College of American Pathologists
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  Clinical Abstracts





August 2009

Michael Bissell, MD, PhD, MPH

Low levels of high-density lipoprotein cholesterol in cardiac transplantation
Low serum retinol levels and renal transplant mortality
Study of hantavirus disease outbreak in Germany
Interstitial norepinephrine concentrations and ischemic heart failure
Antibodies against activated coagulation factor X in antiphospholipid syndrome
Testosterone regulation and plasma homocysteine levels
Tetrahydrobiopterin oxidation and myocardial ischemia
Use of B-type natriuretic peptide to predict mortality in noncardiac surgery patients

Low levels of high-density lipoprotein cholesterol in cardiac transplantation Low levels of high-density lipoprotein cholesterol in cardiac transplantation

The development of atherosclerosis may be associated with compensatory vessel enlargement, which has been termed positive remodeling. The mechanism of positive arterial remodeling remains uncertain, but potential mechanisms include compensatory vessel enlargement owing to increased shear stress in atherosclerotic vessels and disruption of arterial architecture in atherosclerotic vessels. Studies have demonstrated that evidence of positive remodeling can be noninvasively identified by brachial artery ultrasound as an increase in end-diastolic resting brachial artery diameter. Pathologic evidence indicates that subclinical atherosclerotic changes occur in the brachial arteries of people with coronary atherosclerosis, supporting the concept that alterations in the brachial artery occur in the setting of systemic atherosclerosis. Dyslipidemia is common among cardiac transplant recipients and is a risk factor for peripheral atherosclerosis. Abnormal low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride levels can occur in transplant recipients. However, the contribution of specific lipid factors to the atherosclerotic process following transplantation is not fully understood. The use of statins to decrease the risk of allograft vasculopathy is standard clinical practice, but whether specific lipid abnormalities should be targeted for therapy in this population is unknown. Furthermore, although dyslipidemia is common among cardiac transplant recipients, its effects on vascular remodeling in this population are unknown. The authors conducted a study to examine the relationship between brachial artery diameter and dyslipidemia in cardiac transplant recipients. Thirty-five stable cardiac transplant recipients underwent high-resolution brachial artery ultrasound to evaluate resting brachial artery diameter. Levels of HDL-C, LDL-C, and triglycerides were determined and the presence of other cardiac risk factors assessed. The authors found that brachial artery diameter was larger (4.3±0.1 mm) in subjects with low levels of HDL-C (<40 mg/dL; n=11) than in subjects with high levels of HDL-C (≥40 mg/dL; n=24) who had a mean brachial artery diameter of 3.7±0.1 mm (P=.006). Neither high LDL-C levels (≥100 mg/dL) nor high triglyceride levels (≥200 mg/dL) were associated with differences in brachial artery diameter. Multivariate analysis demonstrated that the relationship between low HDL-C levels and increased brachial artery diameter was independent of body surface area or statin use. The authors concluded that low levels of HDL-C are an independent predictor of brachial artery enlargement in stable cardiac transplant recipients. These findings suggest that suboptimal HDL-C levels may be associated with the development of vascular remodeling and atherosclerosis in this population.

Ambrose MS, DeNofrio D, Kuvin JT, et al. Low levels of high-density lipoprotein cholesterol are associated with vascular remodeling in cardiac transplant recipients. Transplant Proc. 2006;38:3016–3020.

Correspondence: Dr. Ayan R. Patel at

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Low serum retinol levels and renal transplant mortality Low serum retinol levels and renal transplant mortality

