Novel Inflammatory Biomarkers and Their Correlation to Chlamydia pneumoniae Titres in Acute Ischemic Stroke M. V. Padma Srivastava, MBBS, MD, DM, FAMS, FNASc,* Ashu Bhasin, PhD,* Rama Chaudhry, MD,† Sakshi Sharma, MSW,* Vivekanandhan Subbaiah, PhD,‡ Rohit Bhatia, MBBS, MD, DM, DNB,* and Manjari Tripathi, DM*
Background: Young stroke patients constitute 15%-30% of all stroke patients in India as against 3.0%-8.5% reported from the West. The mechanisms for stroke in the young may include unconventional risk factors such as infections. We aimed to investigate the role (if any) of Chlamydia pneumoniae antibodies in young patients with acute ischemic stroke (AIS). Several proinflammatory cytokines and biomarkers are released early after the onset of brain ischemia. We assessed the role of heat shock protein (hsp) 65, neopterin, and myeloperoxidase upregulation after AIS in predicting stroke severity. We also assessed relationship of upregulated inflammatory biomarkers with C pneumoniae antibody titres (IgG, IgA, and IgM). Methods: Eighty acute stroke patients and healthy age- and sex-matched controls were recruited. Blood samples were drawn within 1 week from the onset of stroke. Detection of IgA, IgG, and IgM antibodies to C pneumoniae was done with a validated microimmunofluorescence technique from 5 mL of serum in all subjects. Inflammatory biomarkers such as neopterin, myeloperoxidase and hsp 65 were estimated with sandwich enzyme linked immunosorbent assay (ELISA) method. Results: hsp 65 and neopterin were significantly elevated in all stroke patients with respect to healthy controls (odds ratio [OR], 4.9; 95% confidence interval [CI], 23.5-67.8; P 5 .001 and OR, 4.4; 95% CI, 2.089.4; P 5.04, respectively). Eighty-one percent of cases were seropositive for IgA versus 32% of controls (P 5 .003), and IgG was positive in 52.7% versus 17.3% of controls (P 5 .05). Myeloperoxidase levels were similar in patients and controls. Correlation and multiple regression indicated a high level of predictability and sensitivity of hsp 65 to IgA. C. pneumoniae antibody titres when all other variables were constant (F [4,90] 5 26.8, P 5 .001). Patients with high NIHSS scores (.15) had elevated levels of hsp 65 (mean, 13.2 ng/mL) suggesting correlation with stroke severity. Conclusions: The study demonstrated high levels of hsp 65 and neopterin levels in AIS correlated to significantly elevated IgA titres of C pneumoniae. Elevated levels of hsp 65 were associated with stroke severity. Key Words: Acute ischemia—inflammatory biomarkers— C pneumoniae—stroke severity. Ó 2014 by National Stroke Association
Introduction Stroke in young, including stroke in children and young adults (,45 years), is an important cause of From the *Department of Neurology, AIIMS, New Delhi; †Department of Microbiology, All India Institute of Medical Sciences, New Delhi; and ‡Department of Neurobiochemistry, All India Institute of Medical Sciences, New Delhi, India. Received March 21, 2014; revision received May 9, 2014; accepted May 16, 2014.
morbidity and mortality throughout the world, especially in developing countries.1 Young stroke patients constitute 15%-30% of all stroke patients in India, vis
Address correspondence to M. Vasantha Padma Srivastava, MBBS, MD, DM, FAMS, FNASc, Department of Neurology, All India Institute of Medical Sciences, New Delhi, India. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2014.05.016
Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 9 (October), 2014: pp 2391-2396
2391
M.V.P. SRIVASTAVA ET AL.
2392
a vis 3.0%-8.5% of all stroke patients reported from the West.2 Serologic evidence of past infection with Chlamydia pneumoniae has been found in epidemiologic studies to be associated with risk for atherosclerosis and cardiac disease, although prospective cohort studies have not always confirmed this association.3 Because of heterogeneity of etiopathogenesis of stroke and stroke subtypes, the association of C pneumoniae infection with ischemic stroke can be more complex. Recent bacterial or viral infection has been shown to be associated with acute ischemic stroke (AIS). The link between C pneumoniae and cerebrovascular disease has been investigated in a number of seroepidemiologic and antibiotic intervention studies.4 However, the role of C pneumoniae infection in acute stroke as a cause/effect/trigger remains controversial. Because infectious diseases in general are more common in India, compared with Western countries, we aimed to investigate the role (if any) of C pneumoniae antibodies in patients with AIS. Recent reports from South India suggest positive association of C pneumoniae infection with the occurrence of AIS. Our previous study also confirmed the same.5,6 The present study was undertaken to investigate the possible role of C pneumoniae in young stroke patients. The role of inflammatory markers in ischemic cascade after AIS has been widely studied. All stages of the atherosclerotic plaque—initiation, growth, and complications— might be considered to be an inflammatory response to injury. Potential targets, which could help to identify and monitor the ongoing inflammatory7,8 process are Creactive protein (CRP), interleukins (IL-1,6), tumor necrosis factor-a, neopterin, myeloperoxidase, heat shock protein (hsp) 65 and others. There has been some literature published on the predictor ability of CRP for both prognosis and occurrence of AIS.9,10 Although many blood markers have been investigated to date, no single blood marker has been proven to be clinically useful to predict stroke severity and outcomes. We investigated the role of hsp 65, neopterin, and myeloperoxidase in predicting the severity of AIS and their correlation to the titres of C pneumoniae.
selected for the previously mentioned analysis were healthy age- and sex-matched subjects, who did not have any history of cerebrovascular disease. Blood samples were drawn within 1 week from the onset of stroke. In each case, 5 mL of serum was obtained by centrifugation (699 g for 15 minutes at 14 C) and then stored at 70 C until analysis. Detection of IgA, IgG, and IgM antibodies to C pneumoniae was done with a validated microimmunofluorescence technique. The cutoff point for seropositivity was 1/16 for IgA and 1/64 for IgG. Inflammatory biomarkers such as hsp 65, neopterin, and myeloperoxidase were estimated using commercially available high sensitivity quantitative sandwich enzyme immunoassays according to the manufacturer’s instructions (R & D Systems, Minneapolis, MN). This study was cleared by the institute review board, and written informed consent was obtained from all patients.
Statistical Methods Analysis of data was carried out using SPSS statistical analysis software (SPSS Inc, Chicago, IL). For continuous variables such as age and NIHSS scores, descriptive statistics was calculated and reported as means 6 standard deviation. Normality of distribution of continuous variables was verified using the parametric tests.
Results This study was a case–control design in which 80 AIS patients were recruited with the same number of healthy age-matched controls. The mean age in the study group was 43.6 6 6.7 years and 43.2 6 7.3 years in controls (male:female ratio 5 2:1). Standard stroke care was administered to all patients. The risk factors found in stroke patients were hypertension (38%), diabetes mellitus (23.4%), hypercholesterolemia (29.4%), and smoking (22.3%; Table 1). hsp65 was found elevated significantly (mean of 30.4 ng/mL in 58 patients vs. none in controls, P , .05). Neopterin levels were found elevated in 50% (40 patients vs. 14 controls, with a mean of 19.26 mg/mL Table 1. Risk factors in study and control groups
Methods The criteria for inclusion were patients in the age group of 18-45 years, presenting with AIS within a week of onset. Complete medical history of hypertension, smoking, diabetes mellitus, hypercholesterolemia, ischemic heart disease, stroke, or TIA was recorded. Neurologic evaluation included recording of National Institute of Health Stroke Scale (NIHSS). noncontrast computed tomography head was performed in all, and if needed magnetic resonance imaging/computed tomography angiography/magnetic resonance angiography was performed according to the clinical requirement. Controls
Risk factors
Patients, %
Controls, %
Hypertension Hypercholesterolemia Diabetes mellitus Smoking Alcohol h/o cardiac disease Atrial fibrillation Migraine Family h/o stroke
38 29.4 23.4 22.35 22.3 21.1 1.17 7.05 20.14
11 18 15 12 16 8.5 0 1.5 12.2
Abbreviation: h/o, history of.
