Clinical Abstracts

December 2005

Editors:
Michael Bissell, MD, PhD, MPH
Ronald Domen, MD

The group A Streptococcus metagenome

Group A Streptococcus is a gram-positive bacterial pathogen responsible for a wide range of human infections, including pharyngitis, impetigo, puerperal sepsis, necrotizing fasciitis, scarlet fever, and the post-infection sequelae glomerulonephritis and rheumatic fever. The genomes of four group A Streptococcus (GAS) strains have been characterized, including serotype M1, M3 (two strains), and M18 organisms. Each genome is polylysogenic—that is, each contains four to six prophages or prophage-like elements, most of which encode one or two extracellular secreted proteins thought to enhance fitness or increase virulence. Prophage-related open-reading frames compose a small minority of the total GAS genome, but they are responsible for up to 74 percent of the variation in gene content among different strains. Hence, prophages and related elements have contributed significantly to the evolution of GAS and to diversification of the genome. Given the critical importance of prophage-like elements in GAS biology, it is possible that sequencing the genomes of additional GAS strains will reveal novel attributes bearing on prophages and their role in host pathogen interactions. The authors tested this hypothesis by sequencing the genome of a serotype M6 GAS strain. The genome sequence was used to conduct further analysis of serotype M6 strains, resulting in a more detailed understanding of GAS biology. The authors described the genome sequence of a macrolide-resistant strain (MGAS10394) of serotype M6 GAS. The genome is 1,900,156 bp in length, and eight prophage-like elements or remnants compose 12.4 percent of the chromosome. An 8.3-kb prophage remnant encodes the SpeA4 variant of streptococcal pyrogenic exotoxin A. The genome of strain MGAS10394 contains a chimeric genetic element composed of prophage genes and a transposon encoding the mefA gene conferring macrolide resistance. This chimeric element also has a gene encoding a novel surface-exposed protein, designated R6 protein, with an LPKTG cell-anchor motif located at the carboxyterminus. Surface expression of this protein was confirmed by flow cytometry. Humans with GAS pharyngitis caused by serotype M6 strains had antibody against the R6 protein present in convalescent, but not acute, serum samples. The authors concluded that their studies support the viewpoint that GAS prophage-encoded extracellular proteins contribute to host-pathogen interactions in a strain-specific fashion.

Banks DJ, Porcella SF, Barbian KD, et al. Progress toward characterization of the group A Streptococcus metagenome: complete genome sequence of a macrolide-resistant serotype M6 strain. J Infect Dis. 2004;190:727-738.

Reprints: Dr. James M. Musser, Dept. of Pathology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030; musser@bcm.tmc.edu

Release of tPA in renal patients with hypertension

In the healthy blood vessel, activation of platelets or the coagulation cascade induces a massive release of the key enzyme of the fibrinolytic system, tissue-type plasminogen activator, from the endothelium. This acute tissue-type plasminogen activator (tPA) response probably constitutes a counter-regulatory mechanism to prevent a clotting process from progressing into an occlusive thrombus. If the capacity for acute tPA release is defective, the likelihood of timely spontaneous thrombolysis is reduced. The capacity for tPA release is markedly impaired in patients with untreated primary hypertension, likely due to elevated intraluminal pressure. Chronic kidney disease is frequently accompanied by hypertension and a markedly increased risk of atherothrombotic complications. Recent studies have indicated that chronic kidney disease is associated with an impaired function of the vascular endothelium similar to that observed in hypertension. Indeed, some early studies have shown that chronic kidney disease is associated with an impaired fibrinolysis as assessed by the global clot lysis time test. More specifically, stimulation of V₂-receptor-mediated tPA release by systemic administration of desmopressin has been found to induce a lesser increase in venous tPA levels in patients with renal failure than in healthy control subjects. However, the capacity to respond to a local thrombotic process by acute tPA release cannot be predicted from these studies because mixed venous plasma concentrations do not reflect regional secretion patterns. Furthermore, the short half-life of tPA in plasma (three to five minutes) makes plasma levels sensitive to changes in hepatic clearance rates. In this study, nine nondiabetic, nonsmoking men with chronic kidney disease (glomerular filtration rate, 11 to 28 mL/min. x 1.73 m²; ages 33 to 75 years) were compared with age-matched healthy controls. Intra-arterial infusions of desmopressin, methacholine, and sodium nitroprusside were given locally in the brachial artery. Forearm blood flow was measured by venous occlusion plethysmography and blood collected repeatedly during the desmopressin infusion to determine stimulated net and total cumulated release of tPA. The maximal release rate of active tPA (P<0.05) and the capacity for acute tPA release were markedly impaired in the renal patients as compared with healthy subjects (P=0.013). However, there were no significant differences in vasodilator responses between the groups. Thus patients with advanced chronic kidney disease and hypertension have a markedly impaired capacity for acute release of tPA, despite preserved endothelium-dependent vasodilation. This defect may contribute to a defective local defense against arterial thrombosis.