The success of renal transplantation depends on achieving a balance between immunosuppression sufficient to avoid rejection and immune competence sufficient to protect the recipient from developing infection or cancer. Cardiovascular disease, infection, and cancer are the leading causes of morbidity and mortality after renal transplantation. Because these diseases are associated with oxidative stress and inflammation, dietary antioxidants such as vitamin E, β-carotene, and carotenoids may protect renal transplant recipients from the main causes of morbidity and mortality. Vitamin A is a weak antioxidant, but it also plays a central role in epithelial integrity, immune function, and retinal physiology. Deficiency of vitamin A has been shown to impair innate immunity by impeding normal regeneration of mucosal barriers damaged by infection and by diminishing the function of neutrophils, macrophages, and natural killer cells. Children with mild vitamin A deficiency are at increased risk of infections and death, and several large randomized control trials conducted in developing countries worldwide have shown that vitamin A supplementation reduces child mortality by approximately one-third. Vitamin A deficiency or marginal vitamin A status is often exacerbated by infectious disease, and, conversely, poor vitamin A status may prolong or exacerbate the course of illness. Given the risk of infection following transplantation, adequate vitamin A levels may be of particular importance in patients who undergo renal transplant. Therefore, the authors conducted a study in which they assessed whether an association exists between serum concentrations of retinol or other dietary antioxidants and mortality in renal transplant recipients. They recruited 379 asymptomatic renal transplant recipients between June 2000 and December 2002 and measured their serum retinol levels at baseline. They collected prospective followup data at a median of 1,739 days. The authors found that retinol was significantly decreased in those renal transplant recipients who had died at followup compared with those who were still alive at that time. Kaplan-Meier analysis showed that retinol concentration was a significant predictor of mortality. In multivariate Cox regression analysis, decreased retinol concentration remained a statistically significant predictor of all-cause mortality after adjusting for traditional cardiovascular risk factors, high-sensitivity C-reactive protein, and esti-mated glomerular filtration rate. The authors concluded that serum retinol concentration is a significant independent predictor of all-cause mortality in renal transplantation patients. A higher retinol concentration might impart a survival advantage via an anti-inflammatory or anti-infective mechanism.

Connolly GM, Cunningham R, Maxwell AP, et al. Decreased serum retinol is associated with increased mortality in renal transplant recipients. Clin Chem. 2007;53:1841–1846.

Correspondence: Grainne M. Connolly at

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Study of hantavirus disease outbreak in Germany Study of hantavirus disease outbreak in Germany

Hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome, also named hantavirus pulmonary syndrome, are caused by hantavirus infections. Fatality rates can reach up to 50 percent. The agents, negative-strand RNA viruses forming the genus Hantavirus within the Bunyaviridae family, are transmitted to humans from their respective rodent reservoirs. In Central Europe, hemorrhagic fever with renal syndrome (HFRS) occurs mainly due to infections by Puumala virus (PUUV) transmitted from the red bank vole (Clethrionomys glareolus) and by a Dobrava virus (DOBV) lineage, named DOBV-Aa, carried by the striped field mouse (Apodemus agrarius). Infections by PUUV and DOBV-Aa usually cause mild to moderate HFRS, which is also known as nephropathia epidemica. Germany reports up to 450 cases of HFRS per year. Since viremia in HFRS patients is short termed and characterized by fluctuating RNA levels, molecular genetic approaches for identifying human infections are not common. Instead, laboratory diagnosis is based on serology. Usual methods are ELISA and immunofluorescence assay formats based on the immunodominant nucleocapsid protein (N) antigen and virus-infected cells, respectively, to detect antibodies of immunoglobulin M (IgM) and IgG classes. An immunoblot assay, based on the use of N antigen, also has been established. The majority of hantavirus-infected patients present detectable IgM class antibodies at the onset of disease. In only a few cases was the IgM response found to be delayed. IgM disappears within months; however, when using sensitive test formats, it can be detected in single cases as long as two years after the acute phase of infection. The IgG response is long lasting but sometimes delayed; in about 10 percent of acute-phase, IgM-positive sera, no IgG can be found with routine assays. The authors described the serological and molecular epidemiological investi-gation of an outbreak of hantavirus disease in Lower Bavaria, a district in southeast Germany (Federal State of Bavaria), in 2004. They tested the early and, in some cases, followup sera of 31 patients, which showed that the outbreak was caused by Puumala virus. In certain cases, ELISAs, immunofluorescence assays, and immunoblot assays were unable to support the diagnosis of PUUV infection because of extended cross-reaction of IgM and IgG antibodies to other hantavirus antigens. In these cases, focus reduction neutralization testing confirmed PUUV infection. PUUV sequences were amplified from sera from three patients and compared with those from red bank voles trapped in the same area where the three patients resided. The human infections were caused by a novel genetic lineage of the PUUV species, called Bavaria.