NOVEL INFLAMMATORY BIOMARKERS AND C. PNEUMONIAE TITRES IN AIS
2393
7.5mg/mL (r 5 .85, P 5.002; graph 2), whereas moderately affected patients had lower levels of hsps and neopterin levels (mean, 7.1 ng/mL and 32, mg/mL, respectively; Fig 2).
Discussion
Figure 1. Mean values of biomarkers levels in all subjects. hsp 65 and neopterin levels were significantly high in stroke patients compared with controls.
[normal range, ,3 m/mL]; odds ratio [OR], 4.4; 95% confidence interval [CI], 2.08-9.4; P 5 .04). Myeloperoxidase levels were near normal in both patients and controls (OR, 1.9; 95% CI, .21-7.1; P 5 .56; Fig 1). Seropositivity for IgA titers of C pneumoniae was 81% in cases versus 32% of controls (P 5 .003; Table 2). This difference was less striking for IgG, 52.7% of cases were seropositive versus 17.3% of controls (P 5.05). IgM antibodies were found to be similar and negative in patients as well as in controls (P . .05). Correlation and multiple regression analyses were performed with hsp 65 and neopterin as dependent variables to independent antibody titres of IgA, IgG, and IgM. hsp 65 had positive regression weights indicating that patients with a high hsp level predicted a high C pneumoniae IgA antibody (F [4,90] 5 16.8, P 5.001). The results were statistically significant accounting to 21.2% variability of hsp 65 with IgG (P 5.02). The regression model for neopterin also indicated a good level of prediction for IgA antibodies (R2 5 .78, F 5 6.38, P , .05), whereas it was nonsignificant for myleoperoxidase levels. The receiver operating curve (ROC) for hsp 65 showed a high sensitivity with a threshold of .95 for IgA antibody of C pneumoniae compared with IgG and IgM. We divided patients according to their severity as mild to moderately affected (NIHSS score between 0 and 15) and severely affected and critically ill with NIHSS score greater than 15. Bivariate correlation analysis was performed to correlate the severity with the biomarker levels. It was observed that patients with NIHSS score of more than 15 had a mean hsp 65 of 13.2 ng/mL and neopterin
This case–control study provides evidence of an association between acute infection with C pneumoniae (IgA antibodies) and the risk of AIS in young patients (,45 years).11,12 Many studies demonstrated a positive association between C pneumoniae infection and stroke, yielding a OR of 1.71-8.58, and several did not.13,14 Table 3 summarizes the results seen in various studies. C pneumoniae IgG antibodies may be considered as markers of ‘‘chronic’’ infection, and they could be in circulation for 3-5 years after infection.15,16 The IgA antibodies may be considered as markers of a primary, acute, or chronic infection and stay in circulation on an average of 3-5 days or longer. C pneumoniae infection has been implicated to have a role in enhancing systemic inflammation and immune-mediated vascular damage, altering lipid metabolism, inducing the production of cytokines and lipoproteins. These changes may adversely modify the conventional risk factors and aggravate atherosclerosis.17,18 C pneumoniae is phagocytosed by alveolar macrophages and transported by blood stream to the vascular subendothelial region through the injured arterial endothelium. Several studies demonstrated the presence of C pneumoniae in atherosclerotic lesions in coronary arteries, carotid arteries, and aorta obtained from autopsy and endarterectomy specimens. One Indian study, which has shown association of high CRP in stroke patients lends further credence to the hypothesis that infection with C pneumoniae, acute or chronic, may be associated with atherosclerosis through the mechanisms of low-grade inflammation.19 This mechanism has been further supported by the finding of increased carotid intima-media thickness in asymptomatic patients who are seropositive for C pneumoniae. However, 2 large randomized controlled trials did not observe a beneficial effect of antichlamydial antibiotic therapy on the secondary prevention of cardiovascular heart disease.20,21 Neither trial used the serologic marker of chronic C pneumoniae infection during patient selection. Stroke also is far more heterogenous in nature than acute coronary syndrome and both cannot be equated
Table 2. Seropositivity of IgA, IgM, IgG antibodies to Chlamydia pneumoniae IgA
Patients Controls P value
IgG
IgM
Positive, %
Negative, %
Positive, %
Negative, %
Positive, %
Negative, %
81 32
19 63
52.7 33
15.4 62
4.54 3
88.1 96
.003
.05
.12
M.V.P. SRIVASTAVA ET AL.
2394
Hsp
Neopterin
mean scores
20 15 10 5 0 5-15NIHSS
>15NIHSS
Grouped NIHSS according to severity
Figure 2. Association of hsp 65 and neopterin levels with NIHSS scores. Patients with high NIHSS score greater than 15 had higher hsp 65 and neopterin levels compared with patients with NIHSS score between 0 and 15.
