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`
`2 34 776
`
`May 1988
`
`Volume 81
`---
`Number 5
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`Publishedfor The American Society for Clinical Investigation, Inc. by The Rockefeller University Press (cid:9)
`
`I
`
`Ex. 1092 - Page 1
`
`

`

`LUCILLE P MARKEY
`SCHOLAR AWARDS
`IN BIOMEDICAL SCIENCE
`1989
`
`Nominations are invited for the 1989 Markey Scholar
`Awards which provide substantial support in post-
`doctoral years and in the first five years of faculty
`service. The fifth series of awards will be made
`for the twelve-month academic year beginning
`July 1, 1989.
`A maximum of sixteen annual appointments will be
`made to individuals who hold a Ph.D., D.Sc., M.D./
`Ph.D., M.D., D.V.M. or D.D.S. degrees. Awards will
`provide both a stipend/salary and support for the
`Scholar's research. Nominations must be submitted
`by the institutions in which the nominees will be
`pursuing postdoctoral research training when the
`awards are initiated.
`For further information and nomination procedure,
`please write:
`Scholar Awards in Biomedical Science
`Department 510
`Lucille P Markey Charitable Trust
`3250 Mary Street, Suite 405
`Miami, FL 33133
`
`.•,'"
`
`Deadline for nominations is October 3, 1988
`
`SCIENTIFIC WRITING
`FOR
`GRADUATE STUDENTS
`
`This manual is a "must" for those who would introduce courses of in-
`struction in scientific writing into university graduate schools. The first
`nine chapters provide the essentials for "Writing a Journal Article," and
`the remaining five chapters cover "Related Topics" in scientific com-
`munication.
`
`CONTENTS: I. Clearing Away the Underbrush • 2. The Ground
`Plan • 3. The Master Plan • 4. The First Draft • 5. The First
`Revision: Structural Alterations • 6. Further Revision: Polishing the
`Style • 7. Editing Assignments • 8. The Final Steps • 9. Re-
`sponding to the Editor • 10. Design of Tables and Figures • 11. Prep-
`aration for Writing the Doctoral Thesis • 12. Writing a Research Pro-
`ject Proposal • 13. Oral Presentation of a Scientific Paper
`• 14. Principles and Practices in Searching the Scientific Literature
`
`Paperbound; ISBN: 0-914340-01-8; Published 1968, reprinted 1983;
`Trim size: 6 x 9 inches; 190 pages
`
`Regular Price: $9.75 (10% discount on 10 or more copies delivered to
`one address)
`CBE Member Discount Price: $8.75 (single copy paid by personal
`check)
`
`Terms of Sale: All sales final; no returns.
`Prepayment required; U.S. currency drawn on a U.S. bank.
`Price includes BOOK RATE postage.
`For faster delivery--first class, air mail, or UPS available at additional
`charge (book weight, 11.5 oz).
`Maryland residents, please add 5% sales tax.
`
`P UNIVERSITY 12.1 PENNSYLVANIA
`
`CHAIRMAN
`Department of Human Genetics
`The University of Pennsylvania School of Medicine is
`seeking applicants for the position of Professor and
`Chairman of the Department of Human Genetics.
`Application is invited from individuals with
`outstanding achievement in research, demonstrated
`leadership ability, and appreciation for the
`contirwing role of a department of genetics and
`molecular biology in educating both medical and
`graduate students. We are seeking candidates from a
`broad spectrum of disciplines and departments
`related to genetics.
`The University is an equal opportunity, affirmative
`action employer. Women and minority candidates
`are encouraged to apply.
`Interested applicants should send curricula
`vitae to:
`
`Nicholas A. Kefalides, M.D., Ph.D.
`Chairman, Human Genetics
`Search Committee
`Professor, Department of Medicine
`University City Science Center
`3624 Market Street
`Philadelphia, PA 19104
`
`ECONOMICS
`OF
`SCIENTIFIC JOURNALS
`
`"This publication is an in-depth source for anyone who wants to learn
`about the inner workings of scientific journal publishing. Although it
`deals mostly with society, not-for-profit publishing, it provides the
`reader with the general philosophy and methods used by most
`commercial publishers as well."
