September 2008

Editor:
Michael Bissell, MD, PhD, MPH

Screening blood for prions
Elevated prolactin levels in patients with systemic lupus erythematosus
IgA rheumatoid factor in rheumatoid arthritis
Muscle tissue oxygenation in septic patients
Unlabeled probes for herpes simplex virus

Screening blood for prions
Prions are normal host proteins that may also exist in a misfolded proteinase-resistant conformation (PrP^TSE) responsible for disease in animals and humans. The considerable public health consequence of these diseases, which include bovine spongiform encephalopathy, sheep scrapie, chronic wasting disease in deer and elk, and Creutzfeldt-Jakob disease in humans, has led to significant efforts to develop methods of preclinical detection. The transmissibility of transmissible spongiform encephalopathy (TSE) via blood has been established in animal models for Gerstmann-Sträussler-Scheinker (GSS) disease and variant Creutzfeldt-Jakob disease (vCJD) in mice, scrapie in hamsters, and bovine spongiform encephalopathy in sheep. Blood is also infectious in endemic sheep scrapie and human infections with vCJD. There are several challenges to detecting PrP^TSE in blood. The level of PrP^TSE, especially in early disease, is below the detection limits of standard immunoassay methods, and the co-existence of a large amount of normal cellular form of the protein (PrP^C) compromises detection specificity without the use of proteinase enzyme to digest interfering species. PrP^TSE does not elicit a humoral immune response in the infected host, preventing a serologic antibody from being detected. The absence of a foreign nucleic acid component in the infectious particle eliminates the use of polymerase chain reaction. However, immuno-polymerase chain reaction has been used to detect recombinant PrP and PrP^TSE from scrapie-infected brain homogenate at a level that is 1 million-fold more sensitive than antibody-based detection methods, allowing immuno-polymerase chain reaction to detect PrP^TSE in the preclinical phase of disease in blood. These challenges have restricted diagnostic tests to the use of postmortem tissue and, in some cases, antemortem lymphoreticular tissue, but there is an urgent need for a preclinical screening test to prevent secondary transmission of vCJD disease from blood donation. The authors developed a method for detecting PrP^TSE in blood that uses a conformationally sensitive pyrene-labeled peptide from the N-terminal region of PrP that contains conserved amino acid sequences (Pronucleon ligands). The structural features of the peptide have been previously reported—for example, in circular dischroism studies documenting the thermodynamic conditions for its transition from a coiled structure to a β-sheet structure that occurs in the presence of PrP^TSE. This conformational change induces an excimeric signal from the conjugated pyrenes when the conformational change to β-sheet brings the pyrene pair into close proximity. The conformational change is propagated to other peptide molecules, serving to amplify the signal for PrP^TSE. The authors have previously shown that these peptides can detect PrP^TSE in brain during the preclinical and clinical stages of disease in hamsters infected with the 263K strain of scrapie. With this investigation, they have extended those studies to the detection of PrP^TSE in the blood of animals and humans with various forms of TSE. The authors reported that the misfolded protein diagnostic assay employs a pyrene-labeled palindromic sequence of prion peptides that undergoes a cascade of coil to β-sheet conversion in the presence of PrP^TSE. They tested the ability of the assay to detect PrP^TSE in brain, serum, and plasma. The basic protocol involved a several-hour incubation of 200-µL sample volumes with the peptide reagent in 96-well plates, after which fluorescence was monitored by a fluorescence plate reader with an excitation wavelength of 350 nm and emission scanning wavelength range of 365 to 600 nm. Target specificity for PrP^TSE was documented by correlating assay signal with Western blot signals in brain tissue from TSE-infected, normal, and knockout mice and with negative assay signals by use of reagents with different peptide sequences. When applied to plasma or serum, the assay discriminated between samples from a variety of experimental and natural TSE infections compared with uninfected controls, with a sensitivity threshold of approximately one infectious dose/mL in pooled plasma from TSE-infected mice. The authors concluded that the misfolded protein diagnostic assay is a sensitive and specific test for detecting PrP^TSE that may be useful in the preclinical and clinical diagnosis of TSE diseases of animals and humans.

