In the search for novel cancer biomarkers, various proteolytically derived peptides have been proposed to exhibit cancer or cancer-type specificity. As these peptides are presumably also generated after sample collection by tumor-specific proteases, extensive investigation of the involved proteolytic processes is crucial for further research.
Materials and Methods
Using two previously developed and fully validated liquid-chromatography coupled to tandem-mass spectrometry assays, absolute quantification of, in total, 13 proteolytically derived peptides in human serum was accomplished. The analytes included eight peptides derived from inter-α-trypsin inhibitor heavy chain-4 (ITIH4-30, ITIH4-29, ITIH4-28, ITIH4-27, ITIH4-26, ITIH4-25, ITIH4-22, and ITIH4-21), bradykinin, des-Arg9-bradykinin, Hyp3-bradykinin, and fragments from fibrinogen-α-chain (Fib-α [605–629]) and complement component 4a (C4a [1337–1350]). Samples were obtained from different healthy individuals and prepared with variable tube types, clotting times, and temperatures. Furthermore, stabilities in the serum fraction were assessed and compared to stabilities in serum from breast cancer patients.
Results and Discussion
The quantitative analyses showed either increasing or decreasing serum concentrations during blood coagulation, while comparable effects were observed in serum separated from the blood clot. Furthermore, comparisons of inter- and intra-individual variations suggested better reflection of an individual’s protease activity after prolonged ex vivo incubation. This was illustrated for the putative breast cancer marker ITIH4-22, revealing better differentiation after incubation of serum at ambient temperature for 24 h.
The presented study provides suggestions for more specific and optimized sample preparation, as well as extended knowledge necessary to further explore the opportunities of these proteolytic peptides as cancer biomarkers.
Biomarker; Cancer; Protease activity; LC-MS/MS; Peptide; Sample handling
The inter-alpha-trypsin inhibitors (ITI) are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by AMBP – and five homologous heavy chains (encoded by ITIH1, ITIH2, ITIH3, ITIH4, and ITIH5), contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis.
We systematically investigated differential gene expression of the ITIH gene family, as well as AMBP and the interacting partner TNFAIP6 in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas) using cDNA dot blot analysis (Cancer Profiling Array, CPA), semiquantitative RT-PCR and immunohistochemistry.
We found that ITIH genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, ITIH genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose ITIH2 expression in human breast cancer. Loss of ITIH2 expression in 70% of cases (n = 50, CPA) could be confirmed by real-time PCR in an additional set of breast cancers (n = 36). Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p < 0.001) between ITIH2 expression and estrogen receptor (ER) expression indicating that ER may be involved in the regulation of this ECM molecule.
Altogether, this is the first systematic analysis on the differential expression of ITIH genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies.
Epigenetic mechanisms such as DNA hypermethylation and modifications of histone amino acids are known to play an important role in the control of gene expression both in normal human development and tumorigenesis. Hypermethylation of CpG islands within promoter regions of tumor suppressor genes is associated with transcriptional inactivation and represents, in addition to genetic aberrations, an important mechanism of gene silencing in the pathogenesis of human cancer. Inter-α-trypsine inhibitors (ITIs) are a family of serine protease inhibitors consisting of one light chain (bikunin) and two heavy chains (ITI heavy chains, ITIHs). ITIHs stabilize the extracellular matrix (ECM) by interacting with hyaluronic acid, which is a major ECM component. Hypermethylation in the upstream region of the promoter-associated CpG island of ITIH5, the most recently described member of the ITIH family, has been previously detected in breast cancer and was associated with an adverse outcome. In this study, we determined the DNA methylation status of the promoter region near the transcription start site of the ITIH5 tumor suppressor gene in leukemia cell lines and primary samples from patients with acute myeloid leukemia (AML) as well as the potential use of demethylating agents to restore a demethylated state of the promoter. Aberrant ITIH5 promoter hypermethylation occurred in 15 of 104 (14.4%) diagnostic AML samples. There were no statistically significant correlations between the ITIH5 methylation status and clinical prognostic parameters. Our results indicate that aberrant ITIH5 promoter hypermethylation is a novel epigenetic event in AML.
DNA hypermethylation; Epigenetics; Acute myeloid leukemia; Inter-α-trypsine inhibitor; Tumor suppressor gene
Diagnosis of ovarian carcinoma is in urgent need for new complementary biomarkers for early stage detection. Proteins that are aberrantly excreted in the urine of cancer patients are excellent biomarker candidates for development of new noninvasive protocol for early diagnosis and screening purposes. In the present study, urine samples from patients with ovarian carcinoma were analysed by two-dimensional gel electrophoresis and the profiles generated were compared to those similarly obtained from age-matched cancer negative women.
