College of American Pathologists
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  Clinical Abstracts





April 2010

Michael Bissell, MD, PhD, MPH

Inter-alpha-trypsin inhibitor in complement-mediated lung injury Inter-alpha-trypsin inhibitor in complement-mediated lung injury

Tissue damage through an inadequately controlled inflammatory response has been increasingly recognized in the pathogenesis of acute respiratory distress syndrome, multiorgan dysfunction syndrome, and autoimmune diseases. Complement activation is an important element of inflammatory processes. In the complement activation cascade, complement factors are initially activated by immune complexes or microbial elements, leading to a succession of proteolytic cleavage of complement proteins. Complement inhibitors are essential to avoid harmful excess activation. Inter-α-trypsin inhibitor (ITI) is a complex molecule consisting of an L chain protease inhibitor named bikunin or urinary trypsin inhibitor (UTI) and two H chains containing von Willebrand type A (vWA) domains. ITI is assembled in the liver and released in the circulation and can be found in fairly high concentrations in mammalian plasma. Little is known about its exact function in an organism, other than its ability to bind to hyaluronic acid in the extracellular matrix through its H chains. The role of ITI in sepsis was investigated in animal and human studies. ITI levels in the serum of septic patients were significantly lower than levels in healthy controls. Among septic patients, ITI levels were also inversely correlated with physiologic scores and mortality. ITI given prophylactically or therapeutically in animal models of sepsis significantly improved survival, but the mechanism of action remains elusive. Complement and ITI are abundantly present in serum. The authors hypothesized that complement factors and ITI may be able to interact with each other because catalytic complement proteins are serine proteases that activate downstream complement factors through proteolytic cleavage. Further-more, complement factors contain integrin-like sites that are potential binding targets to the vWA domain in the ITI H chains. In contrast, the L chain of ITI is a serine protease inhibitor. In addition, ITI inhibits furin, a serine protease that can cleave complement. Therefore, the authors hypothesized that ITI can inhibit complement activation and attenuate complement-dependent processes. They tested this hypothesis with in vitro complement activation assays as well as in vivo in a murine model of complement-dependent lung injury. They found that ITI inhibited complement activation through the classical and alternative pathways, inhibited complement-dependent phagocytosis in vitro, and reduced complement-dependent lung injury in vivo. The authors concluded that this novel function of ITI provides a mechanistic explanation for its observed salutary effects in sepsis and opens new possibilities for its use as a treatment for inflammatory dis-eases.

Garantziotis S, Hollingsworth JW, Ghanayem RB, et al. Inter-α-trypsin inhibitor attenuates complement activation and complement-induced lung injury. J Immunol. 2007;179:4187–4192.

Correspondence: Dr. Stavros Garantziotis at

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Plasmin as an agent in progressive renal disease Plasmin as an agent in progressive renal disease

