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`MILLENIUM EXHIBIT 2015
`Baxter Healthcare Corp. et. al. v. Millenium Biologix, LLC
`IPR2013-00582,-00583,-00590,-00591
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`164
`
`CA). During sampling, the temperature and dew
`point were measured using a dew point meter (Alnor,
`Niles, ILL) and the per cent relative humidity was
`read from a psychrometric chart. The silica content
`in the sample collected on the FWS-B filter was
`determined by x ray diffraction using first an auto-
`matic 2-theta scan, then by a count at the major and
`minor peaks at 26-7 and 20-85 degrees (Phillips XRG
`3000 with APD controller and printout, Mahwah,
`NJ). Each run was standardised against a minus 5 pm
`“minusil” sample.4
`Enzyme studies were with pulmonary macrophage
`(PM) cultures from six to 12 week old female swiss
`mice (Biolabs, St Paul, MN). All cells were cultured
`in Medium-199 (Gibco, Grand Island, NY) sup-
`plemented with 10% heat inactivated fetal calf serum
`(HIFCS) (Gibco), penicillin (100 units/ml), strep-
`tomycin
`(100 pig/m1)
`enriched with
`dextrose,
`pyruvate, glutamine, and non—essential amino acids
`(M-199 [+] w/HIFCS). The cells were incubated at
`37°C in a humid atmosphere of 5% carbon dioxide.
`Macrophages were obtained from mice by mincing
`their lungs over nylon stocking material and filtering
`down with M-199 [+] w/HIFCS. Cell suspensions
`were centrifuged at 150 g for 10 minutes. The pellet
`was resuspended in 10 ml ammonium chloride red
`blood cell hypotonic lysing buffer for 10 minutes at
`4°C, recentrifuged, and washed twice with MEM
`F-l4 (Gibco). Cell counts were obtained with a
`haemocytometer and adjusted to 10° macrophages/
`ml in M-199 [+] w/HIFCS. A glass cover slip was
`placed in each well of a Costar cluster dish and 1 ml of
`the cell suspension was added per well. After three
`hours of incubation the supernatants were aspirated,
`' the monolayers were washed twice with’warm phos-
`phate buffered saline and 1 ml of M-l99 [+] w/
`HIFCS was added per well. Adherent cells consisted
`of about 85% PMs, as found by non-specific esterase
`and peroxidase stains.5° Respirable particles collec-
`ted on the silver membrane were washed down with
`distilled water to a predetermined volume and kept
`suspended through constant shaking. A measured
`amount of this suspension was added to PM cultures
`in the well,
`in duplicate,
`to make up 0035 mg
`respirable dust addition per 10° PMs. After 24 hours
`the supernatant was analysed for collagenase using
`I“C glycine labelled collagen fibres (courtesy National
`Institute of Health, Bethesda, MD). Two hundred
`microlitres of 50 mM Tris HCl (pH 7-5), 5 mM
`C30,, and 0-5 ml of media from culture were added
`to 300 pg of the labelled material. This was incubated
`for 16 hours at 35°C and the unreacted fibres were
`removed by ultracentrifugation (Beckman Micro—
`fuge). The supernatant was pipettqd off and the
`button suspended in 100 )1] of tris HCl (pH 7-5). This
`was added to 10 ml of scintillation fluid (Packard Inc,
`Chicago, ILL) and the amount of labelled material
`remaining was read off on a liquid scintillation
`
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`
`Bagchi
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`counter (model LS-100C, Beckman). The extent of
`non-specific degradation was checked by adding
`001% trypsin.
`
`Results
`Charge measurements were made on different days
`over a five month period in the cleaning rooms of grey
`iron foundries. Five of these were in the same
`foundry but all used silica sand in their moulding
`process. Measurements were also taken on one day in
`an aluminium foundry that used mainly olivine
`(magnesium orthosilicate) in its process. Table 1
`gives the results.
`Table 2 gives results of the cell culture studies.
`
`Discussion
`Silica is known to be cytotoxic to the alveolar
`macrophage and the death of the macrophage has
`been suggested to be intimately related to the genesis
`of silicotic fibrosis. The pulmonary reaction to silica
`has four distinct events:2
`(1) Necrosis of the macrophage that had taken
`up and liberated the silica particle.
`(2) Continued production of macrophages to
`re—ingest silica particles.
`(3) Formation of collagen.
`(4) Hyalinisation.