Hrafnkelsdóttir T, Ottosson P, Gudnason T, et al. Impaired endothelial release of tissue-type plasminogen activator in patients with chronic kidney disease and hypertension. Hypertension. 2004;44:300-304.

Reprints: Dr. Thórdis Hrafnkelsdóttir, Clinical Experimental Research Laboratory, Sahlgrenska University Hospital/Östra, SE 41685, Göteborg, Sweden; throdis.hrafnkelsdottir@hjl.gu.se

Use of rapid intraoperative parathyroid hormone testing

Parathyroidectomy is the treatment of choice for primary hyperparathyroidism and for secondary hyperparathyroidism that cannot be controlled with medical intervention. It traditionally has consisted of bilateral neck exploration to identify all four parathyroid glands, with removal of the abnormal glands. The parathyroid glands have been identified with the help of the pathologist using intraoperative evaluation with frozen section for confirmation. This approach has been highly successful over the years in the hands of experienced teams of surgeons and pathologists, but it is also time-consuming because it necessitates identifying all glands, not to mention a low but significant number of failures, with recurrent hypercalcemia in patients with primary hyperparathyroidism. The latter situation often necessitates re-exploratory surgery, which is more difficult and is associated with a higher incidence of complications. More recently, however, the aforementioned approach has been challenged by minimally invasive parathyroidectomies in cases of primary hyperparathyroidism. Two reasons for this change in the surgical approach are accurate pre-operative imaging and localization of abnormal parathyroid glands, and the use of rapid intra-operative parathyroid hormone (RI-PTH) measurements. The author reviewed 141 cases of parathyroidectomy aided by RI-PTH from Florida Hospital Medical Center, Orlando. He found that the orientation provided by the intraoperative assay is essential in guiding the surgeon in these minimally invasive procedures and that it helps reveal cases of primary hyperparathyroidism with involvement of more than one gland. Furthermore, it replaces the need for performing frozen sections, except for cases of secondary hyperparathyroidism.

Guarda LA. Rapid intraoperative parathyroid hormone testing with surgical pathology correlations. Am J Clin Pathol. 2004;122:704-712.

Reprints: Dr. Luis A. Guarda, Dept. of Pathology, Florida Hospital Medical Center, 601 E. Rollins St., Orlando, FL 32803