Progressive renal disease is characterized by tubular atrophy and interstitial fibrosis. Interstitial myofibroblasts that accumulate during renal fibrogenesis seem to be the primary source of the extracellular matrix (ECM) proteins that accumulate and ultimately destroy nephron integrity and function. The primary origin of interstitial myofibroblasts is still being debated. It may originate in resident interstitial fibroblasts that activate and proliferate; tubular epithelia that have undergone epithelial-to-mesenchymal transition (EMT) into motile, fibroblast-like cells and migrate into the interstitium; and stem cells that are recruited to the interstitium. Phenotypic features of renal tubular EMT include decreased expression of the cell adhesion receptor E-cadherin and de novo expression of α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1). The molecular pathways that control tubular cell EMT and their interstitial migration during progressive kidney disease remain incompletely delineated. Transforming growth factor-β TGF-β and tissue-type plasminogen activator, a key component of the Plg activation system, have been shown to play important roles. The active serine protease plasmin is generated from the zymogen Plg, a circulating protein that is synthesized in the liver and often extravasates into extravascular sites during inflammation. The zymogen Plg is activated by tissue-type and urokinase-type Plg activators (uPA). Plasmin activity has been associated with a variety of processes that are involved in tissue remodeling or cell migration, or both, including embryonic development, macrophage recruitment, wound healing, angiogenesis, atherosclerosis, and tumor cell invasion. In addition to its fibrinolytic function, plasmin degrades a number of nonfibrin substrates to elicit diverse effects that may modulate the severity of fibrosis. These include its direct proteolytic activity for certain extracellular matrix (ECM) proteins, such as fibronectin and laminin (but not elastin or native helical collagen), and its ability to activate latent matrix metalloproteinases and latent TGF-β. By degrading ECM, plasmin may also liberate sequestered growth factors. Direct degradation of E-cadherin in vitro by plasmin has also been reported. Given its pleiotropic effects in vitro, it is unclear whether the predominant effect of Plg in vivo promotes or inhibits the severity of tissue fibrosis. It has long been speculated that tissue plasmin activity would attenuate fibrosis as a result of its matrix-degrading effects. The authors conducted a study to investigate the role of Plg in renal fibrosis using in vivo and in vitro approaches. For the study, Plg-deficient (Plg–/–) and wild-type (Plg+/+) C57BL/6 mice were subjected to unilateral ureteral obstruction or sham surgery (n=8/group; sham, days 3, 7, 14, and 21). Plg deficiency was confirmed by the absence of Plg mRNA, protein, and plasmin activity. After 21 days of unilateral ureteral obstruction, total kidney collagen was significantly reduced by 35 percent in the Plg–/– mice. EMT was also significantly reduced in Plg–/– mice, by 76 percent and 50 percent, respectively. Attenuation of EMT and fibrosis severity in the Plg–/– mice was associated with significantly lower levels of phosphorylated extracellular signal-regulated kinase (ERK) and active TGF-β. In vitro, the addition of plasmin (20 µg/mL) to cultures of murine tubular epithelial cells initiated ERK phosphorylation within minutes, followed by phenotypic transition to fibroblast-specific protein-1+, α-smooth muscle actin+, fibronectin-producing fibro-blast-like cells. Plasmin-induced ERK activation and EMT were significantly blocked in vitro by the protease-activated receptor-1 (PAR-1) silencing RNA; by pepducin, a specific anti-PAR-1 signaling peptide; and by the ERK kinase inhibitor UO126. Plasmin-induced ERK phosphorylation was enhanced in PAR-1-overexpressing tubular cells. These findings support important profibrotic roles for plasmin that include PAR-1-dependent ERK signaling and EMT induction.

Zhang G, Kernan KA, Collins SJ, et al. Plasmin(ogen) promotes renal interstitial fibrosis by promoting epithelial-to-mesenchymal transition: role of plasmin-activated signals. J Am Soc Nephrol. 2007;18:846–859.

Correspondence: Dr. Allison A. Eddy at

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Lack of antifibrotic activity in endogenous urokinase during progressive renal injury Lack of antifibrotic activity in endogenous urokinase during progressive renal injury

Interstitial fibrosis is a universal feature of progressive kidney disease. Urokinase-type plasminogen activator (uPA) is thought to participate because it is produced predominantly in the kidney; its inhibitor, plasminogen activator inhibitor-1 (PAI-1), is a strong promoter of interstitial fibrosis, whereas its receptor, urokinase plasminogen activator receptor (uPAR), attenuates renal fibrosis; it reduces fibrosis in the liver and lung; and it can activate hepatocyte growth factor (HGF), a potent antifibrotic growth factor. The authors tested the hypothesis that endogenous uPA reduces the severity of fibrosis by investigating the uni-lateral ureteral obstruction (UUO) model in wild-type (WT) and uPA–/– mice. They measured several out-comes, including renal collagen three to 21 days after UUO, macrophage accumulation (F4/80 Western blotting), interstitial myofibroblast density (α-smooth muscle actin immunostaining), and tubular injury (E-cadherin and Ksp-cadherin Western blotting). None of these measures differed significantly between WT and uPA–/– mice. The authors found that uPA genetic deficiency was not associated with compensatory changes in renal uPAR, mRNA levels, PAI-1 protein levels, or tissue plasminogen activator activity levels after UUO. Despite the ability of uPA to activate latent HGF, immunoblotting failed to detect significant differences in levels of the active HGF α-chain and phosphorylated cMET (the activated HGF receptor) between the WT and uPA–/– groups. These findings suggest that the profibrotic actions of PA1-1 are independent of uPA and that an alternative pathway must activate HGF in the kidney. Finally, these results highlight a significant organ-specific difference in basic fibrogenic pathways, as enhanced uPA activity has been reported to attenuate pulmonary and hepatic fibrosis.