in totality. Our findings therefore support the theory that in some stroke patients, inflammation and immune reaction secondary to acute infection with C pneumoniae may trigger an acute stroke.22,23 Raised IgA antibodies indicating probably recurrent acute infections may have set the stage for the acute ischemic event in connivance with other atherosclerotic risk factors in these patients. hsps, also known as ‘‘stress proteins,’’ are among the highly conserved and immunogenic proteins shared among diverse groups of microbial agents and mammals. Heat and other types of stressful stimuli can increase the cellular expression of hsps. These proteins have been categorized into different families according to their molecular weight, such as hsp 90 (85-90 kDa), hsp 70 (68-73 kDa), hsp 60/65 (60-65 kDa), hsp 47, and small hsps (12-43 kDa). hsps are so called because they were first observed in response to hypothermia.24 hsps play an important role in the pathogenesis, prognosis, and treatment of many diseases, although the exact mechanisms of how hsps operate in these processes remain elusive in most cases.25,26 Recent evidence has suggested an association between C pneumoniae infection and coronary atherosclerosis. A significant association has also been detected between hsp 60 antibody and the severity of coronary artery
Table 3. Odd ratio of previous studies and present study of Chlamydia pneumonia with respect to IgA6
Study Wimmer et al Cook et al Elkind et al Anzini et al Johensen et al Rai et al Present study
Age, y
Case /controls, n
18-50 58/52 16-88 1761/1518 .39 89/89 18-46 141/192 50-64 254/254 .18 51/48 18-45 80/80
Odds ratio for IgA .71 (1.08-2.7)13 4.4 (3-6.5)12 4.51 (1.44-14.8)36 .63 (.26-2.47)6 1.54 (.96-2.47)6 4.72 (1.61-13.8)6 4.08 (2.08-8.09)6 Z 5 4.02, P 5 .001
atherosclerosis. In 1 retrospective study, the serum levels of antihuman hsp 60 IgG antibody and antichlamydial IgM, but not IgG or IgA, antibody were significantly higher in acute coronary syndrome patients than in stable ischemic heart disease patients or controls,27 whereas in our study we observed hsp 65 levels and antichlamydial IgA titres to be significantly higher suggesting that acute C pneumoniae infection with hsp 65–related immunological responses may contribute to the pathophysiology of an acute ischemic attack. Studies showed strong association and correlation of serum hsp 65 antibody titers to carotid atherosclerosis and coronary artery disease, which declined after coronary angioplasty. In our study, hsp 65 was found elevated in all patients who had a family history of coronary artery disease and atherosclerosis, which is in confirmation indicating that higher antibody titres to hsp 60,65, and 70 in the dyslipidemic patients may be related to a heightened state of immunoactivation associated with atherosclerosis.28 Zhu et al29 have postuled a role for mycobacterium hsp 65 and the very similar human hsp 60/65 in a series of papers showing an increased hsp 65 expression in ischemic human arterial walls within 30 minutes of external ligation.30 Chlamydia can produce large amounts of hsp 60 and 65 during chronic, persistent infections and stimulate host cells to induce hsps. C. pneumoniae expresses hsp 60, which shows close sequence homology between human counterparts; in this study, we tried to correlate the hsp 65 titres (human antibodies) to C. pneumoniae infectious states. Neopterin is known as a sensitive marker for diseases associated with increased activity of the cellular immune system. It has been reported that neopterin (a by-product of activated macrophage metabolism) is elevated in patients with systemic inflammatory insult at the time of ischemic stroke.31,32 Accumulating evidence has indicated that inflammatory immunologic reactions are involved in the pathogenesis of cerebral ischemia. Neopterin may be a direct participant within the cascade of events leading to oxidative stress and could either induce oxidative stress resulting in apoptotic cell death or superinduce tumor necrosis factor–mediated apoptosis. Antibodies to mycobacterial hsp 65 have been proposed as a diagnostic marker for the presence of atherosclerosis in stroke and in myocardial infarction patients. hsp 65 are chaperonins, and they are released in response to stress, stimuli, or injury. The present study describes the association of 2 inflammatory markers with infectious C. pneumonia titres.33 Levels of hsp 65 and neopterin also correlated well with the stroke severity scores of NIHSS. Lin et al31 demonstrated that neopterin is strongly and independently predictive of 90-day unfavorable clinical outcome in patients after AIS. Multiple logistic regression analysis demonstrated that serum neopterin level was strongly and independently predictive of NIHSS score greater than or equal to 12 (P 5.002) at 48 hours after
NOVEL INFLAMMATORY BIOMARKERS AND C. PNEUMONIAE TITRES IN AIS
AIS and 90-day major adverse clinical outcome (defined as NIHSS $12, recurrent stroke, or death; P 5.003).28,29 From our study, we observed that patients with NIHSS score beyond 15 had had a mean hsp 65 of 13.2 ng/mL and neopterin 7.5 mg/mL (r 5 .85, P 5 .02; graph 3), whereas moderately affected patients had lower levels of hsps and neopterin indicating that these markers may have conjectural role in inflammatory cascade after acute ischemia. Reports have suggested the role of neopterin as a parameter of potential increased monocyte activation in AIS indicating its role in endothelium dependent vascular dysfunction.34 We could not correlate the neopterin levels to monocyte activation, and it was also observed that serum white blood cell counts were within normal range in all patients. Myeloperoxidase is a hemoprotein in neutrophils and monocytes that catalyzes the reaction of hydrogen peroxide and halide ions to form cytotoxic acids and other intermediates; these play a role in the oxygen-dependent killing of tumor cells and microorganisms. It has been proven that presence of myeloperoxidase is an active indicator of atheromatous plaques acting as a catalyst oxidizing low-density lipoproteins. Oxidation of lowdensity lipoproteins by the myeloperoxidase system leads to the uptake by macrophages and further to the formation of foam cells, thus one of the possible mechanisms involved in the initiation of atherosclerotic lesions. Cojocaru et al35 studied myeloperoxidase levels in stroke patients and found that 71 of 78 patients with ischemic stroke presented mean plasma myeloperoxidase levels greater than the upper of normal (425 6 36 pmol/L; P , .0001; risk ratio, 8.188; 95% CI, 4.038-16.600). We did not find any significant increase in myeloperoxidase levels. In a previous post hoc pilot case–control study in northern Manhattan, C pneumoniae IgA antibodies were associated with risk for first ischemic stroke.36 This population-based case–control study in which antibody titer thresholds were chosen before assay performance provides evidence for an association between antibodies against C pneumoniae and risk of ischemic stroke. It further suggests that the association of C pneumoniae IgA antibodies may be stronger than for IgG antibodies. The stronger association for IgA titers reflects the possibility that IgA antibodies, which are produced only 3-5 days after exposure, are a marker of persistent, chronic infection, whereas IgG antibodies, which remain elevated for several years after infection, are a marker of remote, completed infection. Because secretary IgA is an antibody that plays a role in mucosal immunity, found in secretions like tars, mucosa, and saliva, C pneumoniae leads to respiratory tract infection thereby disrupting the smooth muscle wall lining. hsp 65 and neopterin are close associates for infection and atherosclerosis in stroke; it is for this reason C pneumoniae IgA antibodies correlated well with these biomarkers.4
2395
There has been consistent evidence that C pneumoniae is associated with large vessel atherosclerotic and small vessel (‘‘lacunar’’) stroke, consistent with the hypothesis that C pneumoniae contributes to atherosclerosis. Lipohyalinosis, the underlying pathophysiology in the small vessels that leads to lacunar stroke, has been considered to be an early form of atherosclerosis. Several studies identified C pneumoniae in the endothelium, smooth muscle cells, and macrophages within the vascular wall. Although studies have reported its presence in middle cerebral and other large cerebral vessels, no published reports have identified this bacteria in small penetrating vessels of the brain. In our study also, the phenotypic classification of stroke was not done, and hence, we could not correlate this with the C pneumoniae titres. Predictors of stroke severity include various clinical and neuroimaging markers. Role of inflammatory makers may prove to be of added value to the predictability of stroke severity indices. If proven, simple kits at bedside may have a role in future to determine the severity of AIS for tailoring specific treatment strategies. A blood marker must satisfy the proposed criteria of an ideal marker, that is, the marker can classify the patients at high or low risks for poor outcome across clinically relevant threshold, and the marker has statistically independent association with outcome after adjusting for covariates. From our study, we observed that hsp 65 and neopterin showed a higher sensitivity to label them as serum blood marker, which would help improving the discriminating ability of the established clinical and biocellular spectrum of ischemic stroke cascade.10,37
Conclusion Serum blood biomarkers can be a complementary tool for diagnosis, predicting prognosis,38 and therapeutic monitoring of novel treatments in ischemic stroke. In the present study, we measured multiple blood markers (hsp 65, neopterin, and myeloperoxidase)39,40 that may be plausibly related to the pathophysiology of ischemic stroke to predict severity and consequently functional outcomes after stroke. This study would pave way for future larger studies, preferably a prospective population-based study and by using other markers of systemic acute and chronic infection of C pneumoniae along with markers of acute inflammation/thrombosis and acute phase reactants. Future role of chronic antibiotic therapy as a stroke prevention measure in high-risk individuals is a fascinating concept, especially in a developing country where infection and cerebrovascular disease contribute significantly to health mortality and morbidity.
References 1. Banerjee TK, Das SK. Epidemiology of stroke in India. Neurology Asia 2006;11:1-4.
