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`Exhibit 1017
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`
`The Journal of Infectious Diseases
`
`Official Publication of the Infectious Diseases Society of America
`· Louis Weinstein, Associate Editor
`Edward H. Kass, Editor
`Mary M. Eichhorn Adams, Managing Editor
`K. Frank Austen, Associate Editor
`Amy M. Levin, Editorial Assistant
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`Julie McCoy Spaulding, Editorial Assistant
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`Elizabeth C. Krome, Editorial Assistant
`Morton N. Swartz, Associate Editor
`
`EDITORIAL BOARD
`Robert Austrian, Philadelphia, _Pa.
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`
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`Richmond, Va. ·
`·
`
`INTERNATIONAL BOARD OF EDITORS
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`
`The Infectious Diseases Society of America was founded in 1963 to meet the needs for a forutn
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`s. w. 1.
`© 1974 by the University of Chicago. All rights reserved.
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`ISSN0022-1899
`
`Exh. 1017
`
`

`
`• VOL. 129, No. 2
`THE JOURNAL OF INFECTIOUS DISEASES
`© 1974 by the University of Chicago. All rights reserved.
`
`• FEBRUARY 1974
`
`Effects of Tetracycline on Leukotaxis
`
`R. Russell Martin, Glenn A. Warr, Robert B. Couch,
`Henry Yeager, and Vernon Knight
`
`From the Departments of Medicine and of
`Microbiology and Immunology,
`Baylor College of Medicine, Houston, Texas
`
`Tetracycline, in concentrations common during therapy, markedly depressed mi-
`gration of human leukocytes in vitro. Sera from 12 of 13 volunteers who received
`oral tetracycline after infection with Mycoplasma pneumoniae inhibited leukotaxis
`of normal leukocytes. Random migration and chemotaxis of leukocytes from two
`additional subjects were depressed for up to 24 hr after a single 1-g dose of tetra-
`cycline. When tetracycline was tested over a wide range of concentrations, leuko-
`taxis was depressed by lower concentrations (0.01-10 f!glml) but was stimulated
`by higher concentrations (30-300 flg/ml) of the antibiotic. Metabolic studies
`revealed that production of leukocyte lactate was elevated significantly in the pres-
`ence of a high level of tetracycline. The mechanisms by which tetracycline afTects
`lcukotaxis are not known.
`
`Leukotaxis (the migration of leukocytes toward
`an attracting stimulus) is one of the fundamental
`responses of polymorphonuclear leukocytes. This
`property has been examined in vitro with various
`modifications of the micropore filter chamber de-
`veloped by Boyden [1]. Numerous substances have
`been shown to attract leukocytes, including bac-
`terial culture filtrates [2, 3 ], extracts from leuko-
`cytes themselves [ 4 ], components of the serum
`complement system [5, 6], and tissues infected
`with virus [7, 8].
`The effects on polymorphonuclear leukocytes
`of drugs used in treatment of infections are usu-
`ally monitored by the observation of changes in
`counts of total and differential leukocytes in the
`peripheral blood. The possibility that therapy with
`drugs may alter some of the functions of periph-
`eral blood leukocytes has not been extensively
`investigated. However, Ward [9] and Peters et al.
`[1 0] have described the effects of corticosteroids
`on leukotaxis in vitro and in vivo, respectively.
`
`Received for publication March 5, 1973, and in revised
`form August 20, 1973.
`This. investigation was supported by grants from the
`AMA-ERF Committee for Research on Tobacco and
`Health, by the National Tuberculosis and Respiratory
`Diseases Association, by contract no. NAS 9-13139 P/1
`from the N ationai· Aeronautics and Space Administration,
`and by grants no. RR-00350, PH .43-NIAID-68-963, and
`1 TO 1 AI00446-0 1 from the U.S. Public Health Service.
`Please address requests for reprints· to Dr. R. Russell
`Martin, Department of Medicine, Baylor College of Medi-
`cine, 1200 Moursund Avenue, Houston, Texas 77025.
