College of American Pathologists
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  Clinical Abstracts





March 2009

Michael Bissell, MD, PhD, MPH

Urinary podocytes in preeclampsia
Carotenoids and cardiac risk
Tay-Sachs disease risk in French Canadians
Rheumatoid Performance of assays for measuring citrullinated protein/peptide
Effects of nitric oxide on production of Escherichia coli toxin
Use of bacteria to detect antimicrobial peptides
MMP 3 as a biomarker in ankylosing spondylitis
Tracking the evolution of multidrug-resistant S. aureus

Urinary podocytes in preeclampsia Urinary podocytes in preeclampsia

Studies have supported the concept of podocyte (glomerular epithelial cell) damage and loss as proteinuria develops in patients with glom­erular disease. Research indicates that the urinary excretion of podocytes, called podocyturia, occurs during active disease only, whereas proteinuria is present during active and chronic phases of glomerular damage. Given that kidney injury is acute in pre­eclampsia, the authors postulated that urinary podocyte loss occurs concurrently with proteinuria and can be correlated with its severity. The authors conducted a study to test the hypothesis that urinary excretion of viable podocytes, which are identified and quantified on the basis of their expressions of podocyte-specific proteins (podocalyxin, podocin, nephrin, and synaptopodin), occurs in pregnant women with clinically confirmed preeclampsia. The authors also sought to correlate urine podocyte counts with degree of proteinuria. Several lines of evidence have suggested that preeclampsia is associated with elevated levels of fms-like tyrosine kinase receptor-1 (sFlt-1), the soluble receptor for vascular endothelial growth factor, which may bind, neutralize, and therefore decrease free levels of vascular endothelial growth factor and placental growth factor (PlGF), both of which are required for active fetal and placen­tal angiogenesis. More recently, elevated levels of circulating soluble endoglin were reported to interfere with transforming growth factor-®1 signaling and nitric oxide-mediated vasodilation. The authors also compared the test characteristics of podocyturia, sFlt-1, PlGF, and endoglin in patients with a clinically confirmed diagnosis of preeclampsia. They recruited 67 pregnant women at the Mayo Clinic between March and October 2006 who were no more than 24 hours from delivery. Preeclampsia was present in 33 patients, and HELLP (hemolysis, elevated liver enzyme levels, and low platelet count) syndrome was present in 11 patients. Twenty-three normotensive patients served as control subjects. Serum sFlt-1, free PlGF, and soluble endoglin levels were determined from blood samples drawn no more than 24 hours before delivery. Urine samples were collected and tested for podocyturia in a subset of 31 pregnant women (15 women with pre­eclampsia and 16 normotensive, nonproteinuric control subjects). Also serving as controls were 11 patients with hypertension or proteinuria but without clinical evidence of pre­eclampsia. Sediments from random urine samples were incubated with antibodies to one of four podocytespecific proteins: podocalyxin, podocin, nephrin, or synaptopodin. Nucleated, positive-staining cells were considered to be podocytes. The authors also measured free urinary PlGF. The authors found that of 31 patients with urinary measures of podocyturia, podocin-positive cells appeared in the urine of all 15 patients with preeclampsia or HELLP and in none of the 16 normotensive control subjects. The sensitivity and specificity of podocyturia for the diagnosis of preeclampsia were 100 percent. The measurement of podocyturia had lower sensitivity and specificity than did measurements of podocin. Serum sFlt-1 levels were significantly higher in women with preeclampsia or HELLP than in normotensive pregnant controls. Serum soluble endoglin levels were also significantly higher in women with preeclampsia or HELLP than in normotensive pregnant control subjects. Serum-free PlGF levels were lower in women with pre­eclampsia or HELLP than in normotensive pregnant control subjects. The authors’ findings suggest that podocyturia is a highly sensitive and specific marker of pre­eclampsia. The signaling pathways behind podocyte detachment are poorly understood in proteinuric disease in general and pre­eclampsia in particular.

Garovic VD, Wagner SJ, Turner ST, et al. Urinary podocyte excretion as a marker for preeclampsia. Am J Obstet Gynecol. 2007;196:320.e1–320.e7.