Pan T, Sethi J, Nelsen C, et al. Detection of misfolded prion protein in blood with conformationally sensitive peptides. Transfusion. 2007;47:1418–1425

Reprints: Dr. Cindy Orser, Adlyfe Inc., 9430 Key West Ave., Rockville, MD 20850; corser@adlyfe.com

Elevated prolactin levels in patients with systemic lupus erythematosus
The best known biological functions of the polypeptide hormone prolactin are linked to lactation and reproduction, but the hormone is also involved in other physiological processes, including immunoregulation. The first report of the association between prolactin and systemic lupus erythematosus (SLE) was in men, and high serum prolac­tin levels have been associated with lupus activity in humans and in experimental models of SLE. Serum prolactin from healthy subjects and most patients with hyperprolactinemia circulates in several isoforms, of which three major isoforms are identifiable by gel filtration chromatography. The major circulating prolactin isoform is a 23 kDa single-chain polypeptide, called little prolactin (monomeric or free prolactin), which composes up to 80 percent of total prolactin. The two other isoforms—big prolactin (45–50 kDa) and big big prolactin (>100 kDa)—circulate in lesser amounts. These isoforms result from post-translational modifications of the prolactin molecule (aggregates of monomeric prolactin and prolactin bound to binding proteins), which may alter the biological and immunological properties of the hormone. It is well recognized that the molecular heterogeneity of prolactin is present in sera from patients with SLE. The predominant presence of big big prolactin, a phenomenon termed macroprolactinemia, has been reported in approximately 40 percent of SLE patients with idiopathic hyperprolactinemia. The authors used homologous in vitro bioassay systems to measure circulating bioactive prolactin concentrations in samples from patients with SLE. They also attempted to correlate bioactive prolactin levels with disease activity. Serum samples from 98 SLE patients with and without disease activity were tested for immunoreactive and bioactive concentrations of prolactin. Patients with active disease exhibited higher bioactive serum prolactin levels in homologous bioassays (P=.013). In contrast, bioactivity in Nb2 cells was similar between patients with and without activity. The bioactive/ immunoreac­tive prolactin ratio (BA/IA) in ho­mo­logous bioassays was significantly higher in patients with clinical manifestations and serological indicators of lupus disease activity. SLE patients with idiopathic hyperprolactinemia and macroprolactinemia had low SLEDAI scores, and the BA/IA ratio in homologous bioassays was significantly lower than for those with idiopathic hyperprolactinemia and without macroprolactinemia. There was a negative but significant correlation between macroprolactinemia and BA/IA in homologous bioassays (P<.001), but not when the heterologous bioassay was employed. The authors concluded that elevated serum bioactive prolactin levels revealed by homologous bioassays were associated with disease activity and specific organ involvement. Macroprolactin is a prolactin variant with reduced bioactivity towards its homologous receptor, and this altered bioactivity may contribute to the lower disease activity and absence of symptoms related to hyperprolactinemia in SLE patients. These novel data must be considered in future studies to establish a relationship between prolactin and disease activity in SLE.

Cardenas-Mondragon G, Ulloa-Aguirre A, Isordia-Salas I, et al. Elevated serum bioactive prolactin concentrations in patients with systemic lupus erythematosus are associated with disease activity as disclosed by homologous receptor bioassays. J Rheumatol. 2007;34:1514–1521.