2396 2. Dalal PM. Strokes in young and elderly: risk factors and strategies for stroke prevention. J Assoc Phys India 1997;45:125-131. 3. Kalayoglu MV, Libby P, Byrne GI. Chlamydia pneumoniae as an emerging risk factor in cardiovascular disease. J Am Med Assoc 2002;288:2724-2731. 4. Elkind MS, Tondella ML, Feikin DR, et al. Seropositivity to Chlamydia pneumoniae is associated with risk of first ischemic stroke. Stroke 2006;37:790-795. 5. Abraham J, Rao PS, Inbaraj SG, et al. An epidemiological study of hemiplegia due to stroke in South India. Stroke 1970;1:477-481. 6. Rai NK, Chaudhry R, Bhatia R, et al. Chlamydia pneumoniae seropositivity in adults with acute ischemic stroke: a case-control study. Ann Indian Acad Neurol 2011;14:93-97. 7. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005;352:1685-1695. 8. Van Exel E, Gussekloo J, de Craen AJ, et al. Inflammation and stroke: the Leiden 85-Plus Study. Stroke 2002; 33:1135-1138. 9. Casiglia E, Tikhonoff V. Inflammatory and coagulative markers of atherosclerosis. Eur Heart J 2007;28:271-273. 10. Whiteley W, Wardlaw J, Dennis M, et al. Blood biomarkers for the diagnosis of acute cerebrovascular diseases: a prospective cohort study. Cerebrovasc Dis 2011; 32:141-147. 11. Andressen WU, Krakenes J, Ulvestad E, et al. IL-6: an early marker for outcome in acute ischemic stroke. Acta Scand 2005;111:360-365. 12. Cook PJ, Honeybourne D, Lip GY, et al. Chlamydia pneumoniae antibody titers are significantly associated with acute stroke and transient cerebral ischaemia: The West Birmingham Stroke Project. Stroke 1998;29:404-410. 13. Wimmer ML, Sandmann-Strupp R, Saikku P, et al. Association of chlamydial infection with cerebrovascular disease. Stroke 1996;27:2207-2210. 14. Fieschi C, Rasura M, Anzini A, et al. A diagnostic approach to ischemic stroke in young and middle-aged adults. Eur J Neurol 1996;3:325-330. 15. Virok D, Kis Z, Karai L, et al. Chlamydia pneumoniae in atherosclerotic middle cerebral artery. Stroke 2001; 32:1973-1976. 16. Pearson TA, Mensah GA, Wayne AR, et al. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the America Heart Association. Circulation 2003;107:499-511. 17. Emsley HC, Smith CJ, Gavin CM, et al. An early and sustained peripheral inflammatory response in acute ischemic stroke: relationship with infection and atherosclerosis. J Neuroimmunol 2003;139:93-102. 18. Bhatia RS, Gang RK, Gaur SP, et al. Predictive value of routine hematological and biochemical parameters on 30-day fatality in acute stroke. Neurol India 2004; 52:220-223. 19. Planas AM, Gorina R, Chamorro A. Signaling pathways mediating inflammatory responses in brain ischemia. Biochem Soc Trans 2006;34:1267-1270. 20. Cannon CP, Braunwald E, Mc Cabe CH, et al. Antibiotic treatment of Chlamydia pneumoniae after acute coronary syndrome: Pravastatin or Atorvastatin Evaluation and Infection Therapy-Thrombolysis in Myocardial Infarction. N Engl J Med 2005;352:1646-1654. 21. Grayston JT, Kronmal RA, Jackson LA, et al, ACES investigators. Azithromycin for the secondary prevention of coronary events. N Eng J Med 2005;352:1637-1645.
M.V.P. SRIVASTAVA ET AL. 22. Madre JG, Garcia JL, Gonzalez RC, et al. Association between seropositivity to Chlamydia pneumoniae and acute ischaemic stroke. Eur J Neurol 2002;9:303-306. 23. Melnick SL, Shahar E, Folsom AR, et al. Past infection by Chlamydia pneumoniae strain and asymptomatic carotid atherosclerosis. Am J Med 1993;95:499-504. 24. Grundtman C, Kreutmayer SB, Almanzar G, et al. Heat shock protein 60 and immune inflammatory responses in atherosclerosis. Arterioscler Thromb Vasc Biol 2011; 31:960-968. 25. Sharp XR, Xhan X, Liu DZ. Heat shock proteins in the brain: role of Hsp70, Hsp 27, and HO-1 (Hsp32) and their therapeutic potential. Trans Stroke Res 2013;4:685-692. 26. Moharbahn GM, Lamb DJ, Lovell DJ, et al. Plasma antibody titres to heat shock proteins-60,-65 and-70: their relationship to coronary risk factors in dyslipidaemic patients and healthy individuals. Scand J Clin Lab Invest 2005;65:601-614. 27. Hoshida S, Nishino M, Tanouchi J, et al. Acute Chlamydia pneumonia infection with heat shock proteins 60 related response in patient with acute coronary syndrome. Atherosclerosis 2005;183:109-112. 28. Gruden G, Barutta F, Catto I, et al. Serum levels of heat shock proteins 27 in patients with acute ischemic stroke. Cell Stress Chaperones 2013;18:531-533. 29. Zhu J, Katz RJ, Quyyumi A, et al. Association of serum antibodies to heat-shock protein 65 with coronary calcification levels suggestion of pathogen-triggered autoimmunity in early atherosclerosis. Circulation 2004;109:36-41. 30. Birnie DH, Holm ER, McKay IC, et al. Association between antibodies to heat shock protein 65 and coronary atherosclerosis. Possible mechanism of action of Helicobacter pylori and other bacterial infections in increasing cardiovascular risk. Eur Heart J 1998;19:387-394. 31. Lin HS, Tsai TH, Lui CF, et al. Serum level and prognostic value of neopterin in patients with ischemic stroke. Clin Biochem 2012;45:1596-1601. 32. Murr C, Widner B, Wirleitner B, et al. Neopterin as a marker for immune system activation. Curr Drug Metab 2002;3:175-187. 33. Hoffmann G, Schobersberger W, Frede S, et al. Neopterin activates transcription factor nuclear factor-kappa B in vascular smooth muscle cells. FEBS Lett 1996;391:181-184. 34. Cojocaru IM, Cojocaru M, Bursin C, et al. Detection of neopterin as a parameter of potential monocyte activation in patients with acute ischemic stroke. Rom J Intern Med 2007;45:365-369. 35. Cojocaru IM, Cojocaru M, Iliescu I, et al. Plasma myeloperoxidase levels in acute ischemic stroke. Rom J Intern Med 2010;48:101-104. 36. Elkind MS, Lin I-F, Grayston TJ, et al. Chlamydia pneumoniae and the risk of first ischemic stroke: the Northern Manhattan Stroke Study. Stroke 2000;31:1521-1525. 37. Whiteley W, Wardlaw J, Dennis M, et al. The use of blood biomarkers to predict poor outcome after acute transient ischemic attack or ischemic stroke. Stroke 2012;43:86-91. 38. Shaw AC. Serum C-reactive protein and neopterin concentrations in viral and bacterial infections. J Clin Pathol 1991;44:596-599. 39. Syrjanen J, Valtonen VV, Iivanainen M, et al. Preceding infection as an important risk factor for ischemic brain infarction in young and middle aged patients. BMJ 1988;296:1156-1160. 40. Jozwiak PB, Mickielewicz A, Gaciong Z, et al. Elevated levels of anti-Chlamydia pneumoniae IgA and IgG antibodies in young adults with ischemic stroke. Acta Neurol Scand 2007;116:144-149.
