Category Archives: research

A new marker for AMD

A new marker for Neovascular Age-Related Macular Degeneration: Eotaxin-2

 

Role of Eotaxin–CCR3 was reported in choroidal neovascularization (CNV) development in recent studies. It was documented to be better than vascular endothelial growth factor-A treatment when tested in CNV animals. Due to lack of studies on the role of eotaxin in the human age-related macular degeneration (AMD) patients, we sought to determine whether eotaxin-2 (CCL24) has any association with inflammatory processes that occur in CNV.

CCL24 levels were determined by enzyme linked immunosorbant assay (ELISA) after normalization to total serum protein and levels of ELISA were correlated to various risk factors in about 133 AMD patients and 80 healthy controls.

The CCL24 levels were found to be significantly higher in wet AMD patients as compared with dry AMD and normal controls. Further the difference between dry and wet AMD patients (i.e., minimally classic, predominantly classic, and occult) was also significant. We also report significant difference in the CCL24 levels of Avastin-treated and untreated AMD patients. This study shows that CCL24 levels were found to be significantly increased in AMD patients despite Avastin treatment as compared with normal controls and those without Avastin, indicating that CCL24 may have an association with CNV and may be an important target to validate future therapeutic approaches in AMD in tandem with Avastin treatment.

Sharma N K, Prabhakar S, Gupta A, Singh R, Gupta P K,  Gupta P K, and Anand A. New Biomarker for Neovascular Age-Related Macular Degeneration: Eotaxin-2.DNA and Cell Biology. 2012; 31:1618-1627

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ALS Predictive model

Amyotrophic lateral sclerosis (ALS) diagnosis: A predictive model.

 

The clinical diagnosis of amyotrophic lateral sclerosis (ALS) usually takes several months and this delay adversely affects the therapeutic interventions. Hence, earlier and effective diagnosis of ALS for better management of ALS patients needs the development of a statistical model. Therefore, we have developed a statistical model for predicting the risk of ALS at earlier stages. The study recruited 44 sporadic ALS patients along with 29 normal controls. Demographic details (e.g. age, sex, cigarette smoking status, alcohol consumption, diet and bodymass index) of patients at the age of onset of disease was collected using a questionnaire. Thirteen independent variables (predictors) which may be associated with ALS were included in the study. Forward stepwise (likelihood ratio) binary logistic regression was used to find significant variables and probability of disease prediction.

The Serum chemokine ligand-2, chemokine ligand-2 mRNA, vascular endothelial growth factor-A mRNA, smoking and alcohol consumption are the independent variables found significant to predict risk of ALS.  The current model yielded 93.2% sensitivity and 86.2% specificity with 90.4% overall validity of correct ALS prediction. The study suggest the Forward stepwise (likelihood ratio) binary logistic regression model is an accurate method to predict ALS in the presence of serum CCL2, CCL2 mRNA, VEGFA mRNA, smoking and alcohol consumption with high sensitivity and specificity.

Gupta P K,  Prabhakar S, SharmaS and Anand A. A predictive model for amyotrophic lateral sclerosis (ALS) diagnosis. Journal of the Neurological Sciences. 2012;  312:68–72

 

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Stem cell and stroke

Assessment of marrow derived stem cell implantation in experimentally induced brain stroke and retinal ischemia mouse model

 

 

We have established the experimental model of neuronal damage in mice by middle cerebral artery (MCA) occlusion method and assessment of neuronal and ganglion cell damage by infarct area and neurobehavioral measurement, and  investigated the effects of marrow derived mononuclear cell (MNC) and retinal stem cells by transplantation strategies. The adult Swiss albino male mice were used in which the right common carotid artery, external carotid artery and internal carotid artery (ICA) were exposed via a midline incision.

An 8-0 polypropylene suture coated with poly-lysine was inserted into ICA and advanced into the origin of MCA. The animals were subjected to MCA occlusion for 60 minutes followed by 23 h reperfusion. After 24 h, coronal sections as well as retinal sections were obtained and stained with 1% triphenyltetrazolium chloride (TTC), a marker of cerebral ischemia damage in mice brain.

The marrow derived mononuclear cells were injected into ischemic injured mice through tail vein route. The experimental mice showed a contra lateral turning behavior which persisted up to 4 h after MCAO which was considered as a precocious index of neurological deficit and neuronal damage. The ganglion layer depletion results due to retinal ischemia. The retinal stem cells were implanted and followed temporally in order to understand the efficacy of these cells in rescuing the function deficit caused due to ischemia. This is an excellent platform for analysis of the effects of stem cells and drugs on the ischemic injury.