`--Society for Scholarly Publishing Letter
`
`CONTENTS: Member Subscriptions • Single Issues and Back
`Volumes • Reprints • Advertising in Scholarly Journals • Page
`Charges • Journal Income: a Multipublisher's View • Editorial
`Operations • Copy Editing • Purchasing Typesetting Separately from
`Printing • Presswork, Binding, and Paper • Distribution and
`Postage • Subscription Fulfillment • Budgeting, Accounting, and
`Financial Planning • Marketing the Scientific Journal • Subject Index
`
`Paperbound; ISBN: 0-914340-03-4; Publication date: December 1982;
`Trim size: 6 x 9 inches; 106 pages
`
`Regular Price: 511.95 (10% discount on 10 or more copies delivered to
`one address)
`CBE Member Discount Price: $10.75 (single copy paid by personal
`check)
`
`Terms of Sale: All sales final; no returns.
`Prepayment required; U.S. currency drawn on a U.S. bank.
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`COUNCIL OF BIOLOGY EDITORS, INC.
`9650 Rockville Pike, Bethesda, MD 20814
`
`COUNCIL OF BIOLOGY EDITORS, INC.
`9650 Rockville Pike, Bethesda, MD 20814
`
`Ex. 1092 - Page 2
`
`

`

`LUCILLE P. MARKEY
`SCHOLAR AWARDS
`IN BIOMEDICAL SCIENCE
`1989
`
`Nominations are invited for the 1989 Markey Scholar
`Awards which provide substantial support in post-
`doctoral years and in the first five years of faculty
`service. The fifth series of awards will be made
`for the twelve-month academic year beginning
`July 1, 1989.
`A maximum of sixteen annual appointments will be
`made to individuals who hold a Ph.D., D.Sc., M.D./
`Ph.D., M.D., D.V.M. or D.D.S. degrees. Awards will
`provide both a stipend/salary and support for the
`Scholar's research. Nominations must be submitted
`by the institutions in which the nominees will be
`pursuing postdoctoral research training when the
`awards are initiated.
`For further information and nomination procedure,
`please write:
`Scholar Awards in Biomedical Science
`Department 510
`Lucille P Markey Charitable Trust
`3250 Mary Street, Suite 405
`Miami, FL 33133
`
`444.
`
`14/1‘,1 a Ls *c*
`
`Deadline for nominations is October 3, 1988
`
`(cid:9) (cid:9) Revision: Structural Alterations • 6. Further Revision: Polishing the
`
`0
`
`P
`
`WaYERSIT1 of PENN571, 41,14
`CHAIRMAN
`Department of Human Genetics
`The University of Pennsylvania School of Medicine is
`seeking applicants for the position of Professor and
`Chairman of the Department of Human Genetics.
`Application is invited from individuals with
`outstanding achievement in research, demonstrated
`leadership ability, and appreciation for the
`continuing role of a department of genetics and
`molecular biology in educating both medical and
`graduate students. We are seeking candidates from a
`broad spectrum of disciplines and departments
`related to genetics.
`The University is an equal opportunity, affirmative'
`action employer. Women and minority candidates
`are encouraged to apply.
`Interested applicants should send curricula
`vitae to:
`
`M;1"1Inuni "fil
`
`Nicholas A. Kefalides, M.D., Ph.D.
`Chairman, Human Genetics
`Search Committee
`Professor, Department of Medicine
`University City Science Center
`3624 Market Street
`Philadelphia, PA 19104
`
`ECONOMICS
`OF
`SCIENTIFIC JOURNALS
`
`"This publication is an in-depth source for anyone who wants to learn
`about the inner workings of scientific journal publishing. Although it
`deals mostly with society, not-for-profit publishing, it provides the
`reader with the general philosophy and methods used by most
`commercial publishers as well."
`--Society for Scholarly Publishing Letter
`
`CONTENTS: Member Subscriptions • Single Issues and Back
`Volumes • Reprints • Advertising in Scholarly Journals • Page
`Charges • Journal Income: a Multipublisher's View • Editorial
`Operations • Copy Editing • Purchasing Typesetting Separately from
`Printing • Presswork, Binding, and Paper • Distribution and
`Postage • Subscription Fulfillment • Budgeting, Accounting, and
`Financial Planning • Marketing the Scientific Journal • Subject Index
`
`Paperbound; ISBN: 0-914340-03-4; Publication date: December 1982;
`Trim size: 6 x 9 inches; 106 pages
`
`Regular Price: $11.95 (10% discount on 10 or more copies delivered to
`one address)
`CBE Member Discount Price: $10.75 (single copy paid by personal
`check)
`
`Terms of Sale: All sales final; no returns.
`Prepayment required; U.S. currency drawn on a U.S. bank.
`Price includes BOOK RATE postage.
`For faster delivery--first class, air mail, or UPS available at additional
`charge (book weight 8.5 oz).
`Maryland residents, please add 5% sales tax.