Reprints: Dr. A. Leanos-Miranda, Research Unit in Reproductive Medicine, Hospital de Ginecologia y Obstetricia “Luis Castelazo Ayala,” Don Luis 111, Col. Nativitas, Mexico 03500, DF, Mexico; alfredolm@yahoo.com

IgA rheumatoid factor in rheumatoid arthritis
Rheumatoid factor and antibodies to citrullinated proteins are usually considered to be serological markers of rheumatoid arthritis. Classic (IgM) rheumatoid factor is assessed in clinical practice; however, the combined detection of additional isotypes may improve this marker’s diagnostic and prognostic value. Several studies have shown that IgA rheumatoid factor may be strongly linked to a more severe disease. Anti-citrullinated peptide antibodies recognize different citrulline-containing proteins derived from a post-translational modification of arginine residues from peptidyl-arginine deiminase. Tests developed recently allow antibodies that recognize cyclic citrullinated peptides (anti-CCP) to be detected in the serum of most patients with rheumatoid arthritis. Anti-CCP have proved to be highly specific for rheumatoid arthritis and strongly associated with the development of radiographic erosions in the early stages of disease. The role of these antibodies as markers of response to treatment is not yet fully understood. Three tumor necrosis factor (TNF)π-inhibiting agents are used to treat active rheumatoid arthritis, all of which reduce the signs and symptoms of the disease and inhibit the progression of radiographic joint damage. The authors investigated the relationship between serum levels of anti-CCP or different rheumatoid factor isotypes and clinical response to TNFα blockers. They conducted a study involving 132 patients with advanced rheumatoid arthritis refractory to disease-modifying antirheumatic drugs. Patients were treated with infliximab (n=63), etanercept (n=35), or adalimumab (n=34). All patients completed one year of followup, and 126 could be evaluated for clinical response according to the disease activity score (DAS) criteria. IgM, IgA, and IgG rheumatoid factors and anti-CCP antibodies were assessed by enzyme-linked immunosorbent assay before anti-TNFα treatment and one year later. The DAS response was reached in 66 percent of patients who could be evaluated (61% infliximab, 65% etanercept, and 76% adalimumab; P=.354). A significant reduction in the rheumatoid factor level was reported by all treatment groups after one year. The frequency of positive tests for the different antibodies did not vary between responders and nonresponders at baseline. However, significantly higher IgA rheumatoid factor levels were reported by the nonresponder group (130.4 U/mL [interquartile range, 13.8–276.7] versus 24.8 U/mL [10.2–90.8]; P=.003). A significant decrease (P<.001) in the levels of all rheumatoid factor isotypes in the responder group was reported after one year of treatment, whereas anti-CCP antibody levels were not significantly affected. The authors concluded that, according to the clinical response, anti-TNFα agents seem to reduce IgM, IgG, and IgA rheumatoid factor levels. Furthermore, high pretreatment levels of IgA rheumatoid factor are associated with a poor clinical response to TNFα inhibitors.

Bobbio-Pallavicini F, Caporali R, Alpini C, et al. High IgA rheumatoid factor levels are associated with poor clinical response to tumour necrosis factor α inhibitors in rheumatoid arthritis. Ann Rheum Dis. 2007;66:302–307.

Correspondence: C. Montecucco, Cattedra di Reumatologia, Policlinico S Matteo, 27100 Pavia, Italy; montecucco@smatteo.pv.it