Significant reduced levels of CD59, kininogen-1 and a 39 kDa fragment of inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), and enhanced excretion of a 19 kDa fragment of albumin, were detected in the urine of patients with ovarian carcinoma compared to the control subjects. The different altered levels of the proteins were confirmed by Western blotting using antisera and a lectin that bind to the respective proteins.
CD59, kininogen-1 and fragments of ITIH4 and albumin may be used as complementary biomarkers in the development of new noninvasive protocols for diagnosis and screening of ovarian carcinoma.
The pancreatic islet comprises endocrine, vascular, and neuronal cells. Signaling among these cell types is critical for islet function. The extracellular matrix (ECM) is a key regulator of cell-cell signals, and while some islet ECM components have been identified, much remains unknown regarding its composition. We investigated whether hyaluronan, a common ECM component that may mediate inflammatory events, and molecules that bind hyaluronan such as versican, tumor necrosis factor–stimulated gene 6 (TSG-6), and components of inter-α-trypsin inhibitor (IαI), heavy chains 1 and 2 (ITIH1/ITIH2), and bikunin, are normally produced in the pancreatic islet. Mouse pancreata and isolated islets were obtained for microscopy (with both paraformaldehyde and Carnoy’s fixation) and mRNA. Hyaluronan was present predominantly in the peri-islet ECM, and hyaluronan synthase isoforms 1 and 3 were also expressed in islets. Versican was produced in α cells; TSG-6 in α and β cells; bikunin in α, β, and δ cells; and ITIH1/ITIH2 predominantly in β cells. Our findings demonstrate that hyaluronan, versican, TSG-6, and IαI are normal islet components and that different islet endocrine cell types contribute these ECM components. Thus, dysfunction of either α or β cells likely alters islet ECM composition and could thereby further disrupt islet function.
extracellular matrix; hyaluronan; islet; proteoglycan
The pancreatic islet comprises endocrine, vascular, and neuronal cells. Signaling among
these cell types is critical for islet function. The extracellular matrix (ECM) is a key
regulator of cell-cell signals, and while some islet ECM components have been identified,
much remains unknown regarding its composition. We investigated whether hyaluronan, a
common ECM component that may mediate inflammatory events, and molecules that bind
hyaluronan such as versican, tumor necrosis factor–stimulated gene 6 (TSG-6), and
components of inter-α-trypsin inhibitor (IαI), heavy chains 1 and 2 (ITIH1/ITIH2), and
bikunin, are normally produced in the pancreatic islet. Mouse pancreata and isolated
islets were obtained for microscopy (with both paraformaldehyde and Carnoy’s fixation) and
mRNA. Hyaluronan was present predominantly in the peri-islet ECM, and hyaluronan synthase
isoforms 1 and 3 were also expressed in islets. Versican was produced in α cells; TSG-6 in
α and β cells; bikunin in α, β, and δ cells; and ITIH1/ITIH2 predominantly in β cells. Our
findings demonstrate that hyaluronan, versican, TSG-6, and IαI are normal islet components
and that different islet endocrine cell types contribute these ECM components. Thus,
dysfunction of either α or β cells likely alters islet ECM composition and could thereby
further disrupt islet function.
extracellular matrix; hyaluronan; islet; proteoglycan
We have isolated from calf serum a protein with an apparent Mr of 120,000. The protein was detected by using antibodies against major acute-phase protein in pigs with acute inflammation. The amino acid sequence of an internal fragment revealed that this protein is the bovine counterpart of ITIH4, the heavy chain 4 of the inter-alpha-trypsin inhibitor family. The response of this protein in the sera was determined for animals during experimental bacterial and viral infections. In the bacterial model, animals were inoculated with a mixture of Actinomyces pyogenes, Fusobacterium necrophorum, and Peptostreptococcus indolicus to induce an acute-phase reaction. All animals developed moderate to severe clinical mastitis and exhibited remarkable increases in ITIH4 concentration in serum (from 3 to 12 times the initial values, peaking at 48 to 72 h after infection) that correlated with the severity of the disease. Animals with experimental infections with bovine respiratory syncytial virus (BRSV) also showed increases in ITIH4 concentration (from two- to fivefold), which peaked at around 7 to 8 days after inoculation. Generally, no response was seen after a second infection of the same animals with the virus. Because of the significant induction of the protein in the animals in the mastitis and BRSV infection models, we can conclude that ITIH4 is a new positive acute-phase protein in cattle.