`
`LecO (cid:9)
`
`'kz;r6". (cid:9)
`
`ScIENTIFIC WRITING
`FOR
`GRADUATE STUDENTS
`
`I:
`
`This manual is a "must" for those who would introduce courses of in-
`struction in scientific writing into university graduate schools. The first
`nine chapters provide the essentials for "Writing a Journal Article," and
`the remaining five chapters cover "Related Topics" in scientific com-
`munication.
`
`CONTENTS: I. Clearing Away the Underbrush • 2. The Ground
`Plan • 3. The Master Plan • 4. The First Draft • 5. The First
`
`Style • 7. Editing Assignments • 8. The Final Steps • 9. Re-
`sPonding to the Editor • 10. Design of Tables and Figures • 11. Prep-
`aration for Writing the Doctoral Thesis • 12. Writing a Research Pro-
`lect Proposal • 13. Oral Presentation of a Scientific Paper
`• 14. Principles and Practices in Searching the Scientific Literature
`
`Paperbound; ISBN: 0-914340-01-8; Published 1968, reprinted 1983;
`Trim size: 6 x 9 inches; 190 pages
`
`Regular Price: $9.75 (10% discount on 10 or more copies delivered to
`one address)
`CBE Member Discount Price: $8.75 (single copy paid by personal
`check)
`
`Terms of Sale: All sales final; no returns.
`Prepayment required; U.S. currency drawn on a U.S. bank.
`Price includes BOOK RATE postage.
`For faster delivery--first class, air mail, or UPS available at additional
`charge (book weight, 11.5 oz).
`Maryland residents, please add 5% sales tax.
`
`COUNCIL OF BIOLOGY EDITORS, INC.
`9650 Rockville Pike, Bethesda, MD 20814
`
`COUNCII, OF BIOLOGY EDITORS, INC.
`9650 Rockville Pike, Bethesda, MD 20814
`
`Ex. 1092 - Page 3
`
`

`

`LUCILLE P MARKEY
`SCHOLAR AWARDS
`IN BIOMEDICAL SCIENCE
`1989
`
`Nominations are invited for the 1989 Markey Scholar
`Awards which provide substantial support in post-
`doctoral years and in the first five years of faculty
`service. The fifth series of awards will be made
`for the twelve-month academic year beginning
`July 1, 1989.
`A maximum of sixteen annual appointments will be
`made to individuals who hold a Ph.D., D.Sc., M.D./
`Ph.D., M.D., D.V.M. or D.D.S. degrees. Awards will
`provide both a stipend/salary and support for the
`Scholar's research. Nominations must be submitted
`by the institutions in which the nominees will be
`pursuing postdoctoral research training when the
`awards are initiated.
`For further information and nomination procedure,
`please write:
`Scholar Awards in Biomedical Science
`Department 510
`Lucille P Markey Charitable Trust
`3250 Mary Street, Suite 405
`Miami, FL 33133
`
`P
`
`P
`
`1.511ERS111E.N.% S11.14
`
`CHAIRMAN
`Department of Human Genetics
`The University of Pennsylvania School of Medicine is
`seeking applicants for the position of Professor and
`Chairman of the Department of Human Genetics.
`Application is invited from individuals with
`outstanding achievement in research, demonstrated
`leadership ability, and appreciation for the
`continuing role of a department of genetics and
`molecular biology in educating both medical and
`graduate students. We are seeking candidates from a
`broad spectrum of disciplines and departments
`related to genetics.
`The University is an equal opportunity, affirmative
`action employer. Women and minority candidates
`are encouraged to apply.
`Interested applicants should send curricula
`vitae to:
`
`Nicholas A. Kefalides, M.D., Ph.D.
`Chairman, Human Genetics
`Search Committee
`Professor, Department of Medicine
`University City Science Center
`3624 Market Street
`Philadelphia, PA 19104
`
`Deadline for nominations is October 3, 1988
`
`SINE h10,0,15
`
`F
`
`..
`
`,
`
`ECONOMICS
`OF
`'6,,„„, -' SCIENTIFIC JOURNALS
`
`SCIENTIFIC WRITING
`FOR
`GRADUATE STUDENTS
`
`This manual is a "must" for those who would introduce courses of in-
`struction in scientific writing into university graduate schools. The first
`nine chapters provide the essentials for "Writing a Journal Article," and
`the remaining five chapters cover "Related Topics" in scientific com-
`munication.