Muscle tissue oxygenation in septic patients
Sepsis causes microcirculatory derangements characterized by decreased vascular density, a large number of nonperfused and intermittently perfused vessels, and heterogeneity of capillary transit time with an increase in the proportion of fast-flow to normal-flow times. These microcirculatory alterations can be due to various factors, including altered vascular tone, increased adhesion and aggregation of leukocytes, increased aggregation and decreased deformability of red blood cells, tissue edema, microthromboses in vessels, and capillary endothelial cell injury and swelling. Endothelial cell dysfunction can impair vascular reactivity to vasodilating and vasopressor substances and hypoxic stimuli. It has been possible to study these microvascular disturbances in a number of animal models, but this is difficult at the bedside. Near-infrared spectroscopy (NIRS) has been proposed as a tool to quantify microvascular dysfunction in patients with sepsis. Altered reactive hyperemia has been reported in septic patients using various techniques. However, all these studies included a relatively small number of patients. Therefore, the authors conducted a study to quantify the sepsis-induced alterations in changes in muscle tissue oxygenation using NIRS after a calibrated ischemic challenge in a large population of critically ill septic patients and to explore whether these alterations are related to outcome. The prospective study was conducted in the intensive care department of a 31-bed university hospital. Seventy-two patients with severe sepsis or septic shock, 18 hemodynamically stable, acutely ill patients without infection, and 18 healthy volunteers were given three-minute occlusion of the brachial artery using a cuff inflated 50 mmHg above systolic arterial pressure. Thenar eminence StO₂ was measured continuously by NIRS before (StO₂ baseline), during, and after the three-minute occlusion. Changes in StO₂ were assessed by the slope of increase in StO₂ during the first 14 seconds following the ischemic period and by the difference between the maximum StO₂ and StO₂ baseline (Δ). The authors found that the slope was lower in septic patients than in controls and volunteers (2.3 [1.3–3.6], 4.8 [3.5–6.0], and 4.7 [3.2–6.3]%/second; P<.001). Delta was also significantly lower in septic patients than in the other groups. Slopes were lower in septic patients with shock than without shock (2.0 [1.2–2.9] versus 3.2 [1.8– 4.5]%/second; P<.05). In 52 septic patients in whom the slope was obtained every 24 hours for 48 hours, slopes were higher in survivors than in nonsurvivors and tended to increase in survivors but not in nonsurvivors. The authors concluded that altered recovery in StO₂ after an ischemic challenge is frequent in septic patients and more pronounced in the presence of shock. The presence and persistence of these alterations in the first 24 hours of sepsis are associated with worse outcome.

Creteur J, Carollo T, Soldati G, et al. The prog­nostic value of muscle StO2 in septic patients. Intensive Care Med. 2007;33:1549– 1556.

Reprints: J.-L. Vincent, Erasme University Hospital, Free University of Brussels, Dept. of Intensive Care, Route de Lennik 808, 1070 Brussels, Belgium; jlvincen@ulb.ac.be

Unlabeled probes for herpes simplex virus
Real-time polymerase chain reaction (PCR) allows for the amplification, quantification, and detection of nucleic acid in a single-tube reaction. Detection of target can be achieved using synthetic oligonuleotide probes or by adding a double-stranded DNA (dsDNA) binding dye. Specificity may be increased by including a post-amplification melting analysis step to confirm target when using a dsDNA binding dye or certain types of probes. One method of real-time PCR detection and confirmation uses an unlabeled probe combined with asymmetric amplification. Unlabeled probes possess no fluorescent moiety; rather, they produce a characteristic melting signature when used with a dsDNA binding dye, such as LCGreen Plus (Idaho Technology). In its simplest form, the unlabeled probe technique generates two melting curves—one from dissociation of the unlabeled probe off single-stranded DNA (ssDNA) generated by asymmetric PCR and one from the homozygous dsDNA amplicon. Unlabeled probe and amplicon melting analysis have been used to detect small deletions and single-nucleotide polymorphisms in human RET and factor V genes, among others. Human genomic DNA is an ideal target for unlabeled probe assays since target copy number is rarely limited, but the use of unlabeled probes in assays that require low copy number sensitivity has not been reported. The authors described the limits of copy number detection, typing accuracy, and melting reproducibility with the use of unlabeled probes. Herpes simplex virus (HSV) was chosen to develop an unlabeled probe assay based on the availability of quantified DNA targets for HSV-1 and HSV-2, characterized clinical samples, the necessity of low-level detection, and an established in-house reference assay. The authors found that the unlabeled probe technique displayed 98 percent concordance with the reference assay for detecting HSV from a variety of archived clinical samples (n=182). HSV typing using unlabeled probes was 99 percent concordant (n=104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe. The authors concluded that unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detecting HSV and might be useful for other low-copy targets where typing is informative.

Dames S, Pattison DC, Bromley LK, et al. Unlabeled probes for the detection and typing of herpes simplex virus. Clin Chem. 2007; 53: 1847–1854.

Reprints: Shale Dames, ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108; shale.dames@aruplab.com