`
`We became interested in the effects of tetracy-
`cline on leukotaxis when we observed reduced
`migration of leukocytes in a group of volunteers
`receiving this antibiotic [II]. The present studies
`extend these observations by means of a system-
`atic evaluation of the influence of tetracycline on
`leukotaxis, measured in vitro.
`
`Materials and Methods
`Experimental infection with Mycoplasma pneu-
`moniae was produced in normal volunteers by in-
`tranasal and nasopharyngeal inoculation of 2 ml
`of broth containing 1 X 10° cfu of M. pneumo-
`niae. These volunteers had initial low (~ 1 :2) or
`negative titers of growth-inhibiting antibody to M.
`pneumoniae in serum, and all volunteers became
`infected, as evidenced by a fourfold rise in anti-
`body titer in serum and by repeated isolatio~ of
`M. pneumoniae from throat swab specimens.
`Leukocytes were collected after sedimentation
`of anticoagulated blood in dextran, as previously
`described [I 2]. A portion of the separately col-
`lected serum was used on the day obtained, and
`the rest was stored at -70 C for subsequent tests.
`The system used for leukotaxis has been de-
`scribed in detail [11]. Briefly, the modified Boyden
`chambers used were similar to those described by
`Cornel y [ 4] and consisted of two compartments,
`separated by a 13-mm diameter Millipore® filter
`(SSWP 01300) with a mean pore size of 3 f!m.
`The upper chamber held a volume of 0.35 ml
`containing 1.3 X 10° leukocytes, and the lower
`
`110
`
`Exh. 1017
`
`

`
`Tetracycline and Leukotaxis
`
`Ill
`
`chamber contained a total volume of 1.0 mi. To
`test migration of leukocytes toward an antigen,
`I.3 X I 0° leukocytes in 0.35 ml of fluid were
`added to the upper chamber, and 0.1 ml of anti-
`gen was placed in the lower chamber. For tests
`with mycoplasma antigen and influenza vaccine,
`the basic medium was Hanks' balanced salt solu-
`tion with I % gelatin (Hanks' -gel). The influenza
`antigen used was zonally purified, dialyzed mono-
`valent influenza vaccine (AjHong Kong/ Aichi/
`68, Eli Lilly, Indianapolis, Ind.) with a potency
`of I ,600 chick cell-agglutinating units/mi. In tests
`with serum, a final concentration in serum of l 0%
`was used in both upper and lower chambers.
`Preliminary· studies of the time-course of migra-
`tion of leukocytes in the chambers revealed that
`leukocytes counted on the lower surface of the
`filters increased linearly between 60 and 90 min,
`then reached a plateau until 240 min, after which
`a decrease was noted. Incubations were conducted
`for 2 hr, with temperature maintained at 37 C by
`circulating water at a constant temperature. Filters
`were processed for microscopy, and for each filter,
`leukocytes on the bottom surface were counted in
`l 0 random microscopic fields at a magnification
`of X 400 [ I1]. Leukotaxis was expressed as the
`number of leukocytes migrating completely through
`to the bottom surface of the filter. Thus, for each
`condition of incubation for each subject, counts
`were made of a total of 30 random microscopic
`fields. Under the conditions described, virtually
`all of the cells counted were polymorphonuclear
`leukocytes.
`In incubations with· tetracycline, tetracycline
`HCl (USP, kindly supplied by the Upjohn Co.,
`Kalamazoo, Mich.) was used in final concentra-
`tions of 0.01-300 llg/ml. Each of the two volun-
`teers received an oral 1.0-g dose of tetracycline
`(TETREX® bidCAPS®, Bristol Laboratories,
`Syracuse, N.Y.). The volunteers with infection
`due to mycoplasma received 250 mg of tetracy-
`cline four times daily on days 22-28 after inocu-
`lation. Levels of tetracycline in serum were mea-
`sured by the agar diffusion method of Bennett
`et al. [13].