A porcine genomic library was screened for clones containing the promoter of the major acute-phase protein in pigs, inter-α-trypsin heavy chain 4 (ITIH4). Following isolation of the promoter, a functional analysis was performed with Hep3B cells. The promoter was induced by interleukin-6 (IL-6) but not by IL-1β. However, IL-1β was shown to inhibit the IL-6-induced activation of the porcine ITIH4 promoter.
As critical mediators of local and systemic inflammatory responses, cytokines are produced in the brain following ischaemic stroke. Some have been detected in the circulation of stroke patients, but their role and source is unclear. Focusing primarily on interleukin(IL)-1-related mechanisms, we serially measured plasma inflammatory markers, and the production of cytokines by whole blood, from 36 patients recruited within 12 h and followed up to 1 year after acute ischaemic stroke (AIS).
Admission plasma IL-1 receptor antagonist (IL-1ra) concentration was elevated, relative to age-, sex-, and atherosclerosis-matched controls. IL-1β, soluble IL-1 receptor type II, tumour necrosis factor (TNF)-α, TNF-RII, IL-10 and leptin concentrations did not significantly differ from controls, but peak soluble TNF receptor type I (sTNF-RI) in the first week correlated strongly with computed tomography infarct volume at 5–7 days, mRS and BI at 3 and 12 months. Neopterin was raised in patients at 5–7 d, relative to controls, and in subjects with significant atherosclerosis. Spontaneous IL-1β, TNF-α and IL-6 gene and protein expression by blood cells was minimal, and induction of these cytokines by lipopolysaccharide (LPS) was significantly lower in patients than in controls during the first week. Minimum LPS-induced cytokine production correlated strongly with mRS and BI, and also with plasma cortisol.
Absence of spontaneous whole blood gene activation or cytokine production suggests that peripheral blood cells are not the source of cytokines measured in plasma after AIS. Increased plasma IL-1ra within 12 h of AIS onset, the relationship between sTNF-RI and stroke severity, and suppressed cytokine induction suggests early activation of endogenous immunosuppressive mechanisms after AIS.
This review provides a summary of the protein and RNA biomarkers that have been studied for the diagnosis and assessment of ischemic stroke. Many of the biomarkers identified relate to the pathophysiology of ischemic stroke, including ischemia of CNS tissue, acute thrombosis and inflammatory response. These biomarkers are summarized by their intended clinical application in ischemic stroke including diagnosis, prediction of stroke severity and outcome, and stratification of patients for stroke therapy. Among the biomarkers discussed are recent whole genome studies using RNA expression profiles to diagnose ischemic stroke and stroke etiology. Though many candidate blood based biomarkers for ischemic stroke have been identified, none are currently used in clinical practice. With further well designed study and careful validation, the development of blood biomarkers to improve the care of patients with ischemic stroke may be achieved.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-011-0050-4) contains supplementary material, which is available to authorized users.
Ischemic stroke; Biomarker
Blood biomarkers may improve the performance in predicting early stroke outcome beyond well-established clinical factors. We investigated the value of growth-differentiation factor-15 (GDF-15) to predict functional outcome after 90 days in a prospectively collected patient cohort with symptoms of acute ischemic stroke. Two hundred eighty-one patients with symptoms of acute ischemic stroke were prospectively investigated. Serial blood samples for GDF-15 analysis were obtained after the admission of the patient, after 6 and 24 h. Primary outcome was the dichotomized modified ranking scale (MRS) 90 days after the initial clinical event. Within the final study population (264 patients, mean age 70.3 ± 12.7 years, 55.3% male), National Institutes of Health Stroke Scale (NIH-SS) [odds ratio (OR) 1.269, 95% confidence interval (CI) 1.141–1.412, p < 0.001] and initial GDF-15 levels (OR 1.029, 95% CI 1.007–1.053, p = 0.011) were independently associated with a MRS ≥ 2 after day 90 after multiple regression analysis. Growth-differentiation factor-15 levels increase with higher NIH-SS-tertiles (p = 0.005). Receiver-operator characteristic curves demonstrated a discriminatory accuracy to predict unfavourable stroke outcome of 0.629 (95% CI 0.558–0.699), 0.753 (95% CI 0.693–812) and 0.774 (95% CI 0.717–0.832) for GDF-15, NIH-SS and the combination of these variables. The additional use of GDF-15 to NIH-SS ameliorates the model with a net reclassification index of 0.044 (p = 0.541) and integrated discrimination improvement of 0.034 (p = 0.443). Growth-differentiation factor-15 as an acute stroke biomarker independently predicts unfavourable functional 90 day stroke outcome. Discriminatory value in addition to NIH-SS is only modestly distinct.
