Clinical immunology.
`v. 136, no. 3 (Sept 201 O)
`General Collection
`W1 CL715
`2010-09-07 10.14.45
`
`. ·, .. ~
`
`Control cells
`·. 14R': ,:i
`... . .. : ~!
`
`l ·, .~" J ~
`
`f 'l
`oi
`'f . !
`
`Induced cells
`
`stem cells
`
`. .'
`
`Page 1 of 13
`
`CSL EXHIBIT 1005
`
`

`

`Clinical Immunology
`The Official Journal of the Clinical Immunology Society
`
`Editor-in-Chief
`
`Andrew Saxon
`Division of Clinical Immunology
`Department o{Medicine, UCLA School o{ j\;fedicine
`Los Angeles, California 90095-1680
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`Associate Editor-in-Chief
`
`George 'l'sokos
`Harvard Medical School
`Beth Israel Deaconess Medical Center
`Boston, Massachusetts 02115
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`CIS Publication Committee
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`Chair: John Routes
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`Marc D. Natter
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`Andras Perl
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`David P. Richman
`Lanny J. Rosenwasser
`Lawrence B. Schwartz
`Christine M. Seroogy
`Ram Raj Singh
`Jan Storek
`Joris M. Van Montfrans
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`Elke Bergmann-Leitner
`Jay Berzofsky
`Perluigi Bigazzi
`Jose Boucraut
`Katarzyna Bourcier
`Samuele E. Burastero
`Javier Chinen
`Offer Cohavy
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`Jose C. Crispin
`Heather Donaghy
`George Eisenbarth
`Donna Farber
`C. Garrison Fathman
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`Cover photo. Cord blood mesenchymal stem cells (CB-MSCs) in resting condition and after differentiation. See Heview by H. Francese and
`P. Fiorina in this issue.
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`_,,
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`Clinical
`MMUNOLOGY
`as Clinical
`
`The Official Journal of the Clinical Immunology Society
`
`Immunology
`Society
`
`Volume 136 (3)
`2010
`
`ELSEVIER
`
`Amsterdam • Boston • London • New York • Oxford • Paris • Philadelphia • San Diego • St. Louis
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`Clinical Immunology
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`

`

`Volume 136, Issue 3, September 2010
`
`ELSEVIER
`
`CONTENTS
`
`Abstracted/indexed in: Biological Abstracts, Chemical Abstracts, Current Contents/Life Sciences, Excerpta
`Medica, MEDLINE®, PubMed, and SIIC Databases. Also covered in the abstract and citation database SCOPUS®.
`Full text available on ScienceDirect®.
`
`Review
`
`Immunological and regenerative properties of cord blood stem cells Roberto Francese
`and Paolo Fiorina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
`
`Rapid Communication
`
`Immune Regulation Subcutaneous infusion of human Cl inhibitor in swine Haixiang
`.Jiang, Hua-Mei Zhang, and Michael M. Frank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
`
`Regular Articles
`
`Immunotherapy Enhanced suppressive function of regulatory T cells from patie nts
`with immune-mediated diseases following successful ex vivo expa nsion 'l'inghua
`Cao, Sally E. Wenzel, William A. Faubion, Gregory Harriman, and Li Li. . .. . . . . . . . . . 329
`
`Immunotherapy MHC-1-restricted melanoma antigen s pecific TCR-engineered human
`CD4+ T cells exhibit multifunctional effector and helper r esponses, in vitro
`Swagatam Ray, Arvind Chhabra, Nitya G. Chakraborty, Upendra Hegde, David I.
`Dorsky, 'l'hinle Chodon, Erika von Euw, Begonya Comin-Anduix, Richard C. Koya,
`Antoni Ribas, J ames S. Economou, Steven A. Rosenberg, Bijay Mukherji, and UCLA(cid:173)
`CALTECH-CHLA-USC-UCONN Consortium on 'l'ranslational Program in Engineered
`Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
`
`Immunotherapy Histone/prote in deacetylase inhibitors increase s uppressive functions
`of human FOXP3+ Tregs Tatiana Akimova, Guanghui Ge, 'l'atiana Golovina, 'I'atiana
`Mikheeva, Liqing Wang, James L. Riley, and Wayne W. Hancock ........... . .. . . · ·. 348
`
`Immunotherapy Attenuation of invariant Natural Killer T-cell anergy induc tion
`through intradermal delivery of a-galactosylceramide Hetty J. Bontkes, Maria
`Moreno, Basav Hangalapura, J elle J. Lindenberg, Jan de Groot, Sinead Lougheed, Hans
`J.J. van der Vliet, Alfons J.M. van den Eertwegh, Tanja D. de Gruijl, B. Mary E. von
`Blomberg, and Rik J . Scheper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
`
`Immunotherapy Influence of alemtuzumab on the intestinal Paneth cells and
`microflora in m acaques Qiurong Li, Qiang Zhang, Chenyang Wang, Chun Tang,
`Yanmei Zhang, Shaojun Jiang, Ning Li, and Jieshou Li . . . . . . . . . . . . . . . . . . . . . . . . . . 375
`
`Page 5 of 13
`
`

