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  Clinical Abstracts

 

 

 

 

May 2009

Editor:
Michael Bissell, MD, PhD, MPH

Bone marrow as a reservoir for ‘enhanced effector memory’ T cells
Rheumatic disease and smoking as predictors of aortic inflammation
Lipoprotein (a) and stroke risk
Study of neonatal screening for MCAD in Australia
Antibody specificities in autoimmune arthritis
HLA typing and cerebrospinal fluid hypocretin-1 levels in narcolepsy
Rapid identification of bacteria using melting analysis following PCR
Plasma E-selectin levels and acute respiratory distress syndrome

Bone marrow as a reservoir for ‘enhanced effector memory’ T cells Bone marrow as a reservoir for ‘enhanced effector memory’ T cells

Memory T cells are defined by their capacity to mount a rapid response to secondary antigenic challenge and their ability to maintain homeostatic proliferation in the absence of antigenic stimulation. They recently have been categorized into effector memory (TEM), CD45RO+ CD62Llow CCR7low, and central memory (TCM) CD45RO+ CD62Lhigh CCR7high subsets based on their homing characteristics and effector functions. TCM are primarily ­distributed in lymphoid tissue, but TEM can traffic to and reside in diverse nonlymphoid sites, including lung, liver, and intestine. Evidence suggests that residence in a particular anatomic compartment, such as bone marrow, might confer distinct phenotypic or functional properties on indigenous memory T cells. Early studies in tumor models found that live tumor cells in bone marrow were associated with systemic protection from tumor-specific challenge. In addition, tumor cells in bone marrow were controlled in a dormant state by CD8+ T cells. Correlate data from patients with breast cancer demonstrated that, following adoptive transfer, primed T cells from bone marrow, but not peripheral blood, could treat autologous breast cancer xenografts in NOD/SCID mice. Similar findings have been described for pancreatic tumors, myeloma, and melanoma. Finally, in mouse viral infection models, bone marrow was found to harbor virus-specific memory CD8+ T cells that could mediate protection from lymphocytic choriomeningitis virus infection when adoptively transferred into naive SCID hosts. Virusspecific memory CD8+ T cells were also produced in bone marrow in response to vesicular stomatitis virus infection. These results suggest that bone marrow may harbor a subset of memory CD8+ T cells that could be particularly useful in immunotherapy. However, the nature of these cells and their role in immune response to viral infection in humans remains poorly defined. To address these issues, the authors investigated the phenotypic signature and effector function of memory T cells in human bone marrow isolated from a cohort of patients with degenerative joint disease. The results define a distinct population of CD8+ TEM cells that exist in the bone marrow of patients with osteoarthritis and maintain a unique phenotype, expressing high levels of CD27, CD28, CD38, CD69, and HLA-DR. These cells exhibit a profound recall response to viral Ags and display unique patterns of perforin and granzyme B regulation in response to T-cell receptor stimulation. The findings provide a critical step in understanding the role of bone marrow memory cells in immune response to viral Ags and in maintaining memory T cell homeostasis. They reveal that human bone marrow serves as a repository for viral Agspecific TEM with great therapeutic potential in vaccine development.

Zhang X, Dong H, Lin W, et al. Human bone marrow: a reservoir for “enhanced effector memory” CD8+ T cells with potent recall function. J Immunol. 2006;177:6730–6737.

Correspondence: Dr. Scott E. Strome at sstrome@smail.umaryland.edu

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Rheumatic disease and smoking as predictors of aortic inflammation Rheumatic disease and smoking as predictors of aortic inflammation