`
`CONTENTS: I. Clearing Away the Underbrush • 2. The Ground
`Plan • 3. The Master Plan • 4. The First Draft • 5. The First
`Revision: Structural Alterations • 6. Further Revision: Polishing the
`Style • 7. Editing Assignments • 8. The Final Steps • 9. Re-
`sponding to the Editor • 10. Design of Tables and Figures • 11. Prep-
`aration for Writing the Doctoral Thesis • 12. Writing a Research Pro-
`ject Proposal • 13. Oral Presentation of a Scientific Paper
`• 14. Principles and Practices in Searching the Scientific Literature
`
`Paperbound; ISBN: 0-914340-01-8; Published 1968, reprinted 1983;
`Trim size: 6 x 9 inches; 190 pages
`
`Regular Price: $9.75 (10% discount on 10 or more copies delivered to
`one address)
`CBE Member Discount Price: $8.75 (single copy paid by personal
`check)
`
`Terms of Sale: All sales final; no returns.
`Prepayment required; U.S. currency drawn on a U.S. bank.
`Price includes BOOK RATE postage.
`For faster delivery—first class, air mail, or UPS available at additional
`charge (book weight, 11.5 oz).
`Maryland residents, please add 5% sales tax.
`
`"This publication is an in-depth source for anyone who wants to learn
`about the inner workings of scientific journal publishing. Although it
`deals mostly with society, not-for-profit publishing, it provides the
`reader with the general philosophy and methods used by most
`commercial publishers as well."
`--Society for Scholarly Puhlishing Letter
`
`CONTENTS: Member Subscriptions • Single Issues and Back
`Volumes • Reprints • Advertising in Scholarly Journals • Page
`Charges • Journal Income: a Multipublisher's View • Editorial
`Operations • Copy Editing • Purchasing Typesetting Separately from
`Printing • Presswork, Binding, and Paper • Distribution and
`Postage • Subscription Fulfillment • Budgeting, Accounting, and
`Financial Planning • Marketing the Scientific Journal • Subject Index
`
`Paperbound; ISBN: 0-914340-03-4; Publication date: December 1982;
`Trim size: 6 x 9 inches; 106 pages
`
`Regular Price: $11.95 (10% discount on 10 or more copies delivered to
`one address)
`CBE Member Discount Price: $10.75 (single copy paid by personal
`check)
`
`Terms of Sale: All sales final; no returns.
`Prepayment required; U.S. currency drawn on a U.S. bank.
`Price includes BOOK RATE postage.
`For faster delivery--first class, air mail, or UPS available at additional
`charge (book weight 8.5 oz).
`Maryland residents, please add 5% sales tax.
`
`COUNCIL OF BIOLOGY EDITORS, INC.
`9650 Rockville Pike, Bethesda, MD 20814
`
`COUNCIL OF BIOLOGY EDITORS, INC.
`9650 Rockville Pike, Bethesda, MD 20814
`
`Ex. 1092 - Page 4
`
`

`

`4)
`
`
`
`44-
`
`0
`
`0
`
`0°
`
`t‘
`o
`
`00
`
`•
`
`0.
`•
`
`Bowel Necrosis Induced by Tumor Necrosis Factor
`in Rats Is Mediated by Platelet-activating Factor
`
`Xiao-ming Sun and Wei Hsueh
`Department of Pathology, Children's Memorial Hospital, Northwestern University, Chicago, Illinois 60614
`
`Abstract
`
`We have developed a rat model of ischemic bowel necrosis
`associated with shock by injection of platelet-activating factor
`(PAF) or a combination of PAF and endotoxin. Recent inves-
`tigations have shown that tumor necrosis factor (TNF) also
`induces shock and necrosis of the gastrointestinal tract. The
`morphological changes of TNF-induced bowel lesions are in-
`distinguishable from those caused by PAF. The mechanism of
`TNF-induced bowel necrosis is unclear. In the present study,
`we have shown that (a) TNF caused PAF production in bowel
`tissue; (b) the effects of TNF and LPS on PAF production in
`the intestine are additive; (c) TNF and LPS are synergistic in
`inducing bowel necrosis; and (d) TNF-induced bowel necrosis
`is due to PAF release and can be prevented by pretreatment
`with PAF antagonists.