`

`(contents continued)
`
`Immune Regulation Increased HGF and c-Met in muscle tissues of polymyositis and
`dermatomyositis patients: Beneficial roles of HGF in muscle regeneration
`Tomoko Sugiura, Yasushi Kawaguchi, Makoto Soejima, Yasuhiro Katsumata, Takahisa
`Gono, Sayumi Baba, Manabu Kawamoto, Yohko Murakawa, Hisashi Yamanaka, and
`Masako Hara. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
`
`Host Defense In vitro na'ive T cell proliferation failure predicts poor post(cid:173)
`immunization responses to neoantigen, but not recall antigens, in HIV-infection
`Benigno Rodriguez, Hernan Valdez, Christoph G. Lange, Robert Asaad, Kathy Medvik,
`and Scott F. Sieg. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
`
`Host Defense Secondary immunologic consequences in chromosome 22q11.2 deletion
`syndrome (DiGeorge syndrome/velocardiofacial syndrome) R. Zemble, E. Luning
`Prak, K McDonald, D. McDonald-McGinn, E. Zackai, and K Sullivan. . . . . . . . . . . . . . . 409
`
`Immunogenetics!genomics Association of FeRn expression with lung abnormalities
`and IVIG catabolism in patients with common variable immunodeficiency T.
`Freiberger, L. Grodecbi, B. Ravcukova, B. Kuf·ecova, V. Postranecka, J. Vlcek, J.
`Jarkovsky, V. Thon, and J. Litzman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
`
`Immune Regulation Coeliac disease autoantibodies mediate significant inhibition
`of tissue transglutaminase Greg Byrne, Con Feighery, John Jackson, and Jacinta
`Kelly. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
`
`Immune Regulation A physiological role for inducible FOXP3+ TREG cells: Lessons
`from women with reproductive failure Lourdes Arruvito, Ana I. Sotelo, Ariel
`Billordo, and Leonardo Fainboim. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
`
`Immunogenetics/genomics Analysis of JAK2 and STAT3 polymorphisms in patients
`with ankylosing spondylitis in Chinese Han population Chao Chen, Xuesong
`Zhang, and Yan Wang...................................................... 442
`
`Immunogenetics!genomics Differential expression of glycogenes in tonsillar B
`lymphocytes in association with proteinuria and renal dysfunction in lgA
`nephropathy Tatsuyuki Inoue, Hitoshi Sugiyama, Yoshiyuki Hiki, Keiichi Takiue, Hiroshi
`Morinaga, Masashi Kitagawa, Yohei Maeshima, Kunihiro Fukushima, Kazunori Nishizaki,
`Hirofumi Akagi, Y oshiki N arimatsu, Hisashi N arimatsu, and Hirofumi Makino . . . . . . . . 44 7
`
`Letters to the Editor
`
`Management of pregnancy and vaginal delivery by Cl inhibitor concentrate in two
`hereditary angioedema twins Vincenzo Montinaro and Giuseppe Castellano . . . . . . . 456
`
`Duffy-negative is associated with hemolytic phenotype of sickle cell anemia
`Grazielle Mecabo, Debora Yumi Hayashida, Marily Maria Azevedo-Shimmoto, Perla
`Vicari, Martha Mariana de Almeida Santos Arruda, Jose Orlando Bardin, and Maria
`Stella Figueiredo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
`
`Hematological and clinical relevance of erythroid expression of Duffy Antigen
`Receptor of Chemokine in sickle cell anemia Danitza Nebor, Marie Claude Durpes,
`Danielle Mougenel, Martin Mukisi-Mukaza, Jacques Elion, Marie-Dominique Hardy(cid:173)
`Dessources, and Marc Romana. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
`
`Page 6 of 13
`
`