GDF-15; Clinical trial; Ischemic stroke; TIA; Biomarker; Outcome
Background and Purpose
The National Institutes of Health Stroke Scale (NIHSS) score is known to be effective in predicting the likelihood of recovery after stroke. However, the baseline NIHSS score predicts long-term outcomes rather crudely because early changes in stroke scores may influence the stroke outcomes. Therefore, a precise prognostic algorithm or a cutoff point for predicting long-term outcomes based on data from serial NIHSS scores is needed.
We serially assessed 437 patients with acute symptomatic ischemic stroke within the middle cerebral artery territory who presented with nonlacunar stroke and were followed-up for at least 6 months after symptom onset. The NIHSS score was serially checked at 0, 1, 3, 7, and 14 days after admission. In all patients, the Barthel index (BI) and the modified Rankin Scale (mRS) score were checked, with a poor outcome defined as any of the following endpoints: death, modified mRS score of >3, or BI of <60.
A marked neurological improvement or worsening (i.e., a change in the NIHSS score of at least 4) was seen in 13.5% or 5.5% of the patients, respectively, during the first 7 days after admission. About 25% of the 437 patients had poor long-term outcomes. Analysis of receiver operating characteristic curves showed that the NIHSS score at day 7 after admission was better for predicting poor long-term outcomes than was the baseline score (P=0.003). In addition, we analyzed the cutoff point of the 7th-day NIHSS score for predicting a poor outcome at 6 months after symptom onset. An NIHSS score of at least 6 at day 7 after admission predicted poor long-term outcomes with a sensitivity of 84% [95% confidence interval (CI), 76-90%], a specificity of 92% (95% CI, 88-94%), and positive and negative predictive values of 77% and 95%, respectively. A logistic regression analysis revealed that age, diffusion-weighted imaging lesion volume, stroke history, and 7th-day NIHSS score were independently associated with poor outcome. However, no score used in addition to the 7th-day NIHSS score improved the prediction of a poor outcome.
An NIHSS score of at least 6 on day 7 after admission accurately forecasts a poor long-term outcome after stroke. Our data may be helpful in predicting the long-term prognosis as well as in making decisions regarding novel therapeutic applications in subacute-stroke trials.
Ischemic stroke; Outcome; Stroke scale
Background and Purpose
The most common strategy for treating patients with acute ischemic stroke is thrombolytic therapy, though only a few patients receive benefits because of the narrow time window. Inflammation occurring in the central nervous system (CNS) in association with ischemia is caused by immune cells including monocytes and involved in lesion expansion. If the specific roles of monocyte subsets in stroke can be revealed, they may become an effective target for new treatment strategies.
We performed immunological examinations of 36 consecutive ischemic stroke patients within 2 days of onset and compared the results with 24 age-matched patients with degenerative disorders. The stroke patients were repeatedly tested for the proportions of monocyte subsets in blood, and serum levels of pro- and anti-inflammatory cytokines immediately after admission, on days 3-7 and 12-16 after stroke onset, and on the day of discharge. In addition, immunological measurements were analyzed for relationships to stroke subtypes and complications, including progressive infarction (PI) and stroke-associated infection (SAI).
Monocyte count was significantly increased from 0–16 days after stroke as compared to the controls (p<0.05). CD14highCD16- classical and CD14highCD16+ intermediate monocytes were significantly increased from 0-7 and 3-16 days after stroke, respectively (p<0.05), whereas CD14 dimCD16high non-classical monocytes were decreased from 0–7 days (p<0.05). Cardioembolic infarction was associated with a persistent increase in intermediate monocytes. Furthermore, intermediate monocytes were significantly increased in patients with PI (p<0.05), while non-classical monocytes were decreased in those with SAI (p<0.05). IL-17A levels were positively correlated with monocyte count (r=0.485, p=0.012) as well as the percentage of non-classical monocytes (r=0.423, p=0.028), and negatively with that of classical monocytes (r=-0.51, p=0.007) during days 12-16.
Our findings suggest that CD14highCD16+ intermediate monocytes have a role in CNS tissue damage during acute and subacute phases in ischemic stroke especially in relation to cardioembolism.
Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) have neuroprotective effects in experimental stroke models and are commonly prescribed in clinical practice. The aim of this study was to determine if patients taking statins before hospital admission for stroke had an improved clinical outcome.