Yamaguchi I, Lopez-Guisa JM, Cai X, et al. Endogenous urokinase lacks antifibrotic activity during progressive renal injury. Am J Physiol Renal Physiol. 2007;293:F12–F19.

Correspondence: Dr. Allison A. Eddy at allison.eddy@seattle

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Molecular typing of <em>Neisseria meningitidis</em> Rheumatoid arthritis shared epitope triggers immune signaling via calreticulin

Genetic associations with particular class I or class II major histocompatibility complex alleles have been noted in many autoimmune diseases. Based on the role of major histocompatibility complex (MHC) mole-cules in thymic selection and Ag presentation, it has been postulated that the mechanism underlying these associations involves T-cell repertoire selection or presentation of self-Ags, or both. Evidence to support this paradigm exists in some diseases, but in other cases, the basis for MHC disease association remains unclear. For example, more than 90 percent of rheumatoid arthritis patients carry HLA-DRB1 alleles, which encode a 5-aa sequence motif, known as the shared epitope, in position 70–74 in the β1 domain of the HLA-DR molecule. The mechanistic basis of SE-RA association is unknown. In previous studies, the authors found that the shared epitope acts as a ligand that activates nitric oxidemediated prooxidative signaling in opposite cells. For example, cells carrying shared epitopepositive HLA-DRB1 alleles displayed increased constitutive nitric oxide production, which resulted in higher levels of reactive oxygen species and higher suscep-tibility to oxidative DNA damage. The signaling aberration could be transferred to shared epitope-negative cells by cDNA transfection, indicating that shared epitope-associated signaling activity is not due to linkage disequilibrium with another gene. Importantly, recombinant proteins, HLA-DR tetramers, and short synthetic peptides containing the shared epitope motif could mimic the signaling effect of the native shared epitope-positive DR molecule. To better understand the mechanisms involved in shared epitope-triggered signaling, the authors conducted a study to identify the shared epitope-binding receptor. Using peptide affinity chromatography, N-terminal amino acid sequencing, surface plasmon resonance (SPR), time-resolved fluorescence resonance energy transfer (TR-FRET), and cell-binding assays, they determined that cell surface calreticulin (CRT) is a shared epitope-binding molecule. The involvement of CRT in shared epitope-triggered signaling was confirmed by demonstrating an inhibitory effect with anti-CRT Abs and with CRT-targeted antisense or small-interfering RNA oligonucleotide transfection. Embryonic fibroblasts from CRT-deficient mice could not respond to the shared epitope ligand but regained signaling activity when CRT was added back to the cell culture. The authors concluded that the data indicate that cell-surface CRT plays a critical role in shared epitope-triggered signal transduction.

Ling S, Pi X, Holoshitz J. The rheumatoid arthritis shared epitope triggers innate immune signaling via cell surface calreticulin. J Immu-nol. 2007;179:6359–6367.

Correspondence: Dr. Joseph Holoshitz at

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Analysis of insulin-like growth factor-1 and mammography in four geographic locations Analysis of insulin-like growth factor-1 and mammography in four geographic locations