`

`(contents continued)
`
`Corrigendum
`
`Corrigendum to "D-Dimer and CRP levels are elevated prior to antiretroviral
`treatment in patients who develop IRIS" [Clin. Immunol. 136 (2010) 42-50] Brian
`0. Porter, G. Laissa Ouedraogo, Jessica N. Hodge, Margo A. Smith, Alice Pau, Gregg Roby,
`Richard Kwan, Rachel J. Bishop, Catherine Rehm, JoAnn Mican, and Irini Sereti . . . . . . 462
`
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`Page 7 of 13
`
`

`

`Clinical Immunology (2010) 136, 323- 328
`
`avai l able at www.sc i encedirect.com
`
`Clinical Immunology
`
`ELSEVIER
`
`w ww .elsevier .com/locate / yclim
`
`, . . • ~Clinical
`
`~ ... ._ .. 1 .. ~, lm~unology
`
`~
`
`--
`
`Socrety
`
`RAPID COMMUNICATION
`Subcutaneous infusion of human C1 inhibitor in swine
`Haixiang Jiang a, Hua-Mei Zhang a, Michael M. Frank a, b , C,'k
`
`a Department of Pediatrics, Duke University School of Medicine DUMC 2611 Durham NC 27710 USA
`b
`,
`,
`c Department of MedrCJne, Duke University School of Medicine, DUMC 2611, Durham, NC 27710, USA
`Department of Immunology, Duke University School of Medicine, DUMC 2611, Durham, NC 27710, USA
`
`I
`
`I
`
`1
`
`I
`
`Received 25 February 2010; accepted with revision 6 May 2010
`Available online 8 June 2010
`
`KEYWORDS
`Hereditary angioedema;
`C1 inhibitor;
`Pig;
`Subcutaneous
`
`Abstract Hereditary angioedema afflicts patients with unpredictable episodes of swelling that can
`be life threatening. Treatments approved by the Food and Drug Administration for routine
`prophylaxis includedanazol given orally and the nanofiltered human C1 esterase inhibitor, CINRYZE"",
`which is approved for intravenous administ ration. Approved for the treatment of acute attacks are
`the C1 esterase inhibitor, Berinert®, given intravenously, and the kallikrein inhibitor, KALBITOR®,
`given subcutaneously. C1 inhibitor has generally been non-toxic and neither pro-inflammatory nor
`pro-fibrotic, suggesting that it may be suitable for subcutaneous infusion. The current study used a
`swine model to compare blood levels of human C1 inhibitor following intravenous and subcutaneous
`infusion, and the effect of infusion route on heart and skin pathology. levels of C1 inhibitor achieved
`with SC infusion compared favorably with levels achieved after IV infusion and were relatively more
`stable than those after tV infusion. Neither cardiac nor skin toxicity was observed.
`CO 2010 Elsevier Inc. AU rights reserved.
`
`Introduction
`
`Patients with hereditary angioedema (HAE) fail to synthesize
`normal levels of functional C1 esterase inhibitor (C1 inhibit or)
`protein, which can result in episodic attacks of subcutaneous
`(SQJ and submucosal swelling. These attacks may involve any
`part of the body, with the most frequent episodes related to
`swellings of the skin in any peripheral location, gastrointestinal
`(GI) mucosa, and occasionally the larynx [1]. Attacks are
`unpredictable in location, frequency, and severity, and can be
`life threatening in the presence of laryngeal involvement.
`
`• Corresponding author. Department of Pediatrics, Duke University
`School of Medicine, DUMC 2611, Durham, NC 27710, USA. Fax: +1 919
`668 3750.
`£-mail address: frank007@mc.duke.edu (M.M. Frank).
`
`Currently, the Food and Drug Administration (FDA) has approved
`attenuated androgens danazol and stanazolol [2] and CINRYZP'
`(C1 esterase inhibitor [human]; ViroPharma Inc., Exton, PA) [3]
`for prophylactic use in patients with HAE. Danazol, and other
`impeded androgens, are not useful in all situations and
`increasingly in the United States nanofiltered C1
`inhibitor
`protein is being used for prophylaxis. The C1 inhibitor product,
`Berinert® (CSL Behring LLC, Kankakee, IL) [4], and the
`subcutaneously administered kallikrein inhibitor, Ecallantide
`(KALBITOR®; Dyax Corp., Cambridge, MA) [5], were recently
`approved by the US FDA for the treatment of acute attacks of
`HAE. A bradykinin B2 receptor blocker, lcatibant, is also
`administered subcutaneously and is currently under
`investigation.
`Clinical st udies have suggested that C1 inhibitor has a good
`safety profile and is not pro-inflammatory or pro-fibrotic [3].
`Patients with HAE often have a prodrome, during which time a
`C1
`inhibitor infusion might be started. Intravenously-
`
`1521-6616/$ - see front matter ~ 2010 Elsevier Inc. All rights reserved.
`doi:10.1016/j .ctim.2010.05.001
`
`Page 8 of 13
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`