This was an observational study of 436 patients admitted to the National Institutes of Health Suburban Hospital Stroke Program between July 2000 and December 2002. Self-reported risk factors for stroke were obtained on admission. Stroke severity was determined by the admission National Institutes of Health Stroke Scale score. Good outcome was defined as a Rankin score < 2 at discharge. Statistical analyses used univariate and multivariate logistic regression models.
There were 436 patients with a final diagnosis of ischemic stroke; statin data were available for 433 of them. A total of 95/433 (22%) of patients were taking a statin when they were admitted, rising from 16% in 2000 to 26% in 2002. Fifty-one percent of patients taking statins had a good outcome compared to 38% of patients not taking statins (p = 0.03). After adjustment for confounding factors, statin pretreatment was associated with a 2.9 odds (95% CI: 1.2–6.7) of a good outcome at the time of hospital discharge.
The proportion of patients taking statins when they are admitted with stroke is rising rapidly. Statin pretreatment was significantly associated with an improved functional outcome at discharge. This finding could support the early initiation of statin therapy after stroke.
Study aimed at investigation of pathogenic role and prognostic value of several selected cerebrospinal fluid acute phase factors that can reflect the severity of ischemic brain damage.
Ninety five acute ischemic stroke patients were investigated. Ischemic region visualized at the twenty fourth hour by conventional Magnetic Resonance Imaging. Stroke severity evaluated by National Institute Health Stroke Scale. One month outcome of disease was assessed by Barthel Index. Cerebrospinal fluid was taken at the sixth hour of stroke onset. CSF pro- and anti-inflammatory cytokines were studied by Enzyme Linked Immunosorbent Assay. Nitric Oxide and Lipoperoxide radical were measured by Electron Paramagnetic Resonance. CSF Nitrate levels were detected using the Griess reagent. Statistics performed by SPSS-11.0.
At the sixth hour of stroke onset, cerebrospinal fluid cytokine levels were elevated in patients against controls. Severe stroke patients had increased interleukin-6 content compared to less severe strokes (P < 0.05). Cerebrospinal fluid Electron Paramagnetic Resonance signal of nitric oxide was increased in patients against controls. Severe stroke group had an elevated Electron Paramagnetic Resonance signal of lipoperoxiradical compared to less severe stroke. Cerebrospinal fluid nitrate levels in less severe stroke patients were higher than those for severe stroke and control. Positive correlation was established between the initial interleukin-6 content and ischemic lesion size as well as with National Institute Health Stroke Scale score on the seventh day. Initial interleukin-6 and nitrate levels in cerebrospinal fluid found to be significant for functional outcome of stroke at one month.
According to present study the cerebrospinal fluid contents of interleukin-6 and nitrates seem to be the most reliable prognostic factors in acute phase of ischemic stroke.
brain; ischemia; inflammation; oxidative stress
The purpose of the present study was to investigate the association between admission clinical characteristics and outcomes at discharge among acute ischemic stroke patients in the Chinese population. A total of 2,673 patients with acute ischemic stroke were included in the present study. The clinical characteristics at admission and other study variables were collected for all patients. The study outcome was defined as neurological deficiency (National Institute of Health Stroke Scale score ≥10) at discharge or in-hospital death. Compared with the subjects without neurological deficiency at discharge or in-hospital death, the subjects with neurological deficiency at discharge or in-hospital death had a significantly higher prevalence of hyperglycemia or history of atrial fibrillation at admission. Age ≥ 80 years, hyperglycemia, hypertension, and history of atrial fibrillation were significantly associated with neurological deficiency at discharge or in-hospital death after adjustment for other variables. It is concluded that old age (≥80 years), hyperglycemia, hypertension and history of atrial fibrillation are significantly associated with neurological deficiency at discharge or in-hospital death among patients with acute ischemic stroke.
acute ischemic stroke; clinical characteristics; death; neurological deficiency; discharge outcome
Inflammatory cytokines or cytokine inducers can alter basal ganglia activity, including reducing responsiveness to rewarding stimuli that may be mediated by cytokine effects on dopamine function.
To determine whether long-term administration of the inflammatory cytokine interferon alfa reduces the basal ganglia response to reward and whether such changes are associated with decreased presynaptic striatal dopamine function and altered behavior.
Cross-sectional and longitudinal studies.
Outpatient research unit and neuroimaging facilities at Emory University, Atlanta, Georgia.
Medically stable adults with chronic hepatitis C virus (HCV) infection eligible for interferon alfa treatment.
Main Outcome Measures
Neural activity in the ventral striatum during a hedonic reward task as measured by functional magnetic resonance imaging, uptake and turnover of radiolabeled fluorodopa F 18 (18F-dopa) in caudate and putamen using positron emission tomography, and interferon alfa–induced depression, anhedonia, fatigue, and neurotoxicity.