Schilling S, Emmerich P, Klempa B, et al. Hantavirus disease outbreak in Germany: limitations of routine serological diagnostics and clustering of virus sequences of human and rodent origin. J Clin Microbiol. 2007;45:3008–3014.

Correspondence: Detlev H. Kruger at

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Interstitial norepinephrine concentrations and ischemic heart failure Interstitial norepinephrine concentrations and ischemic heart failure

The causes of exercise limitations in ischemic heart failure are poorly understood. However, it appears that reduced blood flow to active muscle may contribute to this response. Blood flow limitations may be due more to exaggerated sympathetic activation than to reductions in the intrinsic ability of the peripheral vasculature to dilate. When the sympathetic nervous system is engaged, norepinephrine is released from the neurovascular junction, evoking vasocon-striction within a given vascular bed. However, the determinants of the neurovascular concentrations of norepinephrine in skeletal muscle during exercise in congestive heart failure have received little attention. The authors conducted an investigation in which they used the microdialysis method to measure interstitial norepinephrine concentration in the hindlimb muscle after electrical stimulation of the lumbar sympathetic nerve in rats with myocardial infarction and in rats used as controls. Interstitial norepinephrine was used as a surrogate of neurovascular norepinephrine. The authors hypothesized that interstitial norepinephrine concentration for a given level of electrical stimulation of the sympathetic nerves would be augmented in ischemic heart failure. They further hypothesized that since norepinephrine uptake-1 represents the major pathway by which released norepinephrine is metabolized and interstitial norepinephrine concentration is regulated, norepinephrine uptake-1 would be impaired in the skeletal muscle of rats with myocardial infarction. To test this hypothesis, the authors performed studies in male Sprague-Dawley rats (150 to 180 g body weight) eight to 10 weeks after induction of myocardial infarction. They collected interstitial norepinephrine samples from microdialysis probes inserted into the hindlimb muscles of the rats. Dialysate concentration of norepinephrine was determined using the high-pressure liquid chromatography method. The authors found that interstitial norepinephrine concentration increased during electrical stimulation of the lumbar sympathetic nerves in eight control rats. An increase in interstitial norepinephrine concentration was significantly greater in 10 rats with severe myocardi-al infarction. In addition, the norepine-phrine uptake-1 inhibitor desipramine (1 µM) was injected into the arterial blood supply of the muscle in six control rats and eight with myocardial infarction. Desipramine increased interstitial norepinephrine concentration by 24 percent in the controls and by only three percent (P<.05 versus control) in the rats with myocardial infarction. The authors concluded that given levels of electrical stimulation of the lumbar sympathetic nerve lead to higher interstitial norepinephrine concentrations in heart failure. This effect is due, in part, to reduced norepinephrine uptake-1 in heart failure.

Xing J, Koba S, Kehoe V, et al. Interstitial norepinephrine concentrations in skeletal muscle of ischemic heart failure. Am J Physiol Heart Circ Physiol. 2007;293:H1190–H1195.

Correspondence: J. Li at

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Antibodies against activated coagulation factor X in antiphospholipid syndrome Antibodies against activated coagulation factor X in antiphospholipid syndrome

Antiphospholipid syndrome is characterized by the presence of antiphospholipid antibody (Ab) and the clinical mani-festations of thrombosis or miscarriage, or both. Studies of anti-phospholipid Ab (aPL) have shown that it represents a heterogeneous group of immunologically distinct antibodies that recognize various phospholipids (PL), PL-binding plasma proteins, and factors involved in hemostasis. The involved proteins include β2-glycoprotein I (β2-GPI), annexin V, prothrombin, thrombin, activated protein C, plasmin, and tissue plasminogen activator (tPA), among others. Importantly, thrombin, activated protein C, plasmin, and tPA are trypsin-like serine proteases. Although aPL has been shown to promote thrombosis and miscarriage in animal studies, the underlying mechanisms remain unclear. The authors hypothesized that anti-FXa Ab are present in some antiphospholipid syndrome (APS) patients, and that some of these autoantibodies interfere with antithrombin inactivation of FXa. To test this hypothesis, the authors studied the reactivity of eight patient-derived monoclonal IgG antiphospholipid Ab with FXa and the presence of IgG anti-FXa Ab in APS patients. They also investigated the effects of FXa-reactive monoclonal antibodies (mAb) on antithrombin inactivation of FXa. The results revealed that all of six thrombin-reactive IgG mAb bound to FXa and that the levels of plasma IgG anti-FXa Ab in 38 APS patients were significantly higher than those in 30 normal control subjects (P<.001). When the mean plus 3 standard deviations of the 30 normal controls were used as the cut-off, five of 38 (13.2 percent) APS patients had IgG anti-FXa Ab. Of note was that three of six FXa-reactive mAb significantly inhibited antithrombin inactivation of FXa. These results indicate that anti-FXa Ab may contribute to thrombosis by interfering with the anticoagulant function of antithrombin on FXa in some APS patients.