`

`324
`
`Rapid Communication
`
`administered C1 inhibitor concentrate has been used in Europe
`for decades for treating acute attacks of HAE, and studies have
`reported its efficacy in terminating the attack of swelling,
`while having few side effects [6-9].
`Because C1 inhibitor is administered intravenously, most
`patients must present to an emergency room or doctor's
`office for treatment. The FDA has recently approved the
`intravenous (IV) nanofiltered and pasteurized human C1
`esterase inhibitor (CINRYZE) for self-administration for
`routine prophylaxis in patients with HAE. Thus, for prophy(cid:173)
`lactic purposes, suitable patients can self-administer med(cid:173)
`ication IV at their home instead of arranging for an office
`visit. The present study is a first step in determining whether
`C1
`inhibitor can be safely and effectively administered
`subcutaneously. Compared with the current IV route, a SQ
`route of administration would be more easily accessible to
`patients. In addition, the extent of swelling might be limited
`by injection of medicine earlier during the course of an
`attack. The change from IV to SQ route of administration has
`found great utility in other areas of medicine, such as
`immunoglobulin therapy [1 0,11 ].
`The objective of this study was to compare the blood
`levels of subcutaneously administered human serum protein
`C1 inhibitor with those following IV administration in pigs.
`The swine model was chosen to assess the feasibility of a SQ
`route of administration because of the similarity in size and
`skin physiology to humans. Pigs have long been used in
`preliminary drug studies for a variety of assessments, such as
`drug absorption [12] or effects in wound repair [13]. In an
`acute pig model of infarction, C1
`inhibitor protein was
`administered intravenously to swine undergoing cardiopul(cid:173)
`monary bypass [14]. Study results suggested that intrave(cid:173)
`nously-administered C1 inhibitor was relatively non-toxic and
`effective in decreasing the formation of edema in tissues. In a
`inhibitor
`controlled study of bypass surgery in swine, C1
`reduced the amount of edema formation and in general
`improved cardiac function [15]. In the case of a patient with
`HAE undergoing cardiopulmonary bypass, pre-operative
`infusion with C1 inhibitor was safe and effective in preventing
`episodes of swelling during the perioperative period [16].
`The current study sought to assess whether reasonable
`levels of functional human C1 inhibitor could be achieved in
`swine plasma following SQadministration, to compare plasma
`levels of human C1 inhibitor administered SQ vs IV, to assess
`the time to peak levels and duration of maintenance, and to
`assess the safety of subcutaneously administered human C1
`inhibitor in an acute-use study. The 50 U/kg of human C1
`inhibitor product (CINRYZE) used for administration, which
`would add to the pig's own C1 inhibitor level, was chosen to be
`in excess of the amount usually administered to patients to
`explore the reported possibility of cardiac toxicity. Patient
`doses range from about 10-25 U/kg.
`
`Materials and methods
`
`The source of human C1 inhibitor used in these studies was
`CINRYZE (ViroPharma Incorporated, Exton, PA). This is a highly