Patients with HCV receiving interferon alfa for 4 to 6 weeks (n=14) exhibited significantly reduced bilateral activation of the ventral striatum in the win vs lose condition of a gambling task compared with patients with HCV awaiting interferon alfa treatment (n=14). Reduced activation of the ventral striatum was, in turn, significantly correlated with anhedonia, depression, and fatigue. In a separate longitudinal study, patients with HCV treated with interferon alfa for 4 to 6 weeks (n=12) exhibited significantly increased 18F-dopa uptake and decreased 18F-dopa turnover in caudate and putamen and in the same ventral striatal regions identified in the functional magnetic resonance imaging study. Baseline and percentage change in 18F-dopa uptake and turnover were correlated with behavioral alterations, including depression, fatigue, and neurotoxicity, during interferon alfa administration.
These data replicate and extend findings that inflammatory stimuli, including inflammatory cytokines, such as interferon alfa, alter basal ganglia activity and behavior in association with significant changes in presynaptic striatal dopamine function consistent with decreased dopamine synthesis or release.
Pneumonia is the most common cause of mortality in stroke patients and it has been demonstrated to contribute to mortality and poor functional outcome following stroke in the majority of clinical studies. The risk of infection may be attributed to stroke-induced immunodepression syndrome (SIDS). Cytokine production is increased in SIDS. However, the correlation between biomarkers and the risk of post-stroke pneumonia in patients with diabetes mellitus is not clear. The aim of this study was to determine the correlation between pneumonia and the levels of C-reactive protein (CRP) and interleukin-6 (IL-6), as well as to identify early predictors of pneumonia in acute ischemic stroke patients with diabetes mellitus. Additionally, we investigated the impact of pneumonia on functional outcome after 1 month. A total of 106 ischemic stroke patients with diabetes mellitus who were admitted after the onset of symptoms were included in the study. They were divided into two groups, the pneumonia and non-pneumonia groups. CRP, IL-6, white blood cells (WBCs), mean body temperature and National Institutes of Health Stroke Scale (NIHSS) score were measured at the time of admission. The modified Rankin Scale score was assessed at 30 days. The levels of IL-6, CRP and WBCs, as well as mean body temperature were significantly higher in the patients with pneumonia than in the patients without pneumonia. There were also significant differences between the pneumonia and non-pneumonia groups in age, admission NIHSS score, length of hospital stay and dysphagia. Pneumonia patients had worse outcomes compared with patients without pneumonia at 1 month. Age, NIHSS score and dysphagia were significantly associated with pneumonia. WBCs and mean body temperature were not significant predictors of pneumonia. Older patients with more severe ischemic stroke are more susceptible to the development of pneumonia during the stay in hospital. Pneumonia contributes to poor functional outcome. IL-6, CRP, age, NIHSS score and dysphagia may predict the occurrence of pneumonia on the day of stroke symptom onset.
acute ischemic stroke; C-reactive protein; interleukin-6; pneumonia; diabetes mellitus
Pyrexia after stroke (temperature ≥37.5°C) is associated with poor prognosis, but information on timing of body temperature changes and relationship to stroke severity and subtypes varies.
We recruited patients with acute ischemic stroke, measured stroke severity, stroke subtype and recorded four-hourly tympanic (body) temperature readings from admission to 120 hours after stroke. We sought causes of pyrexia and measured functional outcome at 90 days. We systematically summarised all relevant previous studies.
Amongst 44 patients (21 males, mean age 72 years SD 11) with median National Institute of Health Stroke Score (NIHSS) 7 (range 0–28), 14 had total anterior circulation strokes (TACS). On admission all patients, both TACS and non-TACS, were normothermic (median 36.3°C vs 36.5°C, p=0.382 respectively) at median 4 hours (interquartile range, IQR, 2–8) after stroke; admission temperature and NIHSS were not associated (r2=0.0, p=0.353). Peak temperature, occurring at 35.5 (IQR 19.0 to 53.8) hours after stroke, was higher in TACS (37.7°C) than non-TACS (37.1°C, p<0.001) and was associated with admission NIHSS (r2=0.20, p=0.002). Poor outcome (modified Rankin Scale ≥3) at 90 days was associated with higher admission (36.6°C vs. 36.2°C p=0.031) and peak (37.4°C vs. 37.0°C, p=0.016) temperatures. Sixteen (36%) patients became pyrexial, in seven (44%) of whom we found no cause other than the stroke.