Breast cancer incidence differs more than four-fold between women in Japan and the United States, but several generations after migration, Japanese-Americans experience a level of risk similar to that of Cauca-sians. Despite inconsistent evidence that dietary factors play a role, nutritional changes are thought to con-tribute to the gradual increase in breast cancer risk among migrants. Insulin-like growth factor-1 (IGF-1) has been proposed as a link between diet and breast cancer risk. Despite some inconsistencies, evidence supports a positive association between premenopausal breast cancer risk and circulating IGF-1 levels measured as absolute concentrations or relative to IGFBP-3. In addition, a modestly elevated breast cancer risk was observed for pre- and postmenopausal women with higher levels of prolactin. Because of its independent association with breast cancer, breast density has been examined in relation to IGF-1 and prolactin. The authors examined the hypothesis that breast density is associated with serum levels of IGF-1, IGFBP-3, and prolactin among women of Caucasian, Japanese, Latina, and native Hawaiian ancestry residing in Ha-waii, Arizona, Japan, and Norway. In a previous analysis of this pooled data set, the authors indicated that mammographic density differed significantly by ethnicity (P<.001), whereas location appeared to have only a weak relation to breast density (P=.19) after adjusting for covariates. The pooled data set included 1,327 pre- and postmenopausal women: Caucasian women from Norway, Arizona, and Hawaii; Japanese women from Hawaii and Japan; Latina women from Arizona; and native Hawaiians from Hawaii. Serum samples were assayed for IGF-1, IGFBP-3, and prolactin levels using enzyme-linked immunosorbent assays. Mammographic density was quantified using a computer-assisted density method. After stratifying the study population by menopausal status, multiple regression models estimated the relation between serum an-alytes and breast density. The authors found that all serum analytes, except prolactin, among postmeno-pausal women differed significantly by location and ethnic group. Among premenopausal women, IGF-1 levels and the molar ratio were highest in Hawaii, intermediate in Japan, and lowest in Arizona. For IGFBP-3, the order was reversed. Among postmenopausal women, Norwegian women had the highest IGF-1 levels and women in Arizona had the lowest, while women in Japan and Hawaii had intermediate levels. No significant relationship was noted between percent density and IGF-1 or prolactin levels among pre- and postmenopausal women. The authors concluded that the significant differences in IGF-1 levels by location but not ethnicity suggest that environmental factors influence IGF-1 levels, whereas percent breast density varies more based on ethnic background than on location. Based on this analysis, the influence of circulating levels of IGF-1, IGFBP-3, and prolactin on percent breast density appears to be very small.

Maskarinec G, Takata Y, Chen Z, et al. IGF-I and mammographic density in four geographic locations: a pooled analysis. Int J Cancer. 2007;121:1786–1792.

Correspondence: Gertraud Maskarinec at

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SiRNA nanoparticles and cancer imaging SiRNA nanoparticles and cancer imaging

Targeted nanoparticles are a promising class of new experimental medicines that have the potential to provide increased efficacy and lower toxicity relative to conventional therapeutics. Through the enhanced permeability and retention (EPR) effect, macromolecular therapeutics tend to accumulate within tumors after systemic delivery. As such, nanoparticles represent an approach for delivering large drug payloads to tumors. However, because many drugs require cellular internalization for efficacy, accumulation within the tumor microenvironment by the EPR effect may not correlate with therapeutic outcome. SiRNA molecules are short double-stranded nucleic acid molecules that can act as mediators of RNAi within the cytoplasm of a cell. Therapeutic application of siRNAs involves delivering them into the target cells of interest because naked siRNAs do not enter most cell types on their own. To address the challenge of nucleic acid delivery, a variety of methodologies have been developed with varying success, including covalent attachment of cholesterol, interaction with antibody-protamine fusion proteins, liposome encapsulation, or nanoparticle formation with cationic lipids or polymers. Targeted delivery approaches involve using cell-specific targeting ligands that can bind to surface receptors on cells. The authors conducted a study in which they used positron emission tomography (PET) and bioluminescent imaging to quantify the in vivo biodistribution and function of nanoparticles formed with cyclodextrin-containing polycations and siRNA. Conjugation of 1,4,7,10-tetra-azacyclo-dodecane-1,4,7,10-tetraacetic acid to the 5’ end of the siRNA molecules allowed la-beling with 64Cu for PET imaging. Bioluminescent imaging of mice bearing luciferase-expressing Neuro2As.c. tumors before and after PET imaging allowed functional efficacy to be correlated with biodis-tribution data. Although nontargeted and transferrin-targeted siRNA nanoparticles exhibited similar bio-distribution and tumor localization by PET, transferrin-targeted siRNA nanoparticles reduced tumor luciferase activity by approximately 50 percent relative to nontargeted siRNA nanoparticles one day after injection. Compartmental modeling was used to show that the primary advantage of targeted nanoparticles is associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization. Therefore, optimizing internalization may be the key to developing nanoparticle-based targeted therapeutics.

Bartlett DW, Su H, Hildebrandt IJ, et al. Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging. Proc Natl Acad Sci USA. 2007;104:15549–15554.

Correspondence: Mark E. Davis at

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Clinical pathology abstracts editor: Michael Bissell, MD, PhD, MPH, professor, Department of Pathology, Ohio State University, Columbus.