Background: Young stroke patients constitute 15%-30% of all stroke patients in India as against 3.0%-8.5% reported from the West. The mechanisms for stroke in the young may include unconventional risk factors such as infections. We aimed to investigate the role (if any) of Chlamydia pneumoniae antibodies in young patients with acute ischemic stroke (AIS). Several proinflammatory cytokines and biomarkers are released early after the onset of brain ischemia. We assessed the role of heat shock protein (hsp) 65, neopterin, and myeloperoxidase upregulation after AIS in predicting stroke severity. We also assessed relationship of upregulated inflammatory biomarkers with C pneumoniae antibody titres (IgG, IgA, and IgM). Methods: Eighty acute stroke patients and healthy age- and sex-matched controls were recruited. Blood samples were drawn within 1 week from the onset of stroke. Detection of IgA, IgG, and IgM antibodies to C pneumoniae was done with a validated microimmunofluorescence technique from 5 mL of serum in all subjects. Inflammatory biomarkers such as neopterin, myeloperoxidase and hsp 65 were estimated with sandwich enzyme linked immunosorbent assay (ELISA) method. Results: hsp 65 and neopterin were significantly elevated in all stroke patients with respect to healthy controls (odds ratio [OR], 4.9; 95% confidence interval [CI], 23.5-67.8; P 5 .001 and OR, 4.4; 95% CI, 2.089.4; P 5.04, respectively). Eighty-one percent of cases were seropositive for IgA versus 32% of controls (P 5 .003), and IgG was positive in 52.7% versus 17.3% of controls (P 5 .05). Myeloperoxidase levels were similar in patients and controls. Correlation and multiple regression indicated a high level of predictability and sensitivity of hsp 65 to IgA. C. pneumoniae antibody titres when all other variables were constant (F [4,90] 5 26.8, P 5 .001). Patients with high NIHSS scores (.15) had elevated levels of hsp 65 (mean, 13.2 ng/mL) suggesting correlation with stroke severity. Conclusions: The study demonstrated high levels of hsp 65 and neopterin levels in AIS correlated to significantly elevated IgA titres of C pneumoniae. Elevated levels of hsp 65 were associated with stroke severity. Key Words: Acute ischemia—inflammatory biomarkers— C pneumoniae—stroke severity. Ó 2014 by National Stroke Association
Introduction Stroke in young, including stroke in children and young adults (,45 years), is an important cause of From the *Department of Neurology, AIIMS, New Delhi; †Department of Microbiology, All India Institute of Medical Sciences, New Delhi; and ‡Department of Neurobiochemistry, All India Institute of Medical Sciences, New Delhi, India. Received March 21, 2014; revision received May 9, 2014; accepted May 16, 2014.
morbidity and mortality throughout the world, especially in developing countries.1 Young stroke patients constitute 15%-30% of all stroke patients in India, vis
Address correspondence to M. Vasantha Padma Srivastava, MBBS, MD, DM, FAMS, FNASc, Department of Neurology, All India Institute of Medical Sciences, New Delhi, India. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2014.05.016
Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 9 (October), 2014: pp 2391-2396
2391
M.V.P. SRIVASTAVA ET AL.
2392
a vis 3.0%-8.5% of all stroke patients reported from the West.2 Serologic evidence of past infection with Chlamydia pneumoniae has been found in epidemiologic studies to be associated with risk for atherosclerosis and cardiac disease, although prospective cohort studies have not always confirmed this association.3 Because of heterogeneity of etiopathogenesis of stroke and stroke subtypes, the association of C pneumoniae infection with ischemic stroke can be more complex. Recent bacterial or viral infection has been shown to be associated with acute ischemic stroke (AIS). The link between C pneumoniae and cerebrovascular disease has been investigated in a number of seroepidemiologic and antibiotic intervention studies.4 However, the role of C pneumoniae infection in acute stroke as a cause/effect/trigger remains controversial. Because infectious diseases in general are more common in India, compared with Western countries, we aimed to investigate the role (if any) of C pneumoniae antibodies in patients with AIS. Recent reports from South India suggest positive association of C pneumoniae infection with the occurrence of AIS. Our previous study also confirmed the same.5,6 The present study was undertaken to investigate the possible role of C pneumoniae in young stroke patients. The role of inflammatory markers in ischemic cascade after AIS has been widely studied. All stages of the atherosclerotic plaque—initiation, growth, and complications— might be considered to be an inflammatory response to injury. Potential targets, which could help to identify and monitor the ongoing inflammatory7,8 process are Creactive protein (CRP), interleukins (IL-1,6), tumor necrosis factor-a, neopterin, myeloperoxidase, heat shock protein (hsp) 65 and others. There has been some literature published on the predictor ability of CRP for both prognosis and occurrence of AIS.9,10 Although many blood markers have been investigated to date, no single blood marker has been proven to be clinically useful to predict stroke severity and outcomes. We investigated the role of hsp 65, neopterin, and myeloperoxidase in predicting the severity of AIS and their correlation to the titres of C pneumoniae.
selected for the previously mentioned analysis were healthy age- and sex-matched subjects, who did not have any history of cerebrovascular disease. Blood samples were drawn within 1 week from the onset of stroke. In each case, 5 mL of serum was obtained by centrifugation (699 g for 15 minutes at 14 C) and then stored at 70 C until analysis. Detection of IgA, IgG, and IgM antibodies to C pneumoniae was done with a validated microimmunofluorescence technique. The cutoff point for seropositivity was 1/16 for IgA and 1/64 for IgG. Inflammatory biomarkers such as hsp 65, neopterin, and myeloperoxidase were estimated using commercially available high sensitivity quantitative sandwich enzyme immunoassays according to the manufacturer’s instructions (R & D Systems, Minneapolis, MN). This study was cleared by the institute review board, and written informed consent was obtained from all patients.
Statistical Methods Analysis of data was carried out using SPSS statistical analysis software (SPSS Inc, Chicago, IL). For continuous variables such as age and NIHSS scores, descriptive statistics was calculated and reported as means 6 standard deviation. Normality of distribution of continuous variables was verified using the parametric tests.
Results This study was a case–control design in which 80 AIS patients were recruited with the same number of healthy age-matched controls. The mean age in the study group was 43.6 6 6.7 years and 43.2 6 7.3 years in controls (male:female ratio 5 2:1). Standard stroke care was administered to all patients. The risk factors found in stroke patients were hypertension (38%), diabetes mellitus (23.4%), hypercholesterolemia (29.4%), and smoking (22.3%; Table 1). hsp65 was found elevated significantly (mean of 30.4 ng/mL in 58 patients vs. none in controls, P , .05). Neopterin levels were found elevated in 50% (40 patients vs. 14 controls, with a mean of 19.26 mg/mL Table 1. Risk factors in study and control groups
Methods The criteria for inclusion were patients in the age group of 18-45 years, presenting with AIS within a week of onset. Complete medical history of hypertension, smoking, diabetes mellitus, hypercholesterolemia, ischemic heart disease, stroke, or TIA was recorded. Neurologic evaluation included recording of National Institute of Health Stroke Scale (NIHSS). noncontrast computed tomography head was performed in all, and if needed magnetic resonance imaging/computed tomography angiography/magnetic resonance angiography was performed according to the clinical requirement. Controls
Risk factors
Patients, %
Controls, %
Hypertension Hypercholesterolemia Diabetes mellitus Smoking Alcohol h/o cardiac disease Atrial fibrillation Migraine Family h/o stroke
38 29.4 23.4 22.35 22.3 21.1 1.17 7.05 20.14
11 18 15 12 16 8.5 0 1.5 12.2
Abbreviation: h/o, history of.