`purified protein, which on SDS gels contains only traces of
`other proteins. The 16-23 kg swine used in these studies were
`obtained by a local supplier. Purified human C4 was obtained
`from Complement Technology, Incorporated (Tyler, TX).
`
`In vitro studies
`
`Detection of human C1 inhibitor in pig serum
`A method to determine human C1
`inhibitor levels in the
`presence of pig C1 inhibitor was developed. Sheep anti-human
`C1 inhibitor (10 ~Lg) (The Binding Site, San Diego, CA) was added
`to the wells of an ELISA plate, incubated overnight at 4 oc for
`1 h, washed x3 with 0.1 MpH 9.0 carbonate buffer, and blocked
`with 5% bovine serum albumin (BSA) at room temperature for
`1 h. After washing multiple times, the wells received the
`addition of pig serum alone, PBS alone, or pig serum with added
`human C1
`inhibitor, and the plate was incubated at room
`temperature for 1 h. Binding of the human C1 inhibitor was
`detected with the same sheep, polyclonal anti-C1 inhibitor
`coupled with horse radish peroxidase (HRP). After again washing
`and incubating with the HRP-detecting substrate (OPD, Sigma,
`StLouis, MO), the plates were read at 405 nm optical density.
`
`Functional studies
`Interaction of human C1 inhibitor with pig C1 was addressed
`using a functional C4 assay. Swine C1 was partially purified from
`pig plasma by precipitation following dialysis against acidified
`water. The presence of activated pig C1 was shown by its ability
`to cleave purified human C4 (Complement Technology,
`Incorporated, Tyler TX). A 1:40 dilution of the solubilized
`partially purified swine C1 was mixed with 0.1 ~Lg of purified
`human C4 and to this was added 1 ~Lg of human C1 inhibitor. The
`activation of C4 by C1 was detected functionally by a loss of C4
`hemolytic activity that was blocked by highly purified C1
`inhibitor. C4 functional activity was measured according to the
`method of Gaither and Frank [18].
`
`In vivo study design
`
`Swine were divided into 2 groups of 3 animals and received
`C1 inhibitor 50 U/kg intravenously or subcutaneously.
`
`Surgical procedure
`
`An indwelling IV catheter (Quinton Permacath Dual Lumen
`-36 em) was placed under general isofluorane anesthesia in the
`right external jugular vein to facilitate blood draws and
`30-minute IV infusion.
`A fitted mesh jacket was placed on the animal for the
`protection of the implanted catheter, and was not used as a
`restraint. Prior studies from this group showed that swine
`jackets caused neither discomfort nor distress to the
`animals. Ampicillin 1 g was given intramuscularly prior to
`surgery and oral enrofloxacin (Baytril®; Bayer HealthCare
`LLC, Shawnee Mission, KS) 2.5 mg/kg was given for 7 days. A
`fentanyl patch was placed on the day of surgery and day 3,
`serving as analgesic and sedative crucial in the number of
`blood draws in the first 24 h. One to 2 additional doses of
`ketamine no less than 6 h apart was available during the first
`postoperative 24 h, if needed. Heart rate, respiratory rate,
`and oxygen were monitored throughout the surgery. A
`heparin lock 5 mL to 6 mL flush (200 U/250 NS) was used
`for catheter patency. Pain, distress and discomfort were
`assessed by diminished food consumption, changes in weight
`or behavior, or signs of infection.