`
`Introduction
`
`Tumor necrosis factor (TNF-a)' (1) or cachectin (2), a product
`of macrophages activated by BCG and LPS (1, 2), is being
`increasingly recognized as an important monokine that plays a
`major role in the inflammatory response (3, 4). Besides causing
`necrosis of tumor cells (1) and cachexia (2), recent investiga-
`tions have shown that TNF can induce shock, tissue injury,
`and necrosis of the gastrointestinal tract (5). It has been re-
`ported that TNF caused release of other mediators such as IL 1
`(6, 7), PGs (4, 7), and collagenase (4), and that the actions and
`interactions of these cytokines and mediators may account for
`the pathophysiological changes in endotoxin shock. Although
`it is well-known that ischemic bowel necrosis is often asso-
`ciated with shock or sepsis, the role of TNF in causing bowel
`necrosis is unclear. We have developed a rat model of ischemic
`bowel necrosis by injection of platelet-activating factor (PAF)
`or a combination of PAF and LPS (8), in which the lesions are
`morphologically indistinguishable from those induced by TNF
`(5). It is possible that PAF is a secondary mediator of TNF or
`vice versa. In the present study we have provided evidence to
`suggest that TNF-induced bowel necrosis is due to PAF re-
`lease. We have also shown that the effects of TNF and LPS on
`
`Address reprint requests to Dr. Wei Hsueh, Department of Pathology,
`Children's Memorial Hospital, 2300 Children's Plaza, Chicago, IL
`60614.
`Received for publication 31 July 1987 and in revised form 18 No-
`vember 1987.
`
`1. Abbreviations used in this paper: Hct, hematocrit; PAF, platelet-ac-
`tivating factor; INF, tumor necrosis factor.
`
`J. Clin. Invest.
`© The American Society for Clinical Investigation, Inc.
`0021-9738/88/05/1328/04 $2.00
`Volume 81, May 1988, 1328-1331
`
`1328
`
`X.-m. Sun and W. Hsueh
`
`-re
`
`PAF production in the intestine are additive, and LPS and
`TNF are synergistic in inducing bowel necrosis.
`
`Methods
`
`Young male Sprague-Dawley rats with body weights of 60-80 g were
`used in all experiments. The carotid artery was catheterized for contin-
`uous recording of blood pressure, and the jugular vein was cannulated
`for injections of 0.5 mg/ml recombinant TNF-« (Genentech, Inc.,
`South San Francisco, CA) and/or bacterial endotoxin (LPS, Salmo-
`nella typhosa; Difco, Detroit, MI) and withdrawing blood samples for
`white blood cell count and hematocrit (Hct) determination. The ani-
`mals were divided into six groups: (a) control, (i.e., sham operated), (6)
`0.5 mg/kg TNF, (c) I mg/kg TNF, (d) 200 µg/kg LPS, (e) 0.5 mg/kg
`TNF followed 1 h later by 200 µg/kg LPS, and (f ) the same as e, except
`for pretreatment with 5 mg/kg SRI 63-119 (divided into two doses,
`injected 20 min before TNF and 10 min before LPS). All chemicals
`were slowly injected into the jugular vein.
`At the end of the experiment (3 h after the first injection), the small
`intestine was removed and examined. The length that appeared ne-
`crotic was recorded and expressed as a percentage of the total length.
`Representative sections were taken and fixed in 10% formalin, and
`were later processed for histological examination as previously de-
`scribed (8). The remaining small bowel was washed with saline,
`minced, homogenized in methanol, and stored at -70°C for later PAF
`extraction.
`Extraction and bioassay of PAF in the intestinal tissue were per-
`formed as previously reported (9). Briefly, 106 cpm of VIRAF
`(Amersham Corp., Arlington Heights, IL) was added to the sample for
`calculation of extraction efficiency, and the total lipid was extracted by
`Bligh and Dyer's method (10). The extracted lipid was plated on a thin
`layer plate coated with silica gel G, and developed in solvent system
`chloroform/methanol/water, 65:35:6 (vol/vol/vol). The zone coal-
`grated with standard PAF was scraped, repeatedly eluted with chloro-
`form/methanol/water, 1:2:0.8 (vol/vol/vol) (9), and dried. An aliquot
`was counted for estimation of recovery, and another aliquot was re.
`constituted in PBS containing 5 mg/ml BSA, and assayed for PAF
`activity. PAF was assayed by platelet serotonin release as previously
`described (9). 10 µg/ml SRI 63-119 was used in the assay to confirm
`that the active compound in the solution was PAF (9).
`
`tr
`
`Results and Discussion
`
`As shown in Table I, 200 µg/kg LPS or a low dose (0.5 mg/kg)
`of TNF alone did not cause any gross or microscopic bowel
`lesions. In contrast, all five animals treated with the combina•
`The
`tion of TNF and LPS developed focal bowel necrosis.
`involvement varied from 10 to 60% of the small bowel, and
`microscopically, from mild to moderate degrees of necrosis t
`(Table I). Analysis of the PAF content in the small intestine
`showed that although both TNF (0.5 mg/kg) and LPS induced
`PAF production in the intestinal tissue (0.86±0.31 ng/g Uste
`and 1.28±0.45 ng/g, respectively), the effect of TNF and IP
`was additive: animals treated with the combination of IN?