NOVEL INFLAMMATORY BIOMARKERS AND C. PNEUMONIAE TITRES IN AIS
2393
7.5mg/mL (r 5 .85, P 5.002; graph 2), whereas moderately affected patients had lower levels of hsps and neopterin levels (mean, 7.1 ng/mL and 32, mg/mL, respectively; Fig 2).
Discussion
Figure 1. Mean values of biomarkers levels in all subjects. hsp 65 and neopterin levels were significantly high in stroke patients compared with controls.
[normal range, ,3 m/mL]; odds ratio [OR], 4.4; 95% confidence interval [CI], 2.08-9.4; P 5 .04). Myeloperoxidase levels were near normal in both patients and controls (OR, 1.9; 95% CI, .21-7.1; P 5 .56; Fig 1). Seropositivity for IgA titers of C pneumoniae was 81% in cases versus 32% of controls (P 5 .003; Table 2). This difference was less striking for IgG, 52.7% of cases were seropositive versus 17.3% of controls (P 5.05). IgM antibodies were found to be similar and negative in patients as well as in controls (P . .05). Correlation and multiple regression analyses were performed with hsp 65 and neopterin as dependent variables to independent antibody titres of IgA, IgG, and IgM. hsp 65 had positive regression weights indicating that patients with a high hsp level predicted a high C pneumoniae IgA antibody (F [4,90] 5 16.8, P 5.001). The results were statistically significant accounting to 21.2% variability of hsp 65 with IgG (P 5.02). The regression model for neopterin also indicated a good level of prediction for IgA antibodies (R2 5 .78, F 5 6.38, P , .05), whereas it was nonsignificant for myleoperoxidase levels. The receiver operating curve (ROC) for hsp 65 showed a high sensitivity with a threshold of .95 for IgA antibody of C pneumoniae compared with IgG and IgM. We divided patients according to their severity as mild to moderately affected (NIHSS score between 0 and 15) and severely affected and critically ill with NIHSS score greater than 15. Bivariate correlation analysis was performed to correlate the severity with the biomarker levels. It was observed that patients with NIHSS score of more than 15 had a mean hsp 65 of 13.2 ng/mL and neopterin
This case–control study provides evidence of an association between acute infection with C pneumoniae (IgA antibodies) and the risk of AIS in young patients (,45 years).11,12 Many studies demonstrated a positive association between C pneumoniae infection and stroke, yielding a OR of 1.71-8.58, and several did not.13,14 Table 3 summarizes the results seen in various studies. C pneumoniae IgG antibodies may be considered as markers of ‘‘chronic’’ infection, and they could be in circulation for 3-5 years after infection.15,16 The IgA antibodies may be considered as markers of a primary, acute, or chronic infection and stay in circulation on an average of 3-5 days or longer. C pneumoniae infection has been implicated to have a role in enhancing systemic inflammation and immune-mediated vascular damage, altering lipid metabolism, inducing the production of cytokines and lipoproteins. These changes may adversely modify the conventional risk factors and aggravate atherosclerosis.17,18 C pneumoniae is phagocytosed by alveolar macrophages and transported by blood stream to the vascular subendothelial region through the injured arterial endothelium. Several studies demonstrated the presence of C pneumoniae in atherosclerotic lesions in coronary arteries, carotid arteries, and aorta obtained from autopsy and endarterectomy specimens. One Indian study, which has shown association of high CRP in stroke patients lends further credence to the hypothesis that infection with C pneumoniae, acute or chronic, may be associated with atherosclerosis through the mechanisms of low-grade inflammation.19 This mechanism has been further supported by the finding of increased carotid intima-media thickness in asymptomatic patients who are seropositive for C pneumoniae. However, 2 large randomized controlled trials did not observe a beneficial effect of antichlamydial antibiotic therapy on the secondary prevention of cardiovascular heart disease.20,21 Neither trial used the serologic marker of chronic C pneumoniae infection during patient selection. Stroke also is far more heterogenous in nature than acute coronary syndrome and both cannot be equated
Table 2. Seropositivity of IgA, IgM, IgG antibodies to Chlamydia pneumoniae IgA
Patients Controls P value
IgG
IgM
Positive, %
Negative, %
Positive, %
Negative, %
Positive, %
Negative, %
81 32
19 63
52.7 33
15.4 62
4.54 3
88.1 96
.003
.05
.12
M.V.P. SRIVASTAVA ET AL.
2394
Hsp
Neopterin
mean scores
20 15 10 5 0 5-15NIHSS
>15NIHSS
Grouped NIHSS according to severity
Figure 2. Association of hsp 65 and neopterin levels with NIHSS scores. Patients with high NIHSS score greater than 15 had higher hsp 65 and neopterin levels compared with patients with NIHSS score between 0 and 15.
in totality. Our findings therefore support the theory that in some stroke patients, inflammation and immune reaction secondary to acute infection with C pneumoniae may trigger an acute stroke.22,23 Raised IgA antibodies indicating probably recurrent acute infections may have set the stage for the acute ischemic event in connivance with other atherosclerotic risk factors in these patients. hsps, also known as ‘‘stress proteins,’’ are among the highly conserved and immunogenic proteins shared among diverse groups of microbial agents and mammals. Heat and other types of stressful stimuli can increase the cellular expression of hsps. These proteins have been categorized into different families according to their molecular weight, such as hsp 90 (85-90 kDa), hsp 70 (68-73 kDa), hsp 60/65 (60-65 kDa), hsp 47, and small hsps (12-43 kDa). hsps are so called because they were first observed in response to hypothermia.24 hsps play an important role in the pathogenesis, prognosis, and treatment of many diseases, although the exact mechanisms of how hsps operate in these processes remain elusive in most cases.25,26 Recent evidence has suggested an association between C pneumoniae infection and coronary atherosclerosis. A significant association has also been detected between hsp 60 antibody and the severity of coronary artery
Table 3. Odd ratio of previous studies and present study of Chlamydia pneumonia with respect to IgA6
Study Wimmer et al Cook et al Elkind et al Anzini et al Johensen et al Rai et al Present study
Age, y
Case /controls, n
18-50 58/52 16-88 1761/1518 .39 89/89 18-46 141/192 50-64 254/254 .18 51/48 18-45 80/80
Odds ratio for IgA .71 (1.08-2.7)13 4.4 (3-6.5)12 4.51 (1.44-14.8)36 .63 (.26-2.47)6 1.54 (.96-2.47)6 4.72 (1.61-13.8)6 4.08 (2.08-8.09)6 Z 5 4.02, P 5 .001