Reprints available through download at

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Carotenoids and cardiac risk Carotenoids and cardiac risk

Several epidemiologic studies have proposed an inverse relationship between serum carotenoids and cardiovascular disease. Inflammation, oxidative stress, and endothelial dysfunction are known to be associated with atherosclerosis and cardiovascular diseases. Although several cross-sectional studies reported that serum carotenoids are related inversely to these markers of the atherosclerotic pathway, the authors do not know of any studies investigating whether carotenoid concentrations predict the future values of these markers. The authors conducted cross-sectional and longitudinal analyses to identify whether circulating carotenoids are associated with markers of oxidative stress, inflammation, and endothelial dysfunction. They also investigated whether the relationship between serum carotenoid concentrations and these variables differed according to smoking status, following the authors’ prior findings of an interaction between smoking and circulating carotenoids and several other variables. For the study, black and white men and women in the Coronary Artery Risk Development in Young Adults study, ages 18 to 30 years at recruitment (1985–1986) and from four U.S. cities, were investigated over 15 years. The study, which involved 2,048 to 4,580 participants, analyzed the sum of serum -carotene, ®-carotene, zeaxanthin/ lutein, and ®-cryptoxanthin concentrations and lycopene at year zero and year seven. The year-zero sum of four carotenoids was inversely associated (all, P<.05) with year-zero leukocyte count (slope per sum carotenoid SD, –0.17), year-seven fibrinogen (slope, –0.10), year-seven and year-15 C-reactive protein (slope, –0.12 and –0.09), and year-15 F2-isoprostanes (slope, –13), soluble P-selectin (slope, –0.48), and soluble intercellular adhesion molecule-1 (sICAM1; slope, –5.1). Leukocyte counts and sICAM1 and F2-isopro­stane concentrations had stronger associations in smokers than in nonsmokers, and sICAM1 concentrations were higher in the highest carotenoid quartile in smokers than in the lowest carotenoid quartile in nonsmokers. Superoxide dismutase was positively associated with the sum of the four carotenoids (slope, 0.12; P<.01). Lycopene was inversely associated only with sICAM1. The year-seven caro­tenoid associations with these markers primarily were similar to those at year zero. The authors concluded that circulating serum caro­tenoids were associated, some interactively with smoking, in apparently beneficial directions with markers of inflammation, oxidative stress, and endothelial dysfunction.

Hozawa A, Jacobs DR, Steffes MW, et al. Relationships of circulating carotenoid concentrations with several markers of inflammation, oxidative stress, and endothelial dysfunction: the Coronary Artery Risk Development in Young Adults (CARDIA)/Young Adult Longitudinal Trends in Antioxidants (YALTA) Study. Clin Chem. 2007;53:447–455.

Correspondence: David R. Jacobs at

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Tay-Sachs disease risk in French Canadians Tay-Sachs disease risk in French Canadians

Tay-Sachs disease is an autosomal recessive lysosomal lipid storage disorder caused by deficiency of the lysosomal enzyme ®-hexosaminidase A (HEXA). The HEXA ­isoenzyme comprises one ®-subunit encoded by HEXA and one ®-subunit encoded by HEXB. Two ®-subunits can combine to form a second isoenzyme with dif­ferent but overlapping substrate speci­ficities, called ®-hexosaminidase B (HEXB). Heterozygotes for deleterious mutations in HEXA and HEXB can be identified with an enzyme-based activity assay. To identify Tay-Sachs disease (TSD) carriers using an enzymatic assay, the combined activities of HEXA and HEXB, as well as the activity of HEXB after heat inactivation of HEXA, are determined. Heterozygotes for TSD have reduced HEXA activity (observed as a reduced percentage of HEXA relative to the total ®-hexosaminidase activities). This enzyme-based assay is used routinely by clinical laboratories that screen for heterozygotes for TSD. Enzyme-based screening programs for HEXA deficiency were initiated in the 1970s for the Ashkenazi Jewish, and later, French-Canadian populations, both of which were recognized to have an increased incidence of TSD. Elucidation of the molecular basis of TSD has made apparent an alternative approach for heterozygosity screening in some populations. In persons of Ashkenazi Jewish background, DNA-based carrier screening is possible because three mutations account for approximately 95 percent to 98 percent of obligate TSD carriers. In contrast, the molecular basis of TSD in most non-Jewish populations is highly heterogeneous, prohibiting DNA-based screening. Using a fluorescence-based assay for ®-hexosaminidase activity, the authors determined the carrier frequencies for TSD in 2,783 Franco-Americans. DNA analysis was used to identify mutations causing enzyme deficiency in TSD carriers. The authors determined the enzyme-defined carrier frequency for TSD at 1:65 (95 percent confidence interval [CI], 1:49 to 1:90). DNA-based analysis of 24 of the enzyme-defined carriers revealed 21 with sequence changes: nine disease-causing, four benign, and eight of unknown significance. Six of the unknowns were identified as c.748G>A p.G250S, a mutation shown by expression analysis to behave similarly to the previously described c.805G>A p.G269S adult-onset TSD mutation. This putative adult-onset TSD c.748G>A p.G250S mutation has a population frequency similar to the common 7.6 kb deletion mutation that occurs in persons of French-Canadian ancestry. The authors estimated the frequency of deleterious TSD alleles in Franco-Americans to be 1:73 (95 percent CI, 1:55 to 1:107). They concluded that these data provide a more complete database from which to formulate policy recommendations regarding TSD heterozygosity screening in people of French-Canadian background.