Several inflammatory rheumatic diseases, such as rheumatoid arthritis and systemic lupus erythematosus, are associated with higher rates of cardiovascular morbidity and mortality, primarily due to coronary artery disease. This may be caused by a greater susceptibility to atherosclerosis and a higher occurrence of unstable plaques. Atherosclerosis is linked to inflammation in healthy people and those with inflammatory rheumatic disease, but the mechanism is unclear. Elevated levels of biomarkers of inflammation, such as C-reactive protein (CRP), predict the occurrence of cardiovascular events. The authors conducted a study to determine whether patients with inflammatory rheumatic disease and coronary artery disease requiring coronary artery bypass graft (CABG) surgery have enhanced chronic inflammatory cell infiltration in the aortic wall compared with control patients without inflammatory rheumatic disease. They also investigated the relationship between chronic aortic inflammatory cell infiltration and other factors thought to play a role in atherosclerosis, such as smoking. Aortic specimens routinely removed during CABG surgery on 66 consecutive patients with inflammatory rheumatic disease and 51 control patients without inflammatory rheumatic disease were examined by light micro­scopy for the occurrence, location, and severity of chronic inflammatory infiltrates and atherosclerotic lesions. The authors found that mononuclear cell infiltrates in the inner adventitia (apart from those localized along the epicardium) were more frequent in the group with inflammatory rheumatic disease (47 percent versus 20 percent; P=.002; odds ratio [OR], 3.6; 95 percent confidence interval [CI], 1.6–8.5), and the extent of these infiltrates was greater. Multivariate analyses revealed that mononuclear cell infiltrates were associated with inflammatory rheumatic disease (OR, 2.99; P=.020) and current smoking (OR, 3.93; P=.012), and they were observed in six of seven patients with a history of aortic aneurysm. Inflammatory infiltrates in the media were seen only in patients with inflammatory rheumatic disease. The frequency of atherosclerotic lesions, inflammation within the plaques, and epicardial inflammatory infiltrates in the two groups was equal. The authors concluded that among aortic samples collected during CABG surgery, those obtained from patients with inflammatory rheumatic disease had more pronounced chronic inflammatory infiltration in the media and inner adventitia than those obtained from control patients. Current smoking was an independent predictor of chronic inner adventitial infiltrates. The infiltrates may represent an inflammatory process that promotes atherosclerosis and the formation of aneurysms.

Hollan I, Scott H, Saatvedt K, et al. Inflammatory rheumatic disease and smoking are predictors of aortic inflammation: a controlled study of biopsy specimens obtained at coronary artery surgery. Arthritis Rheum. 2007;56:2072–2079.

Correspondence: Dr. Ivana Hollan at i-hollan@online.no

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Lipoprotein (a) and stroke risk Lipoprotein (a) and stroke risk

Lp(a) is a low-density lipoprotein particle containing apo(a), a glycoprotein highly heterogeneous in size, covalently linked to apolipoprotein B-100 by a disulfide bridge. Numerous case-control and prospective studies have documented that Lp(a) may be an independent risk factor for coronary heart disease. The atherogenicity of Lp(a) not only relates to its high content of cholesteryl esters and deposition in atherosclerotic plaques but also to the competitive inhibition of plasminogen action due to the similarity between apo(a) and plasminogen. The role of Lp(a) as a risk factor for ischemic stroke is less well documented than for coronary heart disease. Most case-control studies in middle-aged and older subjects have shown higher levels of Lp(a) in patients with ischemic stroke than in controls. However, findings in prospective studies have contradicted these results. An elevated level of Lp(a) at baseline was associated with increased risk of subsequent stroke in some, but not all, cohort studies. Furthermore, in both positive cohort studies, Lp(a) was an independent predictor of stroke in men but not women. How Lp(a) impacts risk of ischemic stroke in young subjects is even less clear. The authors conducted a case-control study in which they sought to determine whether Lp(a) was an independent risk factor for ischemic stroke in young adults. They examined the association of Lp(a) level with ischemic stroke separately in men and women. They prospectively measured Lp(a) concentration in 100 consecutive patients with acute ischemic stroke (58 men and 42 women) aged 18 to 55 years and in 100 control subjects matched for age and gender. The authors found that the distribution of Lp(a) concentration was skewed toward the highest and median tertiles in male patients. In multivariate logistic regression analyses adjusting for classical risk factors for ischemic stroke and lipid variables, Lp(a) concentration in the highest and medium tertiles, when compared with the lowest tertile, was significantly associated with ischemic stroke in men (odds ratio, 3.55; 95 percent confidence interval [CI], 1.33–9.48; P=.012) but not in women (odds ratio, 0.42; 95 percent CI, 0.14–1.26; P=.12). Although large vessel atherosclerosis was more common in men than in women, no differences were noted in Lp(a) concentration according to the cause of ischemic stroke. The authors concluded that in subjects aged 18 to 55 years, a slightly elevated Lp(a) concentration was strongly and independently associated with ischemic stroke in men but not in women. Additional studies are needed to elucidate the mechanisms underlying this gender-specific association