`and LPS produced 2.08±0.44 ng/g of PAF, significantly MI;
`ferent from the group receiving 0.5 mg/kg TNF alone
`
`7
`
`sl
`
`to
`mi
`jec
`T1,
`an
`all(
`
`<(
`tai:
`(O.(
`
`due
`the
`be
`gro!
`cau
`tisst
`higl
`of T
`
`treat
`LPS
`leuk
`(11,
`dose
`syste
`oper
`mg/1
`mark
`dose
`expel
`blooc
`
`-Co 3
`
`o 2
`
`-o
`0
`'a
`LL
`a.
`
`0
`
`Ex. 1092 - Page 5
`
`

`

`Table 1. Effects of LPS, TNF, and PAF on Development of Bowel Necrosis, and the Prevention by PAF Antagonist
`
`Treatment
`
`No. of
`animals
`
`Gross lesions*
`
`Microscopic necrosis*
`
`None
`TNF (0.5 mg/kg)
`TNF (1 mg/kg)
`TNF (2 mg/kg)
`LPS (200 µg/kg)
`TNF (0.5 mg/kg) + LPS (200 Ag/kg)6
`SRI 63-11911 (5 mg/kg) + TNF (0.5 mg/kg) + LPS (200 µg/kg)
`SRI 63-119 + TNF (I mg/kg)
`PAF (1 µg/kg)
`TNF (0.2 mg/kg) + PAF (1 µg/kg)
`
`5
`6
`3
`3
`7
`5
`3
`3
`3
`4
`
`0
`0
`17±8%
`18±5%**
`0
`35±7%
`0
`0
`0
`35±4%
`
`0
`0
`Mild (2), moderate (1)
`Mild (3)
`
`0
`Mild (1), moderate (4)
`0
`
`0
`0
`Mild (1), moderate (3)
`
`* Gross lesions (violaceous, dark, and lusterless areas). t Microscopic lesions are graded as previously reported (8). Mild: necrosis is confined
`to the tips of mucosal villi. Moderate: partial or total loss of villi, but necrosis is confined to the mucosal layer. Since the microscopic involve-
`ment was not uniform, pathology of the most severely involved areas are presented in the table. § TNF (5.02 x 107 U/mg, 0.5 mg/kg) was in-
`jected intravenously 1 h before the intravenous injection of LPS. H 2.5 mg/kg of SRI 63-119 was injected intravenously 20 min before the
`TNF injection; another 2.5-mg/kg dose was injected 10 min before the LPS injection. ** All three animals died within 2 h. (One died within
`an hour.) Extent of gross lesions was less severe than in animals receiving 1 mg/kg TNF, probably because the shorter survival interval did not
`allow development of advanced lesions.
`
`ever, LPS or TNF by itself can directly activate PMNs and
`promote adhesion between PMNs and endothelial cells (13,
`14), thus enhancing the leukopenic effect of PAF. None of the
`five groups showed any changes in Hct. Although hemocon-
`centration is also a well-known systemic effect of PAF, the
`dose required for Hct changes largely exceeds the amount that
`was endogenously generated in our experiments (12).
`The bowel necrosis induced by injection of the combina-
`tion of 0.5 mg/kg TNF and LPS could be completely pre-
`vented by pretreatment with a PAF antagonist, SRI 63-119 (a
`generous gift from Sandoz Research Institute, East Hanover,
`NJ) (15) (5 mg/kg) (Table I). Other systemic changes, e.g.,
`hypotension and leukopenia, were also significantly amelior-
`ated (Fig. 2). Pretreatment of the animal with SRI 63-119 also
`abolished the development of intestinal necrosis caused by the
`high dose of TNF (1 mg/kg) and ameliorated the hypotensive
`and leukopenic effects of TNF (data not shown).
`PAF is an endogenous mediator (16-18) that causes pro-
`found shock (11, 12) and ischemic bowel necrosis (8) in ani-
`mals. It has been shown to be released in endotoxemia (18).
