil - 2 then with cefadri ®. a total of 20 mammary glands from 7 different holstein dairy cattle entering the dry period are treated . . sup . 3 the total dose in mass of protein is indicated . the biological activity of rboil - 2 is 22 , 000 units / μg . . sup . 4 all quarters are stripped at day 10 and their dry secretions plate for the presence of s . aureus . . sup . 5 all quarters are sampled twice 405 days after calving and the milk plated to detect the presence of s . aureus . improved milk production in lactations immediately following dry cow therapy with cytokines the effects of r - boil - 2 on possible milk production after dry - cow therapy are also evaluated . milk production for three dairy cows for the first five days of the lactation cycle ( second or third lactation ) prior to r - boil - 2 treatment is compared to the milk production for the first five days of the lactation cycle ( third or fourth lactation ) immediately following r - boil - 2 treatment , fig6 . no inhibition of milk production and in fact an 80 % improvement in production at the following lactation after r - boil - 2 treatment is observed .
cytokines are useful for the ability to improve the natural defense system of the involuting mammary gland . in addition to the immunopotentiating activity , cytokines accelerate the normal physiology of involution of the mammary gland and thereby afford further protection against new infections of mammals . the involuting mammary gland is very susceptible to infection during the early stages of involution and therefore decreasing the period of highest susceptibility while also improving the natural defense mechanisms is of great advantage over most therapies . used alone , in various cytokine combinations , or in combination with other therapeutics , cytokines protect the mammary gland and cure existing infections and are particularly useful during cessation of lactation and early reinitiating of lactation .