Yang Y-H, Hwang K-K, FitzGerald J, et al. Antibodies against the activated coagulation factor X (FXa) in the antiphospholipid syndrome that inter-fere with the FXa inactivation by antithrombin. J Immunol. 2006;177:8219–8225.

Correspondence: Dr. Kwan-Ki Hwang at

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Testosterone regulation and plasma homocysteine levels Testosterone regulation and plasma homocysteine levels

Homocysteine is a rogue sulfur-containing amino acid that, at elevated concentrations, is correlated with an increased risk for cardiovascular disease, neural tube defects, and Alzheimer’s disease. Homocysteine is a byproduct of methionine metabolism, and its intracellular concentrations are kept low by efficient disposal through the transmethylation and transsulfuration pathways. In the liver and kidney, parallel transmethylation reactions are catalyzed by methionine synthase and betaine homocysteine methyltransferase, whereas cystathionine β-synthase (CBS) catalyzes the first step in the transsulfuration pathway. Mutations in CBS are the single most common cause of congenital hyperhomocysteinemia. Age, gender, and renal function are determinants of total plasma homocysteine (tHcy) levels in humans. The molecular mechanisms underlying these associations are not known. Plasma homocysteine levels are significantly lower in women than in men. This difference narrows with age because tHcy levels increase after menopause. The authors have previously described tissue- and gender-specific differences in murine CBS activity. In the kidney, CBS activity is approximately twofold higher in male versus female mice and is correlated with an increase in steady-state CBS protein levels. The authors conducted a study in which they demonstrated that the higher CBS activity in male mice is associated with lower levels of plasma tHcy. They further reported that renal CBS expression and activity decrease after castration of male mice, which suggests that testosterone is involved in the regulation of renal CBS and that this is related to higher tHcy levels in female versus male mice. However, the situation is reversed in humans, where the higher plasma tHcy level in males is correlated with lower CBS activity in male versus female kidney cortical tissue. The higher renal CBS activity in males (22.7±3.1 mmol cystathionine/hr.-1/kg kidney-1) versus females (8.4±3.4 mmol cystathionine/hr.-1/kg kidney-1; P<10-6) in C57BJ/6J mice was associated with lower plasma tHcy levels in males versus females, and this difference was exacer-bated in Cbs+/– mice (7.7+1.9 µmol/L in males versus 13.8±6.4 µmol/L in females; P=.005). Surprisingly, mammals exhibit a diversity of regulatory patterns for kidney CBS, with females exhibiting lower CBS activity in mice, higher activity in rats and humans, and levels indistinguishable from those of males in rabbits, hamsters, and guinea pigs. These data suggest that testosterone-dependent regulation of human CBS in kidneys may contribute to gender-dependent differences in homocysteine transsulfuration.