`The first and the third steps have been proposed to
`be closely linked in that
`the dying macrophage
`stimulates fibroblasts to lay down collagen.7 An
`examination of the reticular nodules showed these to
`be made up ofmacrophages, fibroblasts, and reticular
`fibrils.8 The reaction of macrophages, in combination
`with fibroblasts, may have evolved as a part of the
`body’s defence mechanism. During the healing of
`wounds macrophages are required for the normal
`ingrowth of fibroblasts and for the production of
`
`Table 1 Measurement of charge and of silica content
`
`Aluminium
`Grey iron
`foundry
`foundry
`________—_———————
`
`Sampling period
`
`Collection time (h)
`Respirable dust (mg/m3)
`Silica (%)
`Charge (coulombs/60 s)
`
`Charge (coulombs/mg)
`
`22 Ian—
`16 June 1980
`4—7-5
`1-08—1 ~52
`18'4—23-0
`10'7—10‘°
`(positive)
`78 x 10‘7-
`9-2 x 10"
`58
`28—39
`Humidity
`____—__————————-'
`
`29 May 1980
`
`4
`0-15
`0-2
`0-5 x 10"
`(negative)
`3-3 x 10'5
`
`One coulomb equals 16 x 10" elementary charges and assuming
`that the respirable particles collected were all 1 um in aerodynamic
`diameter, then each silica particle carried about 10° elementary
`positive charges. The charge readings on silica were verified once
`by measuring on the current mode of the electrometer: it read
`0-02 uA. Because charge is the product of current and time, this
`value of current was equivalent to 1'2 x 10'6 coulombs in a one
`minute period.
`
`

`

`What makes silica toxic?
`
`165
`
`Table 2 Extent of collagenase reaction measured by the radioactivity of.unreacted collagen
`
`Media with macrophage cultures with silica
`Control (0-01 % trypsin)
`
`960
`2000
`
`The standard amount of labelled fibre had 3000 counts.
`
`Radioactivity in unreacted
`“C labelledfibre: (average counts)
`
`Degree offibre
`breakdown
`
`3000—960=2040
`3000—2000=1000
`
`collagen. In interstitial lung diseases—a group of
`nearly 130 different disorders—a derangement of
`alveolar structures normally exists, including fibrosis
`of the adjacent interstitium. This ultimately disturbs
`the structure of the lung parenchyma so that alveoli
`are no longer able to mediate gas exchange. It has
`been reported that most patients with idiopathic
`pulmonary fibrosis have detectable active collagenase
`in their bronchoalveolar lavage fluid.9 This indicates
`' that the disease is progressive. Of special concern is
`the relation between macrophage secretory products
`and the synthesis and degradation of connective
`tissue components. Macrophages are stimulated by
`phagocytosis and are capable of secreting enzymes
`like collagenase that can degrade connective tissue.”
`The enzymes have been detected in fluids from
`macrophage cultures, but not
`interstitially. This
`suggests that they are secreted rapidly and without
`intracellular storage when activated.“ The output of
`free macrophages in the body, however, is in res-
`ponse to the type of particle and the total load.12 In
`the present study, detectable activities of collagenase
`were found in samples when macrophages were
`incubated with silica particles that had positive
`charge (table 2).
`The electrophoretic mobility (a measure of the
`charge) of particles has been shown to be propor—
`tional to the severity of pulmonary damage when
`tested intratracheally in rats.3 Pretreatment of quartz
`particles with polyvinylpyridine—N—oxide (PVPNO)
`decreased the severity.‘3 The improvement resulted
`from insulation of the charge, a purely physical
`phenomenon,
`and
`the
`characteristic
`reaction
`occurred again when PVPNO was removed from the
`surface of the particles. In a second study, the zeta
`potential, another measure of the charge, was found
`to be close to 50 mV for both asbestos and silica and
`
`the zeta potential of asbestos was proportional to the
`haemolysis of erythrocytes.M It was found that
`surfactants reduced the zeta potential and also the
`toxicity of asbestos. It appears reasonable to con-
`clude that the surfactants insulated the charge of the
`asbestos fibres. Both of these experiments indicate
`that charge of the particles is the property that
`imparts toxicity to a substance. Measurements of
`charge made in the present study always showed that
`silica particles carried positive values of a high
`magnitude (table 1). Whereas positive charge on
`
`particles appears to be a property that causes macro-
`phages and fibroblasts to react in the characteristic
`manner, this reaction could be aggravated by the
`current generated by the positively charged particles.