`Ischemic bowel necrosis is often associated with shock or in-
`fection; thus, it is possible that PAF is a mediator for the
`development of bowel necrosis. In our previous investigations,
`we have shown that 7 µg/kg PAF (8) or a high dose of LPS
`alone (> 10 mg/kg) (unpublished observation) could cause
`ischemic bowel necrosis, and that the effects of LPS and PAF
`are synergistic (8): 80 Atg/kg LPS in combination with 0.35
`µg/kg PAF could induce necrotic lesions in the bowel. These
`observations suggest that PAF, which is released in endotox-
`emia (18), acts synergistically with LPS to induce bowel ne-
`crosis during shock or sepsis. Our present study further dem-
`onstrates that TNF, another proposed mediator of shock (5,
`19), which has also been reported to produce bowel necrosis
`(5), acts synergistically with LPS in causing necrotic lesions in
`the bowel. TNF and LPS also acted additively to induce local
`formation of PAF. Furthermore, the mechanism of action of
`TNF was probably via release of PAF, since pretreatment with
`
`Tumor Necrosis Factor and Platelet-activating Factor (cid:9)
`
`1329
`
`< 0.05). Interestingly, control (sham operated) rats also con-
`tained small amounts of PAF in the intestinal tissue
`(0.08±0.07 ng/g). (Fig. 1).
`The effects of TNF seemed to be dose related. However,
`due to the close proximity of the effective dose (1 mg/kg) and
`the lethal dose (2 mg/kg), a definite dose dependency could not
`be demonstrated. At the high dose (1 mg/kg), TNF induced
`gross and microscopic necrosis of the small bowel (Table I) and
`caused a marked increase in PAF production by the intestinal
`tissue (2.03±0.25 ng/g, Fig. 1). This increase is significantly
`higher than the group receiving the low dose (0.5 mg/kg)
`of TNF.
`Other systemic changes were also observed in animals
`treated with a combination of 0.5 mg/kg TNF and 200 tg/kg
`LPS (Fig. 2). These rats developed profound hypotension and
`leukopenia, both of which are known systemic effects of PAF
`(11, 12). In contrast, the animals treated with LPS or a low
`dose (0.5 mg/kg) of TNF alone showed a minimal decrease in
`systemic BP, not significantly different from that of the sham-
`operated group. However, TNF alone, at a high dose (1
`mg/kg), was effective in inducing mild hypotension and
`marked leukopenia (Fig. 2). Animals receiving LPS or low-
`dose TNF alone showed only mild leukopenia at the end of the
`experimental period. It is not known whether the drop in white
`blood cell count in these groups was due to PAF release. How-
`
`Figure 1. Production of
`PAF by the small intes-
`tine. Abbreviations: C,
`control (sham oper-
`ated); TNF (0.5), 0.5
`mg/kg TNF; TNF (1), 1
`mg/kg TNF; TNF
`+ LPS: 0.5 mg/kg TNF
`+ 200 µg/kg LPS.
`(mean±SEM.)
`
`TNF TNF LPS TNF C
`+LPS
`(1) (cid:9)
`(0.5)
`
`3
`
`2
`
`0
`
`PAF production (ng/g)
`
`d
`
`b)
`
`pt
`
`ds
`
`all
`ie-
`[h.
`nd
`ie
`Re,
`
`AF
`
`AF
`for
`,by
`his
`,ent
`
`)ro•
`not
`re.
`'AF
`only
`Ira
`
`)wel
`Tina
`The
`and
`rosin
`sting
`used
`issue
`
`LPS,
`TNF
`), difb
`lf
`
`Ex. 1092 - Page 6
`
`(cid:9)
`(cid:9)
`

`

`these responses were slow; there was sufficient time for ab-
`sorption of LPS from the intestine (after an initial minor mu.
`cosal injury), which may have acted synergistically with TNF,
`It is interesting to note that such a small amount of PAF
`produced in vivo (see Table I) was sufficient to cause necrotic
`lesions in the bowel, whereas a relatively large amount of exog-
`enous PAF was needed to produce similar lesions in our pre-
`vious experiments (8). This is probably, at least in part, due to
`a synergism of TNF and PAF. We have observed gross necrotic
`lesions in the bowel (confirmed by microscopic examination)
`in all four rats treated with 0.2 mg/kg of TNF and 1 µg/kg of
`PAF (Table I). This dose of PAF is less than one-fifth of what
`was needed to produce bowel necrosis, if administered exoge-
`nously.
`There seems to be a great variation in the levels of endoge-
`nous TNF production and responses to exogenous TNF ad-
`ministration among different species. In humans, the level of
`endogenous TNF production is very low (< 30 ng/ml) (19); the
`200 µg/m2) (20) in clinical trials were much
`doses used (cid:9)
`lower than those used in our experiments. In this study a bolus
`dose of TNF was used. Considering the continuous production
`and the rapid degradation of TNF in vivo (21), the dose used
`could be comparable to what was detected in vivo in rodents
`(22) and rabbits (23). Thus, the present study seems relevant to
`the pathophysiology of endotoxin shock and ischemic bowel
`necrosis. It has been suggested that TNF is an initiating factor
`of shock elicited by LPS (24, 25). Because of its activating
`effects on inflammatory cells (14) and endothelial cells (26),
`and its action in releasing other mediators such as IL 1 (6, 7),
`PGs (4, 7), and collagenase (4), TNF is considered a major
`cytokine that may initiate a cascade of inflammatory and co-
`agulative events in the pathogenesis of septic shock and tissue
`injury. Since LPS administration causes production of TNF
`(21) and PAF (18), the synergistic effects between LPS and
`PAF, and between TNF and LPS, may play an important role
`in the development of irreversible shock and bowel necrosis.