Vitvitsky V, Prudova A, Stabler S, et al. Testosterone regulation of renal cystathionine β-synthase: implications for sex-dependent differences in plasma homocysteine levels. Am J Physiol Renal Physiol. 2007;293:F594–F600

Correspondence: R. Banerjee at

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Tetrahydrobiopterin oxidation and myocardial ischemia Tetrahydrobiopterin oxidation and myocardial ischemia

Nitric oxide synthase converts L-arginine and O2 to nitric oxide and L-citrulline. This enzymatic process consumes NADPH and requires Ca2+/calmodulin, flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin (BH4) as nitric oxide synthase (NOS) cofactors. Endothelial nitric oxide synthase (eNOS) contributes to the regulation of vasomotor tone and blood pressure by producing nitric oxide that activates soluble guanylate cyclase in vascular smooth muscle, resulting in vasorelaxation. Endothelial dysfunction is a prognostic marker of cardiovascular disease. It has been suggested that limited availability of BH4 contributes to eNOS dysfunction in hypercholesterolemia, diabetes, atherosclerosis, hypertension, and heart failure. It is not known how BH4 levels are altered by the process of ischemia or whether these al-terations trigger postischemic vascular dysfunction and loss of nitric oxide production from eNOS. Therefore, the authors conducted a study to determine how BH4 is altered after periods of myocardial ischemia and the role these alterations play in ischemia-induced NOS dysfunction and loss of endothelial-mediated vasodilation. For the study, isolated rat hearts were subjected to varying durations of ischemia, and the alterations in NOS-dependent vasodilation were measured and correlated with assays of eNOS activity and cardiac BH4 concentrations. Ischemia time dependently decreased cardiac BH4 content with 85, 95, and 97 percent irreversible degradation after 30, 45, and 60 minutes of ischemia, respectively. Paralleling the decreases in BH4, reductions of eNOS activity were seen in 58, 86, and 92 percent, and NOS-derived superoxide production was greatly increased. Addition of 10 µM BH4 enhanced eNOS activity in non-ischemic hearts and partially restored activity after ischemia. It also suppressed NOS-derived superoxide production. The authors concluded that impaired coronary flow during postischemic reperfusion was improved by BH4 infusion. Consequently, BH4 depletion contributes to postischemic eNOS dysfunction, and BH4 treatment is effective in partial restoration of endothelium-dependent coronary flow. Therefore, supplementation of BH4 may be an important therapeutic approach to reverse endothelial dysfunction in postischemic tissues.

Dumitrescu C, Biondi R, Xia Y, et al. Myocardial ischemia results in tetrahydrobiopterin (BH4) oxidation with impaired endothelial function ameliorated by BH4. Proc Natl Acad Sci US. 2007;104:15081–15086.

Correspondence: Jay L. Zweier at

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Use of B-type natriuretic peptide to predict mortality in noncardiac surgery patients Use of B-type natriuretic peptide to predict mortality in noncardiac surgery patients

Major noncardiac surgery is associated with a high risk for early cardiovascular complications and significant longer term mortality. Some of these late deaths may relate to the underlying pathology, but many are due to cardiovascular disease. Perioperative and early postoperative cardiac events predict excess medium-term mortality. In particular, even small elevations in markers of myocardial cell necrosis, such as cardiac troponin I (cTnI) or cardiac troponin T, are associated with an increased risk for death. Several relatively small studies have also demonstrated that preoperative levels of B-type natriuretic peptide (BNP) and N-terminal pro-BNP are useful predictors of perioperative cardiac complications in this setting. However, these studies relied largely on subjective, short-term end points. The authors conducted a study to assess the ability of preoperative B-type natriuretic peptide levels to predict medium-term mortality in patients who undergo major noncardiac surgery. During a median of 654 days of followup, 33 patients from a cohort of 204 (16 percent) patients died, 17 from cardiovascular causes. The optimal cutoff in this cohort, determined using a receiver operating characteristic curve, was greater than 35 pg/mL-1. This was associated with a 3.5-fold increase in hazard of death (P=.001) and a 6.9-fold increase in hazard of cardiovascular mortality (P=.003). The authors concluded that these results support recent findings that B-type natriuretic peptide levels obtained before major noncardiac surgery can be used to predict perioperative morbidity and indicate that they forecast medium-term mortality, particularly cardiovascular death.

Cuthbertson BH, Amiri AR, Croal BL, et al. Utility of B-type natriuretic peptide in predicting medium-term mortality in patients undergoing major non-cardiac surgery. Am J Cardiol. 2007;100:1310–1313.

Correspondence: Brian H. Cuthbertson at

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