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Efficacy of amplicor kit for TB

Diagnostic efficacy of Amplicor kit for detection of mycobacterium tuberculosis in the patients suspected of TBM

 

 

We used to screen the Mycobacterium tuberculosis in CSF samples of suspected TBM patients using Amplicor detection kit. We had collected about 100 patients suspected of TBM through a redundant program under former HOD and had analyzed this using the commercially available Amplicor kit that used the patented PCR-ELISA method. Out of the 100 patients from whom CSF samples that were collected 79% were diagnosed negative for TBM and 8% were positive for the disease, providing vital diagnosis for Neurologists. Out the 100 patients analyzed for TBM 58 were male and 42 were females.

 

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CCL-2 and VEGF in ALS

Elevated levels of CCL-2 and VEGF in circulating lymphocytes of Indian sporadic ALS patients

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by degeneration of motor neurons. We evaluated the Vascular Endothelial Growth Factor (VEGF) and Monocyte Chemoattractant Protein-1 (CCL-2) levels in cerebrospinal fluid (CSF) and serum of ALS patients and age matched controls of North Indian origin by ELISA.

The ALS patients were included on the basis of El Escorial criteria after obtaining written informed consent. All the patients were of sporadic origin. The levels of VEGF and CCL2 were then normalized to total protein. We also clinically correlated these results as determined by ALSFRS score. The mean age of ALS patients was 46 ±12, out of which 15 were males and 5 were females. There were 17 cases of limb variant and 3 cases of bulbar onset. Majority of the cases were characterized by late onset. The results showed that CSF levels of CCL2 and serum levels of both VEGF and CCL-2 were significantly upregulated as compared to control subjects (p<0.05).The serum CCL2 and VEGF levels increased with severity of disease. The results suggest that increase in Ccl2 and VEGF may be potential markers for assessment of disease burden and for possible monitoring the effects of treatment. The evidence for intrathecal synthesis of CCL-2 suggests the role of microglial activation as previously reported. Although VEGF dysregulation is believed to compromise neuroprotection and predispose the mouse model of ALS and human to ALS, it is possible that the elevated VEGF levels in late onset could be a due to the compensatory response against glutamate mediated toxicity.   << Back to research area

Ischemic stroke biomarkers

The contribution of Vascular endothelial growth receptor (VEGF) and monocyte chemoattractant protein(MCP-1) to symptomatic atherosclerotic carotid plaques

 

 

The role of vascular endothelial growth factor and monocyte chemoattractant protein as a serum biomarker of atherothrombotic disease is currently under investigation. Previous western studies have mostly related VEGF and MCP-1 to ischemic stroke. This is an Indian study and we tried to relate these protein growth factor levels as potential biomarkers to incidence of ischemic stroke or transient ischemic attack since stroke is a principal cause of death in elderly people. We have estimated the VEGF and MCP-1 levels in stroke patients presenting with carotid atherosclerotic plaques in order to explore their regulatory roles in disease progression.

VEGF and MCP-1 levels in serum obtained from study participants were measured using a solid phase sandwich ELISA (R&D). 57 patients with carotid plaques as screened by carotid Doppler were recruited for this study. 38 stroke control patients without carotid plaque and also 15 healthy controls who had no history of serious illness were recruited. We also investigated the role of environment and demography in this disease. The VEGF level was found to be unaltered in these patients as compared to controls. Similarly, there was no significant upregulation of monocyte chemoattractant protein in these patients. There was no positive association with smoking, alcohol consumption, or dietary habit. However a positive association was seen with gender and urban residence. Hypertension was found to be positively associated with stroke when compared to controls. We also found that smokers are four times more susceptible to stroke.

 

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Biomarkers in ALS

Levels of Vascular endothelial growth factor-A (VEGF-A) and chemokine ligand-2 (CCL2) in Serum and CSF of Amyotrophic Lateral Sclerosis patients.

 

 

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with no reliable markers for disease prognosis. Levels of Vascular endothelial growth factor-A (VEGF-A) and chemokine ligand-2 (CCL2) have been examined in Amyotrophic Lateral Sclerosis (ALS) in Western population. We wanted to measure these levels in North Indian ALS patients as these patients have enhanced survival duration.  In the study 50 sporadic ALS patients (25 with definite ALS, 15 probable and 10 possible ALS) were recruited and VEGF-A and CCL2 levels were analyzed in serum and cerebrospinal fluid using enzyme linked immunosorbent assay (ELISA) and compared with normal controls. Their levels were adjusted for possible confounders like cigarette smoking, alcohol and meat consumption.

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VEGF-A and CCL2 levels were found to be elevated significantly in serum and CSF in ALS patient population studied and results were contrary to our earlier studies. Serum and CSF from definite ALS revealed higher VEGF-A as compared to probable and possible ALS while CCL2 level was unaltered. No association was revealed between smoking, alcohol and meat consumption with VEGF-A and CCL2 levels. It can be concluded that VEGF-A upregulation helps in compensatory responses activation in ALS which accounts for increased survival of North Indian ALS patients.