Normothermia is usual within the first 4 hours of stroke. Peak temperature occurs at 1.5 to 2 days after stroke, and is related to stroke severity/subtype and more closely associated with poor outcome than admission temperature. Temperature-outcome associations after stroke are complex, but normothermia on admission should not preclude randomisation of patients into trials of therapeutic hypothermia.
Ischemic stroke; Tympanic body temperature; Pyrexia; Outcome; OCSP
Early seizure (ES) may complicate the clinical course of patients with acute stroke. The aim of this study was to assess the rate of and the predictive factors for ES as well the effects of ES on the clinical outcome at hospital discharge in patients with first-ever stroke.
Patients and methods
A total of 638 consecutive patients with first-ever stroke (543 ischemic, 95 hemorrhagic), admitted to our Stroke Unit, were included in this prospective study. ES were defined as seizures occurring within 7 days from acute stroke. Patients with history of epilepsy were excluded.
Thirty-one patients (4.8%) had ES. Seizures were significantly more common in patients with cortical involvement, severe and large stroke, and in patient with cortical hemorrhagic transformation of ischemic stroke. ES was not associated with an increase in adverse outcome (mortality and disability). After multivariate analysis, hemorrhagic transformation resulted as an independent predictive factor for ES (OR = 6.5; 95% CI: 1.95–22.61; p = 0.003).
ES occur in about 5% of patients with acute stroke. In these patients hemorrhagic transformation is a predictive factor for ES. ES does not seem to be associated with an adverse outcome at hospital discharge after acute stroke.
seizures; stroke; cortical involvement; hemorrhagic transformation
The objectives of this study were to analyze the outcome and hemorrhagic risk of intravenous (IV) argatroban in patients with acute ischemic stroke presenting beyond six hours of ischemic symptom onset.
Eighty patients with acute ischemic stroke who were admitted to the hospital beyond six hours from ischemic symptom onset were retrospectively analyzed. We could not perform IV thrombolysis or intra-arterial thrombolysis because of limited time window. So, IV argatroban was performed to prevent recurrent thrombosis and progression of infarcted area. The outcome was assessed by the National Institute of Health Stroke Scale (NIHSS) score and related hemorrhagic risk was analyzed. Also, each outcome was analyzed according to the initial stroke severity, subtype, and location.
The median NIHSS was 8.0 at admission, 4.1 upon discharge, and 3.3 after three months. A good outcome was achieved in 81% of patients upon discharge and 88% after three months. Symptomatic hemorrhage occurred in only two patients (3%). IV argatroban was effective regardless of initial stroke severity, subtype, and location.
IV argatroban may be an effective and safe treatment modality for acute ischemic stroke presenting beyond six hours of ischemic symptom onset.
Acute ischemic stroke; Intravenous argatroban
Stroke is the third major cause of death and foremost cause of disability worldwide. Cerebrovascular stroke remains largely a clinical diagnosis. The use of biomarkers in diagnosing stroke and assessing prognosis is an emerging and rapidly evolving field. The study aimed to investigate the predictive value of neurobiochemical marker of brain damage (neuron-specific enolase [NSE]) with respect to degree of disability at the time of admission and neurological worsening in acute ischemic stroke patients. We investigated 150 patients with cerebrovascular stroke who were admitted within 72 h of onset of stroke in the Department of Neurology at SAIMS. Venous blood samples were taken after admission and NSE was analyzed by solid enzyme linked immunosorbent assay using Analyzer and microplate reader from Biored: Code 680. In all patients, the neurological status was evaluated by a standardized neurological examination and the National Institutes of Health Stroke Scale on admission and on day 7. Serum NSE concentration was found to significantly correlate with both degree of disability and neurological worsening in acute ischemic stroke cases in the present study. The maximum serum NSE level within 72 h of admission was significantly higher in patients with greater degree of disability at the time of admission. Serum NSE levels were also found to be significantly elevated in patients with bad neurological outcome. Our study showed that serum NSE has high predictive value for determining severity and early neurobehavioral outcome after acute stroke.
Ischemic stroke; Neuron specific enolase (NSE); National Institute of Health Stroke Scale (NIHSS); Degree of disability; Neurological worsening
Systems of stroke care delivery have been promoted as a means of improving the quality of stroke care, but little is known about their effectiveness. We assessed the effect of the Ontario Stroke System, a province-wide strategy of regionalized stroke care delivery, on stroke care and outcomes in Ontario, Canada.