`Metabolic studies of leukocytes. Determina-
`tions were made on three separate occasions with
`leukocytes from the same individual. Leukocytes
`were placed in Hanks'-gel solution with 10% au-
`tologous serum and 0.050 ml of D-glucose-U-14C
`(final specific activity, 0.023-0.064 [tCi/mol, New
`
`England Nuclear, Boston, Mass.). Triplicate incu-
`bations were performed in 25-ml flasks with plastic
`wells (Kontes Glass, Vineland, N.J.) under three
`experimental conditions: with no added antibiotic,
`with 1 [tg of tetracycline phosphate/ml, and with
`300 [tg of tetracycline phosphate/mi. Additional
`controls included flasks without leukocytes and
`flasks containing acid-treated leukocytes. The mix-
`tures were kept at 4 C in the interval between
`the addition of radioisotope and the beginning of
`incubation. The flasks were gassed for 10 min
`with 95% o~-5% co~, then incubated for 2 hr
`at 37 C at 68 oscillations/min in a Dubnofi met-
`abolic shaker. After incubation, 1.0 ml was with-
`drawn for determination of glucose. Hydroxide of
`hyamine, 0.4 ml (Packard Instrument Co., Down-
`ers Grove, III.), was added to the plastic wells to
`trap 14 CO~, which was measured by pipetting
`0.100 ml of hyamine solution into 15 ml of tolu-
`ene containing 1.0 rrig of 2,5 diphenzloxazole/ml
`and 0.25 mg of 1,4-bis-2-(5-phenyloxazolyl)-bcn-
`zene/ml. Samples were cowated in a Packard Tri-
`Carb model 3375 liquid scintillation counter and
`were corrected for quenching by the standard ex-
`ternal technique.
`The glucose remaining after incubation was de-
`termined by measurement of glucose enzymatically
`(Sigma Chemical, St. Louis, Mo.) in flasks with
`and without cells; production of lactic acid was
`measured enzymatically (Sigma Chemical) as the
`difference between content of lactic acid in super-
`natant fluid of flasks with tissue that had been in-
`cubated and that in those that had been initially
`killed with acid.
`
`Results
`
`The inhibitory effects of tetracycline on migration
`of leukocytes were discovered during a serial study
`of leukotaxis in which white blood cells and sera
`from 13 adult volunteers with experimental M.
`pneumoniae infection were used [ 11]. In the final
`week of that study, on days 22-28 after infection,
`13 volunteers received 1.0 g of tetracycline each
`day in an attempt to reduce carriage of M. pneu-
`moniae. The random migration of leukocytes in-
`cubated in autologous serum obtained on day 29
`(one day after completion of therapy with oral
`tetracycline) was 36% lower Cx2, P < 0.01) than
`random migration of the same leukocytes in con-
`trol serum collected before infection (figure I).
`
`Exh. 1017
`
`

`
`112
`
`Martin et al.
`
`NO ANTIGEN
`
`20
`
`MYCOPLASMA
`GRADIENT
`
`u. a. 15
`:I: o-m
`3:
`a:
`w m 10
`::=!:
`:::;) z
`z
`<t
`w
`::=!:
`
`5
`
`DAY 29
`
`DAYO
`
`DAYO
`DAY29
`SERA
`Figure 1. Random migration (no chemotactic agent
`in lower chamber) and chemotaxis (mycoplasma an-
`tigen in lower chamber) of autologous leukocytes
`collected from 13 volunteers on day 29 after infec-
`tion with mycoplasmas. Clear bars show mean ± SE
`of migration in serum before infection (day 0),
`while dark bars show migration in serum taken on
`day 29.
`
`Although chemotaxis of leukocytes toward myco-
`plasma was maintained in sera from day 29 (mi-
`gration was greater with mycoplasma than with
`no antigen present, z2 , P < 0.01 ), migration was
`lower in magnitude than expected, based on other
`observations [ 11]. The possibility that sera from
`day 29 might interfere with leukotaxis was con-
`sidered.