Martin DC, Mark BL, Triggs-Raine BL, et al. Evaluation of the risk for Tay-Sachs disease in individuals of French Canadian ancestry living in New England. Clin Chem. 2007;53:392–398.

Correspondence: Marvin R. Natowicz at

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Rheumatoid Performance of assays for measuring citrullinated protein/peptide Rheumatoid Performance of assays for measuring citrullinated protein/peptide

Arthritis is a chronic, systemic inflammatory disease that affects approximately 0.8 percent of the world population. It is characterized by an inflammation of synovial joints, which often leads to progressive joint destruction and disability. This poor prognosis has led to an emphasis on early treatment, but early diagnosis is difficult. Autoantibody formation is a common manifestation of rheumatoid arthritis (RA). The best-known antibody is rheumatoid factor, the presence of which is an American College of Rheumatology classification criteria for RA. Despite its lack of specificity, rheumatoid factor is widely used as a diagnostic marker. A variety of other antibodies specific for RA have been discovered and reported to be of diagnostic value. Sensitivity was enhanced substantially without loss of specificity using synthetic cyclic peptides derived from the sequence of human filaggrin with a high content of citrullin (cyclic citrullinated peptides [CCP]) as antigen. The first available CCP (first generation) was further optimized by screening dedicated peptide libraries (second generation). The authors conducted a study to compare the diagnostic accuracies of six enzyme-linked immunosorbent assays for detecting anticitrullinated protein/peptide antibodies (ACPA): one anticitrullinated rat filaggrin antibody assay, one antimutated citrullinated vimenten antibody assay, and four anti-CCP antibody assays. The ELISA reagent sets were Citrullinated Protein Antibodies (Genesis), Anti-MCV (Orgentec), Immunoscan RA (Euro-Diagnostica), Anti-CCP IgG ELISA (Euroimmun), EliA CCP (Phadia), and Quanta Lite CCP3 IgG ELISA (Inova). The authors determined ACPA in 298 serum samples using the aforementioned assays. Of these samples, 102 were from RA patients, including patients with early and established RA, and 196 were from controls. The latter included patients with psoriatic arthritis, connective tissue diseases, organ-specific autoimmune diseases, and a group of consecutive patients for whom a rheumatologist ordered CCP antibodies. The authors compared technical performance (imprecision, linearity, correlation, and agreement) and diagnostic accuracy (sensitivity and specificity). They noted variable technical performance among the different ACPA assays, some of which displayed poor reproducibility and poor linearity. ACPA results were well correlated among assays with the same antigen specificity, but the numerical values reported for each assay differed widely. Using cutoff values proposed by the manufacturer, diagnostic sensitivities ranged from 69.6 percent to 77.5 percent and specificities from 87.8 percent to 96.4 percent. The areas under the receiver operating characteristic curves were comparable among the various assays. The authors concluded that the overall diagnostic performance of ACPA assays is comparable among the different assays but standardization is needed. Analytical characteristics could be improved for some of the assays.

Coenen D, Verschueren P, Westhovens R, et al. Technical and diagnostic performance of 6 assays for the measurement of citrullinated protein/ peptide antibodies in the diagnosis of rheu­matoid arthritis. Clin Chem. 2007;53:498–504.