We used population-based provincial administrative databases to identify all emergency department visits and hospital admissions for acute stroke and transient ischemic attack from Jan. 1, 2001, to Dec. 31, 2010. Using piecewise regression analyses, we assessed the effect of the full implementation of the Ontario Stroke System in 2005 on the proportion of patients who received care at stroke centres, and on rates of discharge to long-term care facilities and 30-day mortality after stroke.
We included 243 287 visits by patients with acute stroke or transient ischemic attack. The full implementation of the Ontario Stroke System in 2005 was associated with an increase in rates of care at stroke centres (before implementation: 40.0%; after implementation: 46.5%), decreased rates of discharge to long-term care facilities (before implementation: 16.9%; after implementation: 14.8%) and decreased 30-day mortality for hemorrhagic (before implementation: 38.3%; after implementation: 34.4%) and ischemic stroke (before implementation: 16.3%; after implementation: 15.7%). The system’s implementation was also associated with marked increases in the proportion of patients who received neuroimaging, thrombolytic therapy, care in a stroke unit and antithrombotic therapy.
The implementation of an organized system of stroke care delivery was associated with improved processes of care and outcomes after stroke.
Inflammatory cytokines including the IL-1 family, TNF-α and IL-6 mediate the formation of thrombosis on the luminal surface of atherosclerotic plaques. Gene polymorphisms that regulate these cytokines’ expression may explain part of the variation in susceptibility to stroke in patients with carotid atherosclerosis. The aim of this study is to evaluate the role of single-nucleotide polymorphisms (SNPs) and haplotypes in inflammatory genes as they relate to symptomatic carotid atherosclerosis.
The study included 95 subjects with symptomatic (transient ischemic attacks (TIA) or stroke) and 113 subjects with asymptomatic carotid atherosclerotic disease. A panel of evenly spaced SNPs including previously reported functionally significant polymorphisms were genotyped for IL-1β (10 SNPs), IL-1α (9 SNPs), IL-1RN (11 SNPs), IL-6 (7 SNPs) and TNF-α and TNF-β (7 SNPs).
Using single SNP analysis, IL-1RN rs315934 (p=0.025), IL-1RN rs315946 (p=0.042), IL-1RN rs315921 (p=0.035), IL-6 rs1180243 (p=0.018), and IL-1α rs2071373 (p=0.025) were associated with decreased odds of symptomatic carotid disease. Additionally, two diplotypes of the IL-1RN gene (p=0.023 and p=0.0064) and one diplotype in the IL-1α gene (p=0.02) were associated with a protective affect from cerebral ischemic events. Logistic analysis for interaction of the protective SNPs reveal an additive effect of all SNP pair combinations.
These results suggest genetic polymorphisms in pro-inflammatory genes may contribute to inter-individual differences in the development of symptomatic carotid atherosclerotic disease.
stroke; atherosclerosis; inflammatory; immune genes; single nucleotide polymorphism; haplotype
Insulin resistance (IR) may not only increase stroke risk, but could also contribute to aggravate stroke prognosis. Mainly through a derangement in endogenous fibrinolysis, IR could affect the response to intravenous thrombolysis, currently the only therapy proved to be efficacious for acute ischemic stroke. We hypothesized that high IR is associated with more persistent arterial occlusions and poorer long-term outcome after stroke thrombolysis.
RESEARCH DESIGN AND METHODS
We performed a prospective, observational, longitudinal study in consecutive acute ischemic stroke patients presenting with middle cerebral artery (MCA) occlusion who received intravenous thrombolysis. Patients with acute hyperglycemia (≥155 mg/dL) receiving insulin were excluded. IR was determined during admission by the homeostatic model assessment index (HOMA-IR). Poor long-term outcome, as defined by a day 90 modified Rankin scale score ≥3, was considered the primary outcome variable. Transcranial Duplex-assessed resistance to MCA recanalization and symptomatic hemorrhagic transformation were considered secondary end points.
A total of 109 thrombolysed MCA ischemic stroke patients were included (43.1% women, mean age 71 years). The HOMA-IR was higher in the group of patients with poor outcome (P = 0.02). The probability of good outcome decreased gradually with increasing HOMA-IR tertiles (80.6%, 1st tertile; 71.4%, 2nd tertile; and 55.3%, upper tertile). A HOMA-IR in the upper tertile was independently associated with poor outcome when compared with the lower tertile (odds ratio [OR] 8.54 [95% CI 1.67–43.55]; P = 0.01) and was associated with more persistent MCA occlusions (OR 8.2 [1.23–54.44]; P = 0.029).
High IR may be associated with more persistent arterial occlusions and worse long-term outcome after acute ischemic stroke thrombolysis.