`The effects of these sera on migration of homol-
`ogous leukocytes were then studied. Leukocytes
`from a single normal donor were tested simulta-
`neously with sera obtained from the 13 volunteers
`at the start of the study (day 0) and at the com-
`pletion of therapy with tetracycline (day 29).
`Levels of tetracycline in the sera from day 29
`were ::;;; 5 ~tg/ml. The random migration of leuko-
`cytes (no chemotactic agent in the lower compart-
`ment of the Boyden chamber) was a mean value
`
`of 33 leukocytes per high-power field in serum
`from day 0, decreasing to 21 leukocytes per high-
`power field in serum from day 29 (figure 2). In
`12 of the 13 individual serum pairs, migration of
`the homologous leukocytes was lower in serum
`from day 29 than in serum from day 0 from the
`same volunteer (paired t-test, P < 0.01). That
`this was not a result of the handling and storage
`of the sera was demonstrated by the unchanged
`values for migration of leukocytes in the serum
`from a normal control subject who did not receive
`tetracycline, but whose serum was collected and
`similarly processed.
`There was a similar suppressive effect by sera
`from day 29 on chemotaxis toward mycoplasma
`antigen of homologous leukocytes from the same
`donor (figure 3). In I 0 of the 13 serum pairs,
`chemotaxis was reduced in serum from day 29
`compared with migration in serum from day 0
`(paired t-test, P < 0.05). In one instance migra-
`tion of leukocytes was the same in both sera, while
`chemotaxis of leukocytes was higher in sera from
`day 29 in two instances. Leukotaxis was reduced
`
`RANDOM MIGRATION
`
`50
`
`45
`
`40
`
`35
`
`0
`
`-' 30
`"-' -u..
`
`0
`
`0 :::: u
`a:>
`s:
`
`25
`
`20
`
`15
`
`10
`
`SERUM 0
`SERUM 29
`Figure 2. Random migration of homologous leuko- ·
`cytes from a normal donor in sera collected before
`illness (day 0) and on day 29 after infection with
`mycoplasmas. Migration in individual serum pairs is
`represented by 0--0. Migration in a normal con-
`trol semm is represented by 0--0. Brackets show
`mean± SE.
`
`Exh. 1017
`
`

`
`Tetracycline and Leukotaxis
`
`113
`
`CHEMOTAXIS
`
`70
`
`65
`
`60
`
`55
`
`50
`
`45
`
`>< 35
`0
`0 ::: u
`"' 30
`:;::
`
`25
`
`0 ~-------L----------------~--------
`SERUM 29
`SERUM 0
`Figure 3. Chemotaxis toward mycoplasma antigen
`of homologous leukocytes from a normal donor, in-
`cubated in 10% sera collected on day 0 and day 29
`after infection with mycoplasmas. Migration of leu-
`kocytes in individual serum pairs is represented by
`•--•. Chemotaxis in serum from an untreated
`control is represented by 0--0. Brackets show
`mean± SE.
`
`from a mean of 35 leukocytes per high-power
`field in sera taken before illness to 24 in sera from
`day 29. Leukotaxis was not significantly different
`in serum from the control subject taken on day 0
`or day 29, collected and processed in the same
`way as the patient sera.
`To investigate further the inhibition of leuko-
`taxis by serum containing tetracycline, serial dilu-
`tions of tetracycline were incubated with 10%
`serum and autologous leukocytes from a single
`normal donor. Random migration of leukocytes
`and chemotaxis of leukocytes toward mycoplasma
`were tested as before. The effects of tetracycline
`were dependent on the concentration of antibiotic.
`In the range from 10 ng to 10 ~tg/ml, both ran-
`
`dom migration (t-test, P < 0.001) and chemo-
`taxis of leukocytes toward mycoplasma (t-test,
`P < 0.01) were depressed (figure 4). Leukotaxis
`was reduced by as much as 60% compared with
`control migration without the presence of tetra-
`cycline. With concentrations of tetracycline rang-
`ing from 30 to 300 ~tg/ml, migration of leukocytes
`was increased to as much as 600% of control
`levels (t-test, P < 0.001). Thus leukotaxis was
`depressed when concentrations were in the range
`found in the serum during therapy but was stim-
`ulated by higher concentrations of tetracycline.