 

Gupta P K, Prabhakar S, Sharma S and Anand A. Vascular endothelial growth factor-A (VEGF-A) and chemokine ligand-2 (CCL2) in Amyotrophic Lateral Sclerosis (ALS) patients. Journal of Neuroinflammation.  2011, 8:47

 

 

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CCL2 gene upregulation in ALS

Upregulation of chemokine ligand (CCL2) genes in peripheral blood mononuclear cells in Amyotrophic Lateral Sclerosis.

 

 

Protein levels of vascular endothelial growth factor-A (VEGF-A) and chemokine ligand-2 (CCL2) were shown to be elevated in Indian Amyotrophic Lateral Sclerosis (ALS) patients in our previous studies. Here, we report the mRNA levels of VEGF-A and CCL2 in peripheral blood mononuclear cells (PBMCs) of 50 ALS patients. Their levels were adjusted for possible confounders like cigarette smoking, alcohol and meat consumption.

Real Time PCR indicates that VEGF-A expression is 77- fold higher in ALS than controls while CCL2 mRNA has shown an increment of 9.5-fold in ALS PBMCs. Definite ALS revealed higher VEGF-A mRNA expression as compared to probable and possible ALS while CCL2 mRNA levels showed no significant difference. In patients with respiratory dysfunction VEGF-A and CCL2 levels were elevated as compared to patients without respiratory dysfunction. No association of cigarette smoking, alcohol and meat consumption with VEGF-A and CCL2 mRNA was observed upon univariate and multivariate analysis. The investigations of these molecules in cross ethnic groups can help in  determining their role in enhancing the mean survival time of Indian ALS patients.

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Gupta P K, Prabhakar S, Abburi C, Sharma N K, and Anand A. Vascular endothelial growth factor-A and chemokine ligand (CCL2) genes are upregulated in peripheral blood mononuclear cells in Indian amyotrophic lateral sclerosis patients. Journal of Neuroinflammation. 2011; 8:114.

 

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Role of CCR2 in ALS disease

Association between Expression of Chemokine Receptor-2 (CCR2) and Amyotrophic Lateral Sclerosis (ALS) Patients of North India.

 

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which is characterized by participation of inflammatory cascade. These inflammatory changes are strongly regulated by interaction of chemokine ligand-2 (CCL2), a small chemokine belongs to C-C subfamily with its receptor chemokine receptor-2 (CCR2). In our earlier studies we have reported elevated chemokine ligand-2 (CCL2) in Indian ALS patients. We further analysed chemokine receptor-2 (CCR2) in these ALS patients. Indian sporadic ALS patients (n = 50) and control (40) were included on the basis of El Escorial criteria. CCR2 expression in peripheral blood mononuclear cells (PBMCs) was valuated using Flow Cytometry and Real Time Polymerase Chain Reaction (PCR). Flow Cytometry revealed significantly reduced CCR2 expressing PBMCs in the ALS patients, decline was more significant in limb onset ALS when compared to bulbar onset ALS. PBMCs from ALS patients showed substantial down-regulation of CCR2 mRNA. When compared with bulbar onset ALS CCR2 mRNA expression was found to be decreased among limb ALS patients. Further, the count of CCR2+ PBMCs and CCR2 mRNA transcript in PBMCs was significantly lower in severe and moderate ALS as compared to ALS patients with mild impairments. This downregulation of CCR2 expression in PBMCs suggests its role in etiology of ALS pathogenesis. It can be postulated that reduction in PBMCs CCR2 may result in decreased infiltration of leukocytes at the site of degeneration as a compensatory response to ALS.

Gupta P K, Prabhakar S, Sharma N K, Anand A. Possible Association between Expression of Chemokine Receptor-2 (CCR2) and Amyotrophic Lateral Sclerosis (ALS) Patients of North India. PLoS ONE 7(6): e38382. doi:10.1371/journal.pone.0038382

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New target for AMD

CC chemokine receptor-3 as new target for age-related macular degeneration.

 

 

Age-related macular degeneration (AMD) is a complex disease, which is influenced by genetic and environmental factors. CCR3 is a chemokine receptor present on basophils and eosinophils. The main role of CCR3 is to activate and recruit eosinophils to the site of inflammation and stimulate macrophage activation. These activated eosinophils can release reactive oxygen species which contribute to host tissue damage during chronic inflammatory responses. Although CCR3 is known for inflammation but its studies are lacking in AMD patients. Therefore, we sought to determine any possible association between CCR3 and inflammatory processes that occur in choroid neovascularization. In the study a total of 176 subjects were recruited on the basis of inclusion criteria. 115 AMD patients and 61 normal controls were used for the comparison. Real time PCR was carried out to analyze the single nucleotide polymorphism in CCR3 of AMD patients and normal controls. Genotype frequency was adjusted for possible confounders like cigarette smoking, alcohol, meat consumption and other risk factors to find the association of these with AMD. Chi-square test was used to determine the polymorphism of CCR3 in human AMD patients.  The genotype distribution of CCR3 (rs3091250) polymorphism was found to be significantly different in AMD patients in the