Rigal M, Ruidavets JB, Viguier A, et al. Lipoprotein (a) and risk of ischemic stroke in young adults. J Neurol Sci. 2007;252:39–44.

Correspondence: V. Larrue at larrue.v@chu-toulouse.fr

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Study of neonatal screening for MCAD in Australia Study of neonatal screening for MCAD in Australia

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is frequently diagnosed by neonatal screening with tandem mass spectrometry. Reports of patients clinically diagnosed with the disorder indicated that 20 percent to 25 percent died, usually during a first episode, and an additional 20 percent sustained neurological damage. A retrospective neonatal screening study of 100,000 stored samples reported that one in eight patients had died and one survivor had learning difficulties. Some individuals with MCAD deficiency could remain healthy, with no episodes of decompensation. After diagnosis, management plans to avoid catabolism during fasting or illness generally prevent adverse episodes. This success with intervention implies the probable advantage of newborn screening for MCAD deficiency, which is the disorder thought to most justify neonatal screening by tandem mass spectrometry. However, there have been few reports of outcome after screening. Data on patient outcomes after screening for rare disord­ers are scarce and difficult to interpret because of small numbers, variable definitions and phenotypes of individual disorders, increased detection by screening, differing mutation ranges in screened and unscreened patients, and often insufficient followup. Therefore, the authors conducted a nationwide study in Australia of the overall effectiveness of neonatal screening by tandem mass spectrometry. They identified MCAD-deficient patients from a total population of 2,495,000 Australian neonates (810,000 screened) born between April 1, 1994 and March 31, 2004. Those from a cohort of 1,995,000 (460,000 screened) were followed up for at least four years. The authors recorded number of deaths and severe episodes, medical and neuropsychological outcome, and hospital admissions within the screened and unscreened groups. They found that in cohorts that were at least four years of age, there were 35 MCAD-deficient patients in those not screened (2.28 per 100,000 total population) and 24 in the screened population (5.2 per 100,000). The authors estimated that patients with this disorder in the unscreened cohort remained undiagnosed. Before four years of age, three screened patients had an episode of severe decompensation (including one neonatal death) versus 23 unscreened patients (including five deaths). At the most conservative estimate, relative risk of an adverse event was 0.44 (95 percent confidence interval [CI], 0.13–1.45). In the larger cohort, the relative risk (screened versus unscreened) of an adverse event by age two years was 0.26 (95 percent CI, 0.07–0.97), which was also a conservative estimate. Thirty-eight of 52 living patients had neuropsychological testing, and there was no suggestion of significant differences in general cognitive outcome between the groups. The authors concluded that screening is effective in patients with MCAD deficiency because early diagnosis reduces the number of deaths and severe adverse events in children up to four years of age.

Wilcken B, Haas M, Joy P, et al. Outcome of neonatal screening for medium-chain acyl-CoA dehydrogenase deficiency in Australia: a cohort study. Lancet. 2007;369:37–42.