`
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`Rapid Communication
`
`Non-surgical procedure
`
`The total amount of C1 inhibitor infused was 50 U/kg. This
`amount was chosen because it is in excess of the 10-25 U/kg
`usually given in man. The 50 U/kg dose was expected to
`approximately double the level of C 1 inhibitor in the pig's blood.
`A 30·minute IV injection time was chosen to mimic IV infusion in
`man. In animals receiving SC infusion, infusions were delivered
`by continuous pump over a 60·minute period. Infusions were
`given on days 0, 3, and 6 to allow comparison to the reported
`human data. The animal's last bleed was on Day9, at which time
`the production of antibody to human C1 inhibitor was expected.
`Six milliliters of blood was obtained at time 0, 1, 3, 6, 9, 12, 24,
`48, and 72 h after each infusion through the external jugular
`catheter. All animals received ketamine 22 mg/kg and acepro·
`mazine 1.1 mg/kg during all infusions, and animals were
`subject to additional doses as needed. At the end of the
`study, animals were sacrificed to study various tissues for
`pathological changes. Animals were sacrificed by the injection
`of ketamine 22 mg/kg SQ plus IV Euthasol® (Virbac Corp., Fort
`Worth, TX) 1 mL/10 lb IV.
`Local tissues were assessed for pathological changes and
`examined for cellular degeneration and fibrosis. Cardiac
`t issues were also examined .
`
`Histopathology studies
`
`Samples collected for pathology included skin/muscle tissue
`from the injection site as well as hearts from all animals.
`Samples were fixed in 10% formalin for histological exami·
`nation. The hearts were bisected, and valves, coronary and
`descending vessels were observed. A section for embedding
`was taken at the following sites in each animal:
`
`1. Left atrioventricular junction, to include a portion of the
`left atrium, left ventricle, coronary artery, and left
`atrioventricular valve
`2. Apex of heart to include the left and right free wall and
`interventricular septum
`3. Right atrioventricular junction, to include a portion of the
`right atrium, right ventricle, coronary artery, and right
`atrioventricular valve
`4. Aorta with aortic ostia
`5. Vena cava
`6. Skin and muscle, if provided.
`
`The histopathology was performed on 2 to 3 seri al sections
`stained wit h hematoxylin and eosin (H & E), as well as 2 to 3
`serial sections stained with Masson's Trichrome. Histologic
`examination was performed by an experienced veterinary
`pathologist with no connection to the study and with no
`connection to the pharmaceutical industry.
`
`Results
`
`325
`
`inhibitor was then detected using a
`inhibi tor. Human C1
`sandwich technique with enzyme-linked sheep anti·C1
`inhibitor antibody. As demonstrated in Figure 1, when
`phosphate-buffered saline (PBS) alone or pig serum alone
`were used in this assay, there was no positive signal. The
`sheep anti-human C1 inhibitor did not detect the pig protein.
`However, incubation of pig serum with added human C1
`inhibitor led to a progressive increase in ELISA signal. This
`increase occurred in a dose-responsive manner, and reached
`a plateau at human C1 inhibitor concentrations of 3~tg/mL.
`These results indicate that this approach can be used to