atherosclerosis. In 1 retrospective study, the serum levels of antihuman hsp 60 IgG antibody and antichlamydial IgM, but not IgG or IgA, antibody were significantly higher in acute coronary syndrome patients than in stable ischemic heart disease patients or controls,27 whereas in our study we observed hsp 65 levels and antichlamydial IgA titres to be significantly higher suggesting that acute C pneumoniae infection with hsp 65–related immunological responses may contribute to the pathophysiology of an acute ischemic attack. Studies showed strong association and correlation of serum hsp 65 antibody titers to carotid atherosclerosis and coronary artery disease, which declined after coronary angioplasty. In our study, hsp 65 was found elevated in all patients who had a family history of coronary artery disease and atherosclerosis, which is in confirmation indicating that higher antibody titres to hsp 60,65, and 70 in the dyslipidemic patients may be related to a heightened state of immunoactivation associated with atherosclerosis.28 Zhu et al29 have postuled a role for mycobacterium hsp 65 and the very similar human hsp 60/65 in a series of papers showing an increased hsp 65 expression in ischemic human arterial walls within 30 minutes of external ligation.30 Chlamydia can produce large amounts of hsp 60 and 65 during chronic, persistent infections and stimulate host cells to induce hsps. C. pneumoniae expresses hsp 60, which shows close sequence homology between human counterparts; in this study, we tried to correlate the hsp 65 titres (human antibodies) to C. pneumoniae infectious states. Neopterin is known as a sensitive marker for diseases associated with increased activity of the cellular immune system. It has been reported that neopterin (a by-product of activated macrophage metabolism) is elevated in patients with systemic inflammatory insult at the time of ischemic stroke.31,32 Accumulating evidence has indicated that inflammatory immunologic reactions are involved in the pathogenesis of cerebral ischemia. Neopterin may be a direct participant within the cascade of events leading to oxidative stress and could either induce oxidative stress resulting in apoptotic cell death or superinduce tumor necrosis factor–mediated apoptosis. Antibodies to mycobacterial hsp 65 have been proposed as a diagnostic marker for the presence of atherosclerosis in stroke and in myocardial infarction patients. hsp 65 are chaperonins, and they are released in response to stress, stimuli, or injury. The present study describes the association of 2 inflammatory markers with infectious C. pneumonia titres.33 Levels of hsp 65 and neopterin also correlated well with the stroke severity scores of NIHSS. Lin et al31 demonstrated that neopterin is strongly and independently predictive of 90-day unfavorable clinical outcome in patients after AIS. Multiple logistic regression analysis demonstrated that serum neopterin level was strongly and independently predictive of NIHSS score greater than or equal to 12 (P 5.002) at 48 hours after
NOVEL INFLAMMATORY BIOMARKERS AND C. PNEUMONIAE TITRES IN AIS
AIS and 90-day major adverse clinical outcome (defined as NIHSS $12, recurrent stroke, or death; P 5.003).28,29 From our study, we observed that patients with NIHSS score beyond 15 had had a mean hsp 65 of 13.2 ng/mL and neopterin 7.5 mg/mL (r 5 .85, P 5 .02; graph 3), whereas moderately affected patients had lower levels of hsps and neopterin indicating that these markers may have conjectural role in inflammatory cascade after acute ischemia. Reports have suggested the role of neopterin as a parameter of potential increased monocyte activation in AIS indicating its role in endothelium dependent vascular dysfunction.34 We could not correlate the neopterin levels to monocyte activation, and it was also observed that serum white blood cell counts were within normal range in all patients. Myeloperoxidase is a hemoprotein in neutrophils and monocytes that catalyzes the reaction of hydrogen peroxide and halide ions to form cytotoxic acids and other intermediates; these play a role in the oxygen-dependent killing of tumor cells and microorganisms. It has been proven that presence of myeloperoxidase is an active indicator of atheromatous plaques acting as a catalyst oxidizing low-density lipoproteins. Oxidation of lowdensity lipoproteins by the myeloperoxidase system leads to the uptake by macrophages and further to the formation of foam cells, thus one of the possible mechanisms involved in the initiation of atherosclerotic lesions. Cojocaru et al35 studied myeloperoxidase levels in stroke patients and found that 71 of 78 patients with ischemic stroke presented mean plasma myeloperoxidase levels greater than the upper of normal (425 6 36 pmol/L; P , .0001; risk ratio, 8.188; 95% CI, 4.038-16.600). We did not find any significant increase in myeloperoxidase levels. In a previous post hoc pilot case–control study in northern Manhattan, C pneumoniae IgA antibodies were associated with risk for first ischemic stroke.36 This population-based case–control study in which antibody titer thresholds were chosen before assay performance provides evidence for an association between antibodies against C pneumoniae and risk of ischemic stroke. It further suggests that the association of C pneumoniae IgA antibodies may be stronger than for IgG antibodies. The stronger association for IgA titers reflects the possibility that IgA antibodies, which are produced only 3-5 days after exposure, are a marker of persistent, chronic infection, whereas IgG antibodies, which remain elevated for several years after infection, are a marker of remote, completed infection. Because secretary IgA is an antibody that plays a role in mucosal immunity, found in secretions like tars, mucosa, and saliva, C pneumoniae leads to respiratory tract infection thereby disrupting the smooth muscle wall lining. hsp 65 and neopterin are close associates for infection and atherosclerosis in stroke; it is for this reason C pneumoniae IgA antibodies correlated well with these biomarkers.4
2395
There has been consistent evidence that C pneumoniae is associated with large vessel atherosclerotic and small vessel (‘‘lacunar’’) stroke, consistent with the hypothesis that C pneumoniae contributes to atherosclerosis. Lipohyalinosis, the underlying pathophysiology in the small vessels that leads to lacunar stroke, has been considered to be an early form of atherosclerosis. Several studies identified C pneumoniae in the endothelium, smooth muscle cells, and macrophages within the vascular wall. Although studies have reported its presence in middle cerebral and other large cerebral vessels, no published reports have identified this bacteria in small penetrating vessels of the brain. In our study also, the phenotypic classification of stroke was not done, and hence, we could not correlate this with the C pneumoniae titres. Predictors of stroke severity include various clinical and neuroimaging markers. Role of inflammatory makers may prove to be of added value to the predictability of stroke severity indices. If proven, simple kits at bedside may have a role in future to determine the severity of AIS for tailoring specific treatment strategies. A blood marker must satisfy the proposed criteria of an ideal marker, that is, the marker can classify the patients at high or low risks for poor outcome across clinically relevant threshold, and the marker has statistically independent association with outcome after adjusting for covariates. From our study, we observed that hsp 65 and neopterin showed a higher sensitivity to label them as serum blood marker, which would help improving the discriminating ability of the established clinical and biocellular spectrum of ischemic stroke cascade.10,37
Conclusion Serum blood biomarkers can be a complementary tool for diagnosis, predicting prognosis,38 and therapeutic monitoring of novel treatments in ischemic stroke. In the present study, we measured multiple blood markers (hsp 65, neopterin, and myeloperoxidase)39,40 that may be plausibly related to the pathophysiology of ischemic stroke to predict severity and consequently functional outcomes after stroke. This study would pave way for future larger studies, preferably a prospective population-based study and by using other markers of systemic acute and chronic infection of C pneumoniae along with markers of acute inflammation/thrombosis and acute phase reactants. Future role of chronic antibiotic therapy as a stroke prevention measure in high-risk individuals is a fascinating concept, especially in a developing country where infection and cerebrovascular disease contribute significantly to health mortality and morbidity.
References 1. Banerjee TK, Das SK. Epidemiology of stroke in India. Neurology Asia 2006;11:1-4.