Correspondence: Bridget Wilcken at bridgetw@chw.edu.au

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Antibody specificities in autoimmune arthritis Antibody specificities in autoimmune arthritis

The effector mechanisms involved in the induction or propagation, or both, of the immuno­pathology in various automimmune diseases are generally categorized as predominantly T cell or Ab mediated. For example, multiple sclerosis, insulin-dependent diabetes mellitus, and rheumatoid arthritis are considered to be prototypes of primarily T cell-driven diseases, whereas systemic lupus erythematosus and myasthenia gravis are typical Ab mediated disorders. The authors conducted a study in which they examined the kinetics, Ag/ epitope specificity, and functional attributes of Abs directed against the disease-related Ags in the rat adjuvant-induced arthritis (AA) model of human rheumatoid arthritis. AA is inducible in Lewis rats (RT.11) by s.c. immunization with heat-killed Mycobacterium tuberculosis H37Ra (Mtb). T cells directed against mycobacterial heat shock protein (hsp) 65 (Bhsp65) are believed to play a pivotal role in disease induction. The authors’ study as well as those of others have defined various T cell epitopes within Bhsp65 that are involved in the induction or regulation of this disease. There is limited information in the AA model regarding the Ab response to Bhsp65 in arthritic Lewis rats. Medical literature has reported on the kinetics, peptide reactivity, and disease-modulating activity of anti-Bhsp65 Abs in naive versus arthritic Lewis rats and compared the Ab response of Lewis (RT.11) rats with that of disease-resistant Brown Norway (BN) (RT.1n) rats. However, additional examination is needed pertaining to the quantitative or qualitative differences, if any, in the antiBhsp65 and anti-self (rat) hsp65 (Rhsp65) Abs produced in AA-susceptible versus AA-resistant rat strains possessing identical MHC haplotype and the implication of such differences in disease susceptibility. Consequently, the authors comprehensively tested the Ab response of arthritic Lewis rats to Bhsp65 as well as to its self homolog, Rhsp65. Also tested were Ab responses to the control hsp65 from Escherichia coli (GroEL) and to another arthritis-related Ag, type II collagen (CII). Furthermore, the Ab responses of Lewis rats were compared with those of AA-resistant WKY rats, which have the same MHC class II haplotype (RT.11) as the Lewis rats. Finally, the physiologic significance of Abs induced during AA was tested by serum adoptive transfer. The authors found that the AA-resistant WKY (RT.11) rat responded to the heat-killed M. tuberculosis immunization with a rapid burst of Abs to Bhsp65 and Rhsp65. These Abs reacted with numerous peptide epitopes; however, this response was reduced to a few epitopes with time. On the contrary, the susceptible Lewis (RT.11) rat developed a relatively lower Ab response to Bhsp65, and Abs to Rhsp65 did not appear until recovery from the disease. The Ab response in Lewis rats diversified with progression of AA, and an intriguing overlap was noted between the repertoire of Bhsp65-reactive B and T cells during the recovery phase of AA. Nonetheless, subsets of the repertoire of the late Abs in both rat strains became focused on the same epitope regions of Bhsp65 and Rhsp65. The functional relevance of these Abs was evident from the results showing that sera from recovery phase Lewis or WKY rats, but not that of naive rats, afforded protection against subsequent AA. The auth­ors concluded that these results shed light on the role of humoral immunity in the pathogenesis of autoimmune arthritis.

Kim HR, Kim EY, Cerny J, et al. Antibody responses to mycobacterial and self heat shock protein 65 in autoimmune arthritis: epitope specificity and implication in pathogenesis. J Immunol. 2006;177:6634–6641.

Correspondence: Dr. Kamal D. Moudgil at kmoud001@umaryland.edu

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HLA typing and cerebrospinal fluid hypocretin-1 levels in narcolepsy HLA typing and cerebrospinal fluid hypocretin-1 levels in narcolepsy