`specifically and quantitatively detect human C1 inhibitor in
`pig serum. Another experiment sought to determine whether
`the pigs· own C1 inhibitor could partially obscure the human
`C1 inhibitor signal. In this case, serial dilutions of human C1
`inhibitor (starting at 1 ~tg/mL) were incubated with PBS or
`pig serum (Fig. 2). Normal human serum containing C1
`inhibitor was incubated alone as a control. In human serum,
`in the absence of inflammation, ( 1 inhibitor is present at a
`concentration of 0.15 to 0.35 g/L [17]. Results show
`detection of native C1
`inhibitor in human serum, and
`overlapping curves for the detection of human C1 inhibitor
`in both PBS and pig serum, demonstrating that pig serum
`does not obscure the detection of human C 1 inhibitor. Taken
`together, results from these experiments confirm the utility
`and specificity of the ELISA assay to detect human C1
`inhibitor in pig serum.
`
`In vitro functional activity
`
`A functional C4 assay was used to determine whether human
`C1 inhibitor is functionally active in inhibiting activated pig
`C1 in pig serum. Mixture of partially purified pig C1 protein
`with purified human C4 led to complete loss of C4 functional
`activity in a hemolytic assay (Fig. 3, column 2). Human C1
`inhibitor prevented the loss of C4 functional activity induced
`by active pig C1 in a dose-dependent manner (Fig. 3, columns
`4- 7). These results demonstrate that human C1 inhibitor is
`functionally active in the pig and successfully binds and
`
`!
`
`0.6
`
`0.5
`
`ci OA
`0
`E 0.3
`c:
`"' 0
`
`0.2
`
`'<I'
`
`-·& .. PBS
`- • - Pig serum
`_._ Pig serum
`+C11NH
`
`0.1
`
`0.0
`
`:.:.:~..:_:.:~.=-::.~.::.:.::_::_:_:::.::.:.:.:.-:.:.:.!
`
`3
`2
`C1 INH {~1g/ml)
`
`4
`
`In vitro detection of human C1 inhibitor in pig serum
`
`An ELISA assay was developed to speci fically measure human
`C1 inhibitor in pig serum. Sheep anti·human C1
`inhibitor
`antibody was used to capture human but not swine C1
`
`Figure 1 Detection of human C1 inhibitor in pig serum. Pig
`serum plus various concentrations of human C 1 inhibitor were
`incubated on ELISA plates bound with sheep anti-human C1
`inhibitor, blocked, rinsed, and incubated with a second anti·
`human antibody. PBS and pig serum alone were used as controls.
`
`T'4..: - __ ... _,..,;_. , - ~- - .. __ ; .. "'
`
`Page 10 of 13
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`

`326
`
`0.6
`
`0.5
`
`ci 0.4
`0
`E 0.3 .
`c
`"' 0
`"'"
`
`0.2
`
`0.1
`
`0.0
`
`-<>· C1 INH in PBS
`..... C1 INH in pig serum
`·*· NHS
`·-·-····-------· --·-·-------...... ~::~: .....
`...............
`
`2
`3
`Serum dilution
`
`4
`
`Figure 2 Signal of human C1 Inhibitor in pig serum. Serial
`dilutions of human C1 inhibitor (starting at 1 ~tg/ ml) in pig serum or
`PBS were subject to the ELISA assay. Serial dilutions of normal
`human serum (NHS) containing native human C1 were used as
`controls.
`
`inhibits pig C1, which allows us to determine whether high
`plasma C1 activity causes acute toxicity in this acute model.
`
`In vivo protein levels
`
`In the 3 animals receiving IV infusion of human C1 inhibitor,
`blood levels of human C1 inhibitor were at their peak by 1 h
`post infusion (Fig. 4, left column) . Peak levels of C1 inhibitor
`gradually declined over the next 3 days prior to the next
`infu