`Although not shown here, leukotaxis was similarly
`affected in incubations in which serum was omitted.
`To confirm the original observation that oral
`administration of tetracycline might affect leuko~
`taxis, two volunteers received 1 g of tetracycline
`as a single oral dose, and sera and leukocytes were
`collected serially for quantitation of leukotaxis. In
`the first volunteer, random migration of leuko-
`cytes and chemotaxis toward two different chemo-
`tactic stimuli (0.1% casein and M. pneumoniae)
`were measured (table 1). Chemotaxis toward
`0.1% casein and mycoplasma was reduced to
`50% or less of control by 2 hr and to 10% or
`less of control by 4 hr after administration of
`
`500
`400
`300
`200
`
`""f
`
`2
`~ 120
`t-«
`"' :: 100
`::;:
`--'
`0
`
`80 1-
`
`"' t-
`2
`0
`u
`
`60
`
`"' 40
`
`20
`
`100
`
`300
`
`NONE
`
`. 01
`
`. 1
`
`. 3
`30
`10
`TETRACYCLINE I,Ugimll
`Figure 4. Migration of normal leukocytes in 10%
`serum containing various concentrations of tetracy-
`cline. Values for random migration ( 0) and chemo-
`taxis (•) toward mycoplasma are expressed as a
`percentage of migration in incubations without tet-
`racycline (mean of two experiments).
`
`Exh. 1017
`
`

`
`114
`
`Martin et al.
`
`Table 1. Effects of oral tetracycline (TC) on in vitro leukotaxis.
`Level of
`Random
`TC in
`migration:
`serum
`Subject Time after
`0.1% casein
`no antigen
`TC (hr)
`Utg/ml)
`no.
`58* (100)
`16* (100)
`()
`()
`29 (50)
`22 (137)
`6.2
`2
`6 (10)
`16 (100)
`10.2
`4
`3 (5)
`4 (25)
`24
`1.7
`180 ( 100)
`108 (100)
`40 ( 100)
`0
`0
`50 ( 46)
`91 (50)
`15 (37)
`4.0
`2
`26 (14)
`43 ( 40)
`20 (50)
`7.6
`4
`52 ( 48)
`165 (92)
`38 (95)
`2.5
`24
`< 0.3
`192 ( 107)
`158 (146)
`48 (120)
`48
`*Number of leukocytes per x400 microscopic field (percentage of migration in serum before tetracycline).
`
`A~ influenza
`virus
`
`Chemotaxis
`Mycoplasma
`pneumoniae
`48* (100)
`16 (33)
`4 (8)
`7 (14)
`
`2
`
`tetracycline, at which time a peak level of tetra-
`cycline in s~rum of 10.2 ftg/ml was present. ~cu­
`kotaxis remained depressed 24 hr after the smglc
`dose of tetracycline.
`In a second volunteer, observations of lcuko-
`taxis were extended through 48 hr after the oral
`ingestion of tetracycline. Preliminary studies had
`shown that this volunteer's leukocytes were not
`responsive to mycoplasma antigen; thus, A~ influ-
`enza virus, to which the leukocytes were respon-
`sive, was used as a chcmotaxin. Casein was used
`as a second chemotactic agent. The degree of de-
`crease in chemotaxis after administration of tetra-
`cycline was similar to that observed in th~ first
`volunteer. Maximal depression of lcukotaxis oc-
`curred at 4 hr, when the serum contained 7.6 flg
`of tetracycline/mi. Random migration of lcuk?-
`cytcs and chemotaxis of leukocytes t~":'ard _cascm
`returned to levels found before admimstratwn of
`.
`b"
`t
`tetracycline between 4 and 24 hr. In this. su Jec ,
`leukotaxis did not return to normal until . 24-48
`hr after the administration of oral tetracyclmc.