2396 2. Dalal PM. Strokes in young and elderly: risk factors and strategies for stroke prevention. J Assoc Phys India 1997;45:125-131. 3. Kalayoglu MV, Libby P, Byrne GI. Chlamydia pneumoniae as an emerging risk factor in cardiovascular disease. J Am Med Assoc 2002;288:2724-2731. 4. Elkind MS, Tondella ML, Feikin DR, et al. Seropositivity to Chlamydia pneumoniae is associated with risk of first ischemic stroke. Stroke 2006;37:790-795. 5. Abraham J, Rao PS, Inbaraj SG, et al. An epidemiological study of hemiplegia due to stroke in South India. Stroke 1970;1:477-481. 6. Rai NK, Chaudhry R, Bhatia R, et al. Chlamydia pneumoniae seropositivity in adults with acute ischemic stroke: a case-control study. Ann Indian Acad Neurol 2011;14:93-97. 7. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005;352:1685-1695. 8. Van Exel E, Gussekloo J, de Craen AJ, et al. Inflammation and stroke: the Leiden 85-Plus Study. Stroke 2002; 33:1135-1138. 9. Casiglia E, Tikhonoff V. Inflammatory and coagulative markers of atherosclerosis. Eur Heart J 2007;28:271-273. 10. Whiteley W, Wardlaw J, Dennis M, et al. Blood biomarkers for the diagnosis of acute cerebrovascular diseases: a prospective cohort study. Cerebrovasc Dis 2011; 32:141-147. 11. Andressen WU, Krakenes J, Ulvestad E, et al. IL-6: an early marker for outcome in acute ischemic stroke. Acta Scand 2005;111:360-365. 12. Cook PJ, Honeybourne D, Lip GY, et al. Chlamydia pneumoniae antibody titers are significantly associated with acute stroke and transient cerebral ischaemia: The West Birmingham Stroke Project. Stroke 1998;29:404-410. 13. Wimmer ML, Sandmann-Strupp R, Saikku P, et al. Association of chlamydial infection with cerebrovascular disease. Stroke 1996;27:2207-2210. 14. Fieschi C, Rasura M, Anzini A, et al. A diagnostic approach to ischemic stroke in young and middle-aged adults. Eur J Neurol 1996;3:325-330. 15. Virok D, Kis Z, Karai L, et al. Chlamydia pneumoniae in atherosclerotic middle cerebral artery. Stroke 2001; 32:1973-1976. 16. Pearson TA, Mensah GA, Wayne AR, et al. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the America Heart Association. Circulation 2003;107:499-511. 17. Emsley HC, Smith CJ, Gavin CM, et al. An early and sustained peripheral inflammatory response in acute ischemic stroke: relationship with infection and atherosclerosis. J Neuroimmunol 2003;139:93-102. 18. Bhatia RS, Gang RK, Gaur SP, et al. Predictive value of routine hematological and biochemical parameters on 30-day fatality in acute stroke. Neurol India 2004; 52:220-223. 19. Planas AM, Gorina R, Chamorro A. Signaling pathways mediating inflammatory responses in brain ischemia. Biochem Soc Trans 2006;34:1267-1270. 20. Cannon CP, Braunwald E, Mc Cabe CH, et al. Antibiotic treatment of Chlamydia pneumoniae after acute coronary syndrome: Pravastatin or Atorvastatin Evaluation and Infection Therapy-Thrombolysis in Myocardial Infarction. N Engl J Med 2005;352:1646-1654. 21. Grayston JT, Kronmal RA, Jackson LA, et al, ACES investigators. Azithromycin for the secondary prevention of coronary events. N Eng J Med 2005;352:1637-1645.
M.V.P. SRIVASTAVA ET AL. 22. Madre JG, Garcia JL, Gonzalez RC, et al. Association between seropositivity to Chlamydia pneumoniae and acute ischaemic stroke. Eur J Neurol 2002;9:303-306. 23. Melnick SL, Shahar E, Folsom AR, et al. Past infection by Chlamydia pneumoniae strain and asymptomatic carotid atherosclerosis. Am J Med 1993;95:499-504. 24. Grundtman C, Kreutmayer SB, Almanzar G, et al. Heat shock protein 60 and immune inflammatory responses in atherosclerosis. Arterioscler Thromb Vasc Biol 2011; 31:960-968. 25. Sharp XR, Xhan X, Liu DZ. Heat shock proteins in the brain: role of Hsp70, Hsp 27, and HO-1 (Hsp32) and their therapeutic potential. Trans Stroke Res 2013;4:685-692. 26. Moharbahn GM, Lamb DJ, Lovell DJ, et al. Plasma antibody titres to heat shock proteins-60,-65 and-70: their relationship to coronary risk factors in dyslipidaemic patients and healthy individuals. Scand J Clin Lab Invest 2005;65:601-614. 27. Hoshida S, Nishino M, Tanouchi J, et al. Acute Chlamydia pneumonia infection with heat shock proteins 60 related response in patient with acute coronary syndrome. Atherosclerosis 2005;183:109-112. 28. Gruden G, Barutta F, Catto I, et al. Serum levels of heat shock proteins 27 in patients with acute ischemic stroke. Cell Stress Chaperones 2013;18:531-533. 29. Zhu J, Katz RJ, Quyyumi A, et al. Association of serum antibodies to heat-shock protein 65 with coronary calcification levels suggestion of pathogen-triggered autoimmunity in early atherosclerosis. Circulation 2004;109:36-41. 30. Birnie DH, Holm ER, McKay IC, et al. Association between antibodies to heat shock protein 65 and coronary atherosclerosis. Possible mechanism of action of Helicobacter pylori and other bacterial infections in increasing cardiovascular risk. Eur Heart J 1998;19:387-394. 31. Lin HS, Tsai TH, Lui CF, et al. Serum level and prognostic value of neopterin in patients with ischemic stroke. Clin Biochem 2012;45:1596-1601. 32. Murr C, Widner B, Wirleitner B, et al. Neopterin as a marker for immune system activation. Curr Drug Metab 2002;3:175-187. 33. Hoffmann G, Schobersberger W, Frede S, et al. Neopterin activates transcription factor nuclear factor-kappa B in vascular smooth muscle cells. FEBS Lett 1996;391:181-184. 34. Cojocaru IM, Cojocaru M, Bursin C, et al. Detection of neopterin as a parameter of potential monocyte activation in patients with acute ischemic stroke. Rom J Intern Med 2007;45:365-369. 35. Cojocaru IM, Cojocaru M, Iliescu I, et al. Plasma myeloperoxidase levels in acute ischemic stroke. Rom J Intern Med 2010;48:101-104. 36. Elkind MS, Lin I-F, Grayston TJ, et al. Chlamydia pneumoniae and the risk of first ischemic stroke: the Northern Manhattan Stroke Study. Stroke 2000;31:1521-1525. 37. Whiteley W, Wardlaw J, Dennis M, et al. The use of blood biomarkers to predict poor outcome after acute transient ischemic attack or ischemic stroke. Stroke 2012;43:86-91. 38. Shaw AC. Serum C-reactive protein and neopterin concentrations in viral and bacterial infections. J Clin Pathol 1991;44:596-599. 39. Syrjanen J, Valtonen VV, Iivanainen M, et al. Preceding infection as an important risk factor for ischemic brain infarction in young and middle aged patients. BMJ 1988;296:1156-1160. 40. Jozwiak PB, Mickielewicz A, Gaciong Z, et al. Elevated levels of anti-Chlamydia pneumoniae IgA and IgG antibodies in young adults with ischemic stroke. Acta Neurol Scand 2007;116:144-149.