Hypertensive stress response in IL-6-deficient mice

Inflammatory cytokines such as interleukin-6 and C-reactive protein are increased in patients at greatest risk for cardiovascular disease, but it is not known if the cytokines are merely markers that correlate with increased risk for cardiovascular disease or if they play a mechanistic role. Emerging evidence indicates that inflammatory cytokines may contribute to the atherosclerotic process, and they correlate positively with blood pressure. Although evidence shows that the sympathetic nervous system and angiotensin II (Ang II) can stimulate interleukin (IL)-6 release and that IL-6 may be able to cause or facilitate vasoconstriction, there is little experimental evidence indicating that it plays a role in blood pressure control. To test this involvement, the authors chose an experimental model of psychosocial stress in mice. This model capitalizes on the central role of the sympathetic nervous system in the acute hypertensive response to stress and on the availability of knockout mouse models to test the role of IL-6 in blood pressure control. Thus, if IL-6 is important in mediating acute, sympathetic-driven hypertensive responses, a blunted increase in blood pressure should be measured in IL-6 knockout mice. The authors used a cage-switch model of psychosocial stress, in which a male mouse is placed in a cage previously occupied by another male mouse, and tested the role of IL-6 in mediating an acute increase in blood pressure by comparing responses in IL-6 mice versus wild-type mice. Male C57BL6 (wild type) and IL-6 knockout mice were implanted with biotelemetry devices to monitor mean arterial pressure, heart rate, and motor activity in the unrestrained state. Baseline mean arterial pressure was 98±1 and 103±1 for wild-type and IL-6 knockout mice. Cage switch increased mean arterial pressure by 41±2 mm Hg in wild-type mice, but this was blunted significantly in knockout mice (31±3 mm Hg peak increase). Area under the curve for the first 90 minutes was also significantly less. Heart rate and motor activity increased similarly, and there were no differences in the increases in plasma renin activity or plasma norepinephrine concentration between wild-type and knockout mice. The authors concluded that acute hypertensive response to psychosocial stress depends significantly on IL-6, and the effect appears to be specific to blood pressure rather than to a global impairment in response to stress. However, because perfusion of the isolated mesenteric bed with phenylephrine and chronic infusion of angiotensin II caused similar response in wild-type and IL-6 knockout mice, it is clear that additional studies are needed to determine to what extent the acute blood pressure effect of IL-6 is stress specific.

Lee DL, Leite R, Fleming C, et al. Hypertensive response to acute stress is attenuated in interleukin-6 knockout mice. Hypertension. 2004;44:259-263.

Reprints: Dr. Michael W. Brands, Dept. of Physiology, Medical College of Georgia, 1120 15th St., Augusta, GA 30912-3000; mbrands@mcg.edu

Cytokine and chemokine receptors in active rheumatoid arthritis

The inflamed synovium of patients with rheumatoid arthritis is characterized by massive leukocytic infiltration, primarily consisting of macrophages, T lymphocytes, and plasma cells. These cells express many proinflammatory cytokines, chemokines, and growth factors and are considered responsible for the degradation of cartilage and erosion of juxta-articular bone. To study the mechanisms in tumor necrosis factor alpha (TNFα), the authors examined the expression of chemokine receptors (CCR3, CCR5) and the secretion of rheumatoid arthritis interleukin (IL)-4, IL-5, interferon (IFN)γ, and TNFα in the peripheral blood T cells and monocytes of patients with active rheumatoid arthritis or other arthritides during early treatment with the monoclonal antibody infliximab. The mRNA expression of IFNγ, IL-4, IL-5, and TNFα in peripheral blood mononuclear cells was assessed with real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The authors also followed the changes in plasma levels of acute-phase reactants, such as C-reactive protein (CRP), serum amyloid protein A, rheumatoid factor, antifilaggrin antibodies, and antibodies to filaggrin-derived cyclic citrullinated peptide. Twenty-five patients with rheumatoid arthritis and five patients with other arthritides were studied during the first six weeks of treatment with infliximab. At the start of treatment and after two and six weeks, flow cytometry was used to study the spontaneous expression of CCR3 and CCR5 on peripheral blood T cells and monocytes. The secretion and mRNA expression of IFNγ, IL-4, IL-5, and TNFα from phytohemagglutinin-stimulated peripheral blood mononuclear cells was measured with an enzyme-linked immunosorbent assay and RT-PCR. Plasma levels of C-reactive protein, serum amyloid protein A, rheumatoid factor, and antibodies to filaggrin and citrullinated cyclic peptide were measured with an ELISA. The authors found that the number of CD4 T cells and CD14 monocytes expressing CCR3 (P=0.013, P=0.009, respectively) and CD8 T cells expressing CCR5 (P=0.040) as well as phytohemagglutinin-stimulated secretion of IL-4 and IFNγ (P<0.05) increased during treatment in patients with rheumatoid arthritis. Fifteen patients (60 percent) with rheumatoid arthritis achieved clinical response (at least ACR20) during the first two weeks. The number of T cells expressing CCR3 and CCR5 was higher before treatment in nonresponders than in responders (P<0.05). The number of T cells increased in responders. The authors concluded that an increase in secretion of Th1 and Th2 cytokines together with induced expression of chemokine receptors on T cells and monocytes suggests restoration of peripheral cell-mediated immunity and blockade of the accumulation of inflammatory cells in joints as a response to treatment.