`Current in the range of 0-01 to 15 ”A has been found
`to promote synthesis of collagen by fibroblasts.15 A
`measurement of current was made in the present
`study; 0-02 [1A was generated by the positively
`charged particles. Further, it has been reported that
`crysotile asbestos caused fibroblasts to lay down
`collagen directly without the activation by macro-
`phages.” This could be because the charge they
`carried generated a current in the critical range to
`elicit this reaction, possibly a potentiating mechan-
`ism of the disease.’7
`
`It has been known for some years that olivine,
`unlike silica, does not cause lung disease and the
`substitution of silica by olivine has been rec-
`ommended as a method of control in foundries.” If
`positive charge on particles was indeed the causative
`factor in silicosis, there should be a distinct difference
`in this property for the two substances. When
`measured in the present study the charge from
`olivine was found to be highly negative but of the
`same magnitude as that measured for silica bearing
`dusts in iron foundries.
`
`The body appears to have low tolerance for
`positive charge or electron deficiency. Most chemical
`carcinogens require metabolic activation in vivo. The
`final active forms ofchemical carcinogens are electron
`deficient,
`electrophilic,
`or
`positively
`charged
`reagents. These electrophils combine with numerous
`electron rich components of cellular molecules such
`as nucleic acids or proteins to form covalently bound
`carcinogen residues attached to these macro-
`molecules. The binding of the chemical carcinogens
`to specific informational molecules involved in the
`control of growth appears to initiate the carcinogenic
`process, the positively charged reagents being the
`initiating factors.‘9 Experiments with implanted sur-
`face charge have supported this process of initia—
`tion.20 The mechanism of pathogenesis in the silica
`reaction at the cellular level has been discussed in
`
`detail.2122 Allison’s experiments also pointed out that
`necrosis of the macrophage was not the important
`step in the sequence culminating in the disease but
`that fibrogenesis was dose dependent, being greatest
`when small doses of silica were used. Small doses of
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`166
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`
`
`Charge(coulomb/mg)
`
`1 o— 7
`
`20
`
`30
`
`4o
`
`50
`
`% Humidity
`
`Relation between charge and humidity.
`
`silica stimulated synthesis of macrophage enzymes
`resulting in fibrogenesis, but did not kill the cells.
`Higher doses of silica resulted in death of macro-
`phages with little fibrogenesis. The combination of
`positively charged silica particles with electron rich
`protein components ofmacrophages appears to result
`in the release of collagenase, which activates
`fibroblasts, through factors, to lay down collagen.
`The extent of exposure, a product of the number of
`particles and their charge, could have different kinds
`of effects on macrophages resulting in different dis-
`eases. A higher exposure could kill the macrophages
`and thus have a more serious effect. It is not surpris-
`ing, therefore, that foundry workers also show a
`higher cancer rate than the general population (Dod—
`son V. Personal communication). This could have
`resulted from an excessive exposure. The same
`mechanism may be true of other substances that
`carry a positive charge.
`The figure shows that higher values of positive
`charge were measured when humidity was low. This
`suggests that keeping the humidity high may help in
`controlling the charge picked up by the silica parti-
`cles and thus their toxicity. Humidity would also
`help in the coalescence of smaller particles to larger
`ones, possibly making them non-respirable.
`'
`‘
`
`Conclusions
`
`The experimental work and survey of published data
`suggest that the toxicity of respirable silica particles
`is caused by the large amount of positive charge they
`carry. Low humidity tends to increase the magnitude
`of the charge.
`The proposed ,mechanism is that inhaled silica
`particles that reach the alveolar regions are taken up
`by macrophages. These positively charged silica
`particles combine with specific electron rich protein
`molecules within the macrophage. This results in the
`release of collagenase. Fibroblasts are activated
`through various factors to lay down collagen, an
`
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`4 of 4
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`Bagchi
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`event that could be aggravated by particles carrying
`positive charges of certain magnitude.
`
`I thank Professor V Dodson, Department of Pre-
`ventive Medicine and of Medicine, University
`of Wisconsin at Madison; Mr R Frankenburg,
`Giddings and Lewis Foundry, Madison; Mr L Wahl,
`National Institute of Health, Bethesda, Maryland;
`and Dr Anne Wheldon, University of Reading,
`England.
`
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`
`Accepted 8 July 1991
`
`