`
`tl
`1
`
`1
`d
`
`of
`er
`
`le.
`er
`
`ex
`
`R.
`by
`la)
`
`Eg
`ph
`me
`
`rial
`
`Ga.
`net
`
`son
`ind
`Phc
`
`C
`
`TNF
`
`LPS
`
`Acknowledgments
`
`120
`
`E
`E 100
`a)
`
`80
`
`N
`
`60
`
`40
`
`20
`
`0
`
`90
`
`60
`
`30
`
`0
`
`40
`
`20
`
`0
`
`2
`
`WBC Count (x100)
`
`Hematocrit
`
`-30 0 30 60 90 120 150 180
`
`Time (min)
`
`Figure 2. Effects of TNF and LPS on (A) mean arterial pressure, (B)
`WBC count, (C) Hct, and prevention by SRI 63-119. o, control, i.e.,
`sham operated; A, 0.5 mg/kg TNF injected at time 0; A, 1 mg/kg
`TNF injected at time 0; o, 200 µg/kg LPS injected at time 0; e, 0.5
`mg/kg TNF injected at time 0 + LPS injected at 60 min; and 0, 5
`mg/kg SRI 63-119 divided into two doses, injected 20 min before
`TNF and 10 min before LPS + 0.5 mg/kg TNF, injected at time 0
`+ LPS injected at 60 min.
`
`the PAF antagonist totally prevented bowel necrosis caused by
`TNF and LPS.
`It remains to be demonstrated whether TNF, without in-
`teraction with LPS, can produce shock and bowel necrosis. It is
`not clear if the TNF preparation used by Tracey et al. (5) was
`uncontaminated by LPS. Although the TNF preparation in
`the present study is relatively free of LPS (LPS < 0.125
`EU/ml), and we have observed that injection of high doses of
`TNF alone resulted in hypotension and intestinal necrosis,
`
`1330
`
`X.-m. Sun and W. Hsueh
`
`We are grateful to Dr. H. M. Shepard at Genentech, Inc. for providing
`us with the recombinant human tumor necrosis factor-alpha, and to
`Dr. R. Saunders at Sandoz Research Institute for giving us SRI 63.119.
`This work is partly supported by National Institutes of Health grant
`DK-34574.
`
`References
`
`1. Carswell, E. A., L. J. Old, R. L. Kassel, S. Green, N. Fiore, alla
`Williamson. 1975. An endotoxin-induced serum factor that causes
`necrosis of tumors. Proc. Natl. Acad. Sci. USA. 72:3666-3670.
`2. Beutler, B., J. Mahoney, N. Le Trang, P. Pekala, and A. Cerarni•
`1985. Purification of cachectin, a lipoprotein lipase-suppressing hon
`mone secreted by endotoxin-induced raw 264.7 cells. J. Exp. Med.
`161:984-995.
`tumour ne.
`
`3 320:5. Beulter,
`
`84-588 B., and A. Cerami. 1986. Cachectin and .
`crosis factor as two sides of the same biological coin. Nature (1014
`l
`4. Dayer, J.-M., B. Beutler, and A. Cerami. 1985. Cachectinitoo
`
`necrosis factor stimulates collagenase and prostaglandin E2 production
`
`by human synovial cells and dermal fibroblasts. J. ExP• Me. d'
`162:2163-2168.
`5. Tracey, K. J., B. Beutler, S. F. Lowry, (cid:9)
`Wolpe, I. W. Milsark, R. J. Hariri, T. J. Fahey III,
`
`(cid:9) rZrYenwieealliall,er.I'DS1
`
`Ex. 1092 - Page 7
`
`(cid:9)
`

`

`ent time for ab
`nitial minor nni•
`lically with TNF.
`amount of PM
`to cause necrotic
`amount of exog
`sions in our pro
`1st in part, duels
`'ed gross necrotic
`pic examination
`F and 1 irgikg o1
`one-fifth of whi:
`ministered exogt
`
`levels of endogo
`genous TN