Nissinen R, Leirisalo-Repo M, Peltomaa R, et al. Cytokine and chemokine receptor profile of peripheral blood mononuclear cells during treatment with infliximab in patients with active rheumatoid arthritis. Ann Rheum Dis. 2004;63:681-687.

Reprints: Dr. R. Nissinen, National Public Health Institute, Dept. of Molecular Medicine Biomedicum, P.O. Box 104, 00251 Helsinki, Finland; riikka.nissinen@ktl.fi

Deep vein thrombosis in rehabilitation patients

Deep vein thrombosis with or without pulmonary embolism is a common complication and cause of death in rehabilitation inpatients. These problems frequently interrupt the rehabilitation process. The incidence of deep vein thrombosis (DVT) after stroke, spinal cord injury, or traumatic brain injury in patients who have not received prophylaxis ranges from 16.4 percent to 100 percent. The incidence remains high even when prophylactic measures, such as intermittent pneumatic compression, compressive stockings, early mobilization, and medication, are used. It is difficult to diagnose DVT in rehabilitation patients because the clinical signs and symptoms of this condition are nonspecific. Most cases are identified by Doppler ultrasonography, plethysmography, or contrast venography, all of which are expensive and relatively time-consuming techniques. In many cases of suspected DVT, further investigation reveals no disease. A simple and sensitive screening test would avoid these unnecessary diagnostic procedures. Recent studies have suggested that the latex D-dimer assay can be used to exclude DVT and pulmonary embolism (PE) more quickly and in a less costly manner than other methods. The authors conducted a study to determine the value of D-dimer measurement in the diagnosis and exclusion of DVT and PE in rehabilitation inpatients with stroke, spinal cord injury, hip arthroplasty, or traumatic brain injury. They undertook a blind comparison of 68 consecutive inpatients being rehabilitated after stroke, spinal cord injury, hip arthroplasty, or traumatic brain injury in an inpatient rehabilitation facility in Turkey. A latex D-dimer assay was performed on patients at admission and then weekly throughout their hospital stay. Color Doppler ultrasonography of the lower limbs was also performed for patients at admission and was repeated when indicated by clinical signs and symptoms of DVT or elevated D-dimer levels. Patients' clinical findings, D-dimer test results, and ultrasonography results were recorded. Sensitivity, specificity, and positive and negative predictive values were calculated for the D-dimer test, each clinical finding, and combinations of D-dimer results and clinical findings in relation to DVT diagnosis. The sensitivity and negative predictive value of the D-dimer test were 95.2 percent and 96.2 percent, respectively. The specificity and positive predictive value were 55.3 percent and 48.7 percent, respectively. No single clinical finding was reliably diagnostic for DVT. The authors concluded that the D-dimer assay is a reliable method for ruling out DVT. In the rehabilitation setting, it can be used as a routine screening test or to assess cases of suspected DVT. D-dimer testing may reduce the need for sophisticated, time-consuming, and expensive diagnostic workup of rehabilitation inpatients, a group that is at increased risk for DVT.

Akman MN, Cetin N, Bayramoglu M, et al. Value of the D-dimer test in diagnosing deep vein thrombosis in rehabilitation inpatients. Arch Phys Med Rehabil. 2004;85:1091-1094.

Reprints: Dr. Mahmut Nafiz Akman, Dept. of Physical Medicine and Rehabilitation, Baskent University Faculty of Medicine, 5. sokak No. 48, Bahcelievler, 06490, Ankara, Turkey; nafiza@baskent-ank.edu.tr