Correspondence: Xavier Bossuyt at

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Effects of nitric oxide on production of Escherichia coli toxin Effects of nitric oxide on production of Escherichia coli toxin

Enterohemorrhagic Escherichia coli (EHEC) are pathogens carried by healthy rearing animals. After infection through ingestion of contaminated food, the pathogens colonize the large intestine and cause gastrointestinal diseases ranging from uncomplicated diarrhea to hemorrhagic colitis. Life-threatening complications, such as hemolyticuremic syndrome (HUS), develop in approximately five to 10 percent of EHEC-infected subjects. HUS is defined by a triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure, which can lead to chronic renal failure and death. O157:H7 is the main EHEC serotype implicated in HUS in Europe and North America. Understanding host–EHEC interactions is critical in fighting bacterial infection and the development of HUS. The main EHEC virulence factor associated with severe human diseases is the Shiga toxin. The genes encoding Shiga toxin are carried by a lambdoid phage integrated in the bacterial genome and are fully expressed after a bacterial SOS response induced by DNA-damaging agents. Because nitric oxide is an essential mediator of the innate immune response of ­infected colonic mucosa, the authors aimed to determine its role in Shiga toxin production by EHEC. They demonstrated that chemical or cellular sources of nitric oxide inhibit spontaneous and mitomycin C-induced Shiga toxin mRNA expression and Shiga toxin synthesis without altering the viability of EHEC. The synthesis of Shiga toxin phage is also reduced by nitric oxide. This inhibitory effect apparently occurs through the nitric oxide-mediated sensitization of EHEC because mutation of the nitric oxide sensor nitrite-sensitive repressor results in loss of nitric oxide inhibiting activity on Shiga toxin expression. The authors’ findings identify nitric oxide as an inhibitor of Shiga toxin-expressing-phage propagation and Shiga toxin release and therefore as a potential protective factor limiting the development of hemolytic syndromes.

Vareille M, De Sablet T, Hindré T, et al. Nitric oxide inhibits Shiga-toxin synthesis by enterohemorrhagic Escherichia coli. Proc Natl Acad Sci USA. 2007;104:10199–10204.

Correspondence: Alain P. Gobert at

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Use of bacteria to detect antimicrobial peptides Use of bacteria to detect antimicrobial peptides

Mechanisms of innate host defense play a crucial role in preventing bacterial infection and colonization. The production of antimicrobial peptides (AMPs) is an evolutionarily conserved mechanism of innate host defense found in virtually all groups of organisms, including amphibians, insects, and other vertebrates and invertebrates. Research has also demonstrated that AMPs play a key role in human immune defenses by contributing to the microbicidal activity of neutrophils, platelets, and epithelial cells. Therefore, bacteria need specific mechanisms of resistance to AMPs to colonize or invade the human body. Bacterial-resistance mechanisms to AMPs differ with regard to efficiency, specificity, and distribution among species. Bacteria must have sensors for AMPs and corresponding gene regulatory mechanisms. However, the authors know of only one such example, the regulation of lipid A modification by the Salmonel­la typhimurium PhoP/ PhoQ two-component regulator, homologues of which are widespread among Gram-negative bacteria. The PhoQ membrane histidine kinase part is activated when cationic AMPs displace divalent cations from an extracellular negatively charged loop of the protein. After phosphorylation by activated PhoQ, the PhoP response regulator protein regulates target gene expression. In contrast, although Gram-positive bacteria comprise a series of the most significant pathogens, it is not known whether there are sensors for AMPs in Gram-positive bacteria that trigger a gene regulatory response to combat the activity of AMPs. By determining the genome-wide gene regulatory response to human β-defensin 3 in the nosocomial pathogen Staphylococcus epidermidis, the authors discovered an antimicrobial peptide sensor system that controls major specific resistance mechanisms of Gram-positive bacteria and is unrelated to the Gram-negative PhoP/ PhoQ system. It contains a classical two-component signal transducer and an unusual third protein, all of which are indispensable for signal transduction and antimicrobial peptide resistance. Furthermore, the authors’ data indicate that a very short, extracellular loop with a high density of negative charges in the sensor protein is responsible for antimicrobial peptide binding and the observed specificity for cationic antimicrobial peptides. The authors’ study showed that Gram-positive bacteria have developed an efficient and unique way of controlling resistance mechanisms to antimicrobial peptides, which may provide a promising target for antimicrobial drug development.

Li M, Lai Y, Villaruz AE, et al. Gram-positive three-component antimicrobial peptidesensing system. Proc Natl Acad Sci USA. 2007;104:9469–9474.