In the Revised International Classification of Sleep Disorders (ICSD-2), narcolepsy has been reclassified to accommodate changes in understanding of the condition and a growing clinical practice in which the Multiple Sleep Latency Test (MSLT) is the primary diagnostic tool for narcolepsy. Measuring hypocretin-1 levels in cerebro­spinal fluid (CSF) is also considered diagnostic if levels are below 110 pg/mL and no other pathology is present. The discovery of a tight HLA-DR2 association in narcolepsy, followed by the finding of a DQB1*0602 association, offers the possibility of diagnosing narcolepsy based on genetic testing. The use of human leukocyte antigen (HLA) typing, at best, provides ancillary information that can only slightly reduce the probability of a diagnosis of narcolepsy. The authors undertook a study with the purpose of analyzing DQB1*0602 status and hypocretin-1 levels in the CSF of a cohort of patients with hypersomnolence and testing ICSD-2 criteria for hypersomnia of central origin. They studied 163 consecutive patients with unexplained sleepiness and 282 control subjects recruited at St. Vincent’s Hospital, in Suwon, Korea. The gold standard for diagnosis was ICSD-2 criteria. Patients and controls completed the Stanford Center for Narcolepsy Sleep Inventory (a previously validated questionnaire predictive of cataplexy and that evaluates for the presence and severity of various other narcolepsy symptoms) and agreed to HLA typing. Patients were subjected to poly­somnography (87 percent), Multiple Sleep Latency Test (MSLT; 96 percent), and CSF hypocretin-1 measurements (53 percent). Most patients (80 percent) could be classified using the ICSD-2. The 33 patients who could not be classified were without cataplexy (four with low CSF hypocretin-1). These could not be included because of sleep apnea (apnea-hypopnea index of five hours or more, 84 percent) or because sleep prior to MSLT was less than six hours (27 percent), or both. Narcolepsy with cataplexy cases were 92 percent HLA positive with low hypocretin-1. Cataplexy at interview was predicted by validated Stanford Sleep Inventory questions regarding cataplexy triggers. In contrast, cataplexy-like events were frequently reported in all groups, including controls. Cases with narcolepsy without cataplexy were frequently men (73 percent) and heterogeneous biologically (36 percent HLA positive, 40 percent with low CSF hypocretin-1). None of the controls had low CSF hypocretin-1, whereas 13 percent were HLA positive. The authors concluded that the ICSD-2 was easily applicable in cases with typical cataplexy. In such cases, the MSLT and additional evaluations were almost always positive and therefore may not always be needed. Many patients without cataplexy were difficult to classify because of difficulties in interpreting the MSLT due to sleep apnea or reduced sleep.

Hong S-C, Lin L, Jeon J-H, et al. A study of the diagnostic utility of HLA typing, CSF hypocretin-1 measurements, and MSLT testing for the diagnosis of narcolepsy in 163 Korean patients with unexplained excessive daytime sleepiness. Sleep. 2006;29:1429–1438.

Correspondence: Dr. Emmanuel Mignot at mignot@stanford.edu

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Rapid identification of bacteria using melting analysis following PCR Rapid identification of bacteria using melting analysis following PCR

Two polymerase chain-reaction-based strategies have been developed for the nonculture diagnosis of bacteremia, one targeting species-specific genes for amplification and the other using broad-range PCR amplification of conserved bacterial DNA sequences, such as the 16S rRNA, 23S rRNA, and 16S-23S rRNA interspace regions. Broad-range PCR generates valuable information that complements results of time-consuming and subjective phenotypic tests for detecting bacterial infections and can be used to differentiate bacterial from viral and other infections. A high-resolution melting analysis incorporating the fluorescent dye LCGreen I has been used to detect heterozygous and homozygous sequence variants for genotying and variation scanning. This approach is a closed-tube technique that does not require fluorescently labeled probes or separation steps. In contrast to traditional melting-curve analysis, high-resolution melting with the HR-1 instrument reliably detects single-base differences in homozygous and heterozygous sequences. This technique is cost-effective, has high sensitivity and specificity, and can be completed in less than two minutes after PCR. The authors reported on a novel and powerful scheme combining LightCycler broad-range real-time PCR and high-resolution melting analysis for rapid species identification of clinical bacteria culture colonies. Without multiplexing or hybridization probes, 25 bacterial species can be identified by this method using two or fewer PCRs within 1.5 hours. The authors subjected 46 bacterial culture colonies, representing 25 clinically important bacterial species, to LightCycler real-time PCR amplification of the 16S rRNA gene in the presence of LCGreen I fluorescent dye. They performed high-resolution melting analysis of the PCR products with the HR-1 instrument and used melting profiles as molecular fingerprints for bacterial species identification. They then validated this method by assessing 54 consecutive bacteria culture colonies obtained from a clinical microbiology laboratory. The authors found that the 16S rRNA gene of all 25 bacterial species could be amplified by their test method, with PCR product lengths of 216 or 217 bp. The authors identified 11 of the 25 bacterial species via a one-step post-PCR high-resolution melting analysis. The remaining bacterial species were identified via the high-resolution melting plots obtained by heteroduplex formation between the PCR products of the tested and reference bacterial species or by a second real-time PCR targeting a different region of the 16S rRNA gene. A high-resolution melting database and a working protocol were established for identifying these 25 bacterial species. A 94 percent accuracy rate was achieved in the validation assay when the bacterial species were in the high-resolution melting database. The authors concluded that this assay requires no multiplexing or hybridization probes and provides a new approach for bacterial species identification in a molecular diagnostics laboratory.