`To sec whether tetracycline could induce changes
`in metabolism of leukocyte glucose that might be
`related to the observed changes in leukotaxis, we
`incubated groups of leukocytes from a single donor
`with o-glucose-14C with no antibiotic, and with
`1.0 g of tetracycline/ml and 300 g of tetracycline/
`mi. No significant change in uptake of glucose or
`production of HCO~ was measured at either c~n­
`ccntration of tetracycline, but a 44% ± 11% m-
`crease (P < 0.05) in production of lactic acid
`occurred at 300 pg of tetracycline/ml, suggesting
`that increased use of glucose did occur at higher
`levels of tetracycline.
`
`Discussion
`These studies have shown that the antimicrobial
`agent,
`tetracycline,
`in concentrations usually
`achieved during therapy, inhibits leukotaxis. When
`sera from volunteers convalescing from infection
`with mycoplasma were first noted to depress leuko-
`taxis, several different possibilities were considered
`as explanations of this observation. Circumstantial
`evidence implicated the tetracycline therapy as the
`cause of the observed reduction in leukotaxis. The
`additional studies documented that tetracycline
`can have this effect, even in low concentrations
`such as thuse (:::;; 5 ftg/ml) in sera of these treated
`volunteers.
`The in vitro addition of tetracycline over a
`range of concentrations commonly encountered in
`therapy (0.1-10 pg/ml) depressed both random
`migration and chemotaxis of leukocytes in either
`the presence or the absence of serum. The marked
`stimulation of lcukotaxis at higher concentrations
`of tetracycline (30-300 ftg/ml) was not antici-
`pated. The basis of this stimulation is not known;
`however, the increased production of lactic acid
`by leukocytes in the presence of 300 ftg of tctra-
`cycline/ml may be a manifestation of a metabolic
`effect of the drug.
`The mechanisms by which lower concentrations
`of tetracycline inhibit lcukotaxis have not been
`delineated. Leukocytes in blood have a high rate
`of aerobic glycolysis [14], and several studies have
`indicated that glycolytic energy is necessary for
`leukotaxis [15, 16]. Uptake of glucose was not
`affected measurably by tetracycline, an observa-
`tion which agrees with the studies of McCurrach
`et a!. [17]. Other cellular pathways, such as the
`
`Exh. 1017
`
`

`
`Tetracycline and Leukotaxis
`
`115
`
`synthesis of cytochrome oxidase, may be depressed
`by tetracycline [ 18 J. Another possible mechanism
`for the alterations of lcukotaxis by tetracycline is
`the prolonged effect on membranes of leukocytes
`exposed to this drug [ 19].
`Leukocytes can take up and store tetracycline,
`resulting in intracellular concentrations that are
`considerably higher than concentrations in the
`surrounding medium [20J. The phenomenon of
`uptake and concentration of tetracycline may ex-
`plain the effects on leukotaxis of the in vitro addi-
`tion of very low levels of the antibiotic. The pro-
`longed depression of leukotaxis observed with
`leukocytes collected from volunteers long after the
`peak levels in serum had passed may also be re-
`lated to intracellular retention of the drug.
`The accumulation of leukocytes in inflamed
`tissue is one of the fundamental responses to in-
`fection. Depressed migration of leukocytes has
`been demonstrated in a number of conditions as-
`sociated with an increased incidence of bacterial
`infections, including poorly controlled diabetes
`[21 ], cirrhosis [22], rheumatoid arthritis [23 ], and
`deficiencies in the complement system [24, 25].
`Even though tetracycline is a bacteriostatic an-
`tibiotic, and therefore requires a participation by
`the host defenses to eradicate infection, it is likely
`that the reduction of leukotaxis by tetracycline
`docs not critically impair defenses in most patients
`who are otherwise normal. However, in patients
`with other complicating conditions
`(especially
`those known to be associated with altered func-
`tion of leukocytes), the additional effects of de-
`pression of Ieukotaxis by tetracycline could be of
`clinical importance.
`
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`Exh. 1017
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`Exh. 1017