Correspondence: Michael Otto at

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MMP 3 as a biomarker in ankylosing spondylitis MMP 3 as a biomarker in ankylosing spondylitis

Ankylosing spondylitis is characterized by inflammation in the spine and the development of ankylosis in the facet joints and intervertebral discs, which leads to immobility and functional impairment. The degree and rate of progression of radiographic changes can be assessed using a validated scoring method, the modified Stoke AS Spinal Score (mSASSS), which records structural changes in the anterior vertebral corners of the lumbar and cervical spine. Only one prospective observational cohort study of patients with ankylosing spondylitis (AS), the Outcome Assessments in Ankylosing Spondylitis International Study (OASIS), has been conducted. The study showed that only baseline radiographic damage was an independent predictor of subsequent damage. Findings of several recent studies support the concept that soluble biomarkers can predict structural joint damage in rheumatoid arthritis. These biomarkers typically reflect different aspects of synthesis and degradation of matrix components and include markers of bone formation and resorption; cartilage turnover or degradation, or both; and synovial hyperplasia or inflammation, or both. One or more of a panel of biomarkers implicated in damage progression in rheumatoid arthritis might also predict damage progression in patients with AS. The authors conducted a study to test this hypothesis. They measured a panel of biomarkers reflecting cartilage turnover and osteoclasis. These biomarkers were cartilage oligomeric matrix protein, human cartilage gp-39 (YKL-40), type II collagen epitopes detected by the C2C and C1,C2 degradation assays and the CPII synthesis assay, aggrecan 846 epitope, osteoprotegerin, and matrix metalloproteinase 3 (MMP 3). The authors analyzed a cohort of AS patients from the Netherlands, Belgium, and France who were enrolled in the OASIS study. They examined two-year radiographic progression data scored using the mSASSS. Complete data were available on 97 patients. The authors found that only the biomarkers YKL-40 and MMP 3 showed weak to moderate univariate correlation with two-year progression. After adjusting for gender, age, disease duration, C-reactive protein level, and baseline mSASSS, only MMP 3 was significantly associated with two-year progression (β=0.29; P=.004). Logistic regression analysis revealed MMP 3 (cutoff, 68 ng/mL; odds ratio, 9.4 [95 percent confidence interval, 1.6–56]) and baseline mSASSS (cutoff, 10 mSASSS units; odds ratio, 18.6 [95 percent confidence interval, 2.5–138]) as the only independent predictors of two-year progression (cutoff, 3 mSASSS units; model R2=50 percent). MMP 3 was primarily contributory in patients who already had substantial baseline damage (more than 10 mSASSS units). The authors concluded that the results indicate that MMP 3 is a significant independent predictor of radiographic progression in patients with AS, particularly in those with pre-existing radiographic damage.

Maksymowych WP, Landewé R, Conner-Spady B, et al. Serum matrix metalloproteinase 3 is an independent predictor of structural damage progression in patients with ankylosing spondylitis. Arthritis Rheum. 2007;56:1846–1853.

Correspondence: Walter P. Maksymowych at

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Tracking the evolution of multidrug-resistant S. aureus Tracking the evolution of multidrug-resistant S. aureus

Staphylococcus aureus has remained one of the most frequent causes of a wide range of hospital- and community-acquired infections, from superficial skin and other soft tissue infections to life-threatening toxic shock, pneumonia, endocardititis, and septicemia. Yet little is known about how resistance traits are acquired in vivo. The authors applied the power of whole-genome sequencing to identify steps in the evolution of multidrug resistance in isogenic S. aureus isolates recovered from the bloodstream of a patient with congenital heart disease who was treated extensively with vancomycin without success. Clinical data suggested that the primary infection was endocarditis. In addition to vancomycin, the patient received a single dose of rifampin and a course of therapy with the β-lactam antibiotic imipenem. After approximately 12 weeks of therapy and replacement of a heart valve, the patient died from complications of the underlying disease. The first isolate, JH1, recovered before chemotherapy had begun, was fully susceptible to vancomycin (MIC=1 µg/mL). Vancomycin therapy was begun between the culture isolation of JH1 and JH2. The last isolate, JH9, recovered at the end of chemotherapy, showed decreased susceptibility to vancomycin (MIC=8 µg/mL). Comparison of the series of JH isolates using several genetic typing techniques indicated that they were isogenic. The JH lineage was also related, although more remotely, to the fully sequenced multidrug-resistant S. aureus strains N315 and MU50. The availability of these isogenic isolates offered an opportunity to identify steps in the in vivo evolution of drug resistance by sequencing the genomes of the initial (drug-susceptible) and terminal (drug-resistant) isolates. The authors concluded that as costs drop, whole-genome sequencing will become a useful tool in elucidating complex pathways of in vivo evolution in bacterial pathogens.

Mwangi MM, Wu SW, Zhou Y, et al. Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing. Proc Natl Acad Sci USA. 2007;104:9451–9456.

Correspondence: Alexander Tomasz at

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Dr. Bissell is professor, Department of Pathology, Ohio State University, Columbus.