Cheng J-C, Huang C-L, Lin C-C, et al. Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR. Clin Chem. 2006;52:1997–2004.

Correspondence: Ching-Ping Tseng at ctseng@mail.cgu.edu.tw

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Plasma E-selectin levels and acute respiratory distress syndrome Plasma E-selectin levels and acute respiratory distress syndrome

Pro-inflammatory cytokines, such as tissue necrosis factor, have been shown to be critically involved in the pathogenesis of various types of organ failure observed in sepsis. Among these types of organ failure, acute respiratory failure frequently develops into acute respiratory distress syndrome (ARDS), a pathologic condition that has a high mortality rate. ARDS has been reported to be associated with septic shock, often leading to multi-organ failure, a major cause of death in patients with ARDS. When activated by tissue necrosis factor (TNF), endothelial cells increase the expression of E-selectin, an endothelial leukocyte adhesion molecule, within a few hours after activation. Plasma soluble E-selectin (sES) is present in the supernatants of cultured endothelial cells activated by TNF. Plasma levels of sES were reported to have increased in patients with severe infection when they were associated with disseminated intravascular coagulation and organ failure. Determination of plasma levels of sES might predict the development of ARDS. However, most laboratory assays of plasma sES level used in previous studies were ELISA-based methods that require several hours to obtain test results. Therefore, if a laboratory method were developed by which plasma sES levels could be determined quickly, the real-time pathological condition could be evaluated in critically ill patients presenting with systemic inflammatory response syndrome (SIRS). To this end, the authors conducted a study in which they examined a newly developed rapid immunoassay method for plasma sES levels to determine whether it can predict the development of ARDS in critically ill patients presenting with SIRS. The new latex agglutination method, which provides results in about 20 minutes, was used to measure plasma levels of sES on admission (day one) to the emergency unit. Development of various types of organ failure, including ARDS, was compared in the first five days of admission (from day one to day five) between patients with normal plasma levels of sES and those with elevated plasma levels of sES. The normal range of the plasma sES level was 4.8 to 29.7 ng/mL using the latex agglutination method. Twenty-two of the patients examined showed elevated sES levels (DAE group) and 28 showed normal sES levels (DAN group). Development of ARDS was significantly higher in the DAE group (15 of 22, 68.2 percent) than in the DAN group (4 of 28, 14.3 percent; P<.001). Development of cardiovascular system failure, renal failure, and coagulation system failure was also significantly higher in the DAE group than in the DAN group. The mortality rate at 28 days after admission was also higher in the DAE group (27.3 percent) than in the DAN group (zero; P<.05). The authors concluded that ­determination of sES levels by this new rapid assay method might be useful for predicting the development of ARDS in critically ill patients with SIRS, a pathological condition that can lead to multiple organ failure.

Okajima K, Harada N, Sakurai G, et al. Rapid assay for plasma soluble E-selectin predicts the development of acute respiratory distress syndrome in patients with systemic inflammatory response syndrome. Transl Res. 2006;148:295–300.

Correspondence: Dr. Keniji Okajima at whynot@med.nagoya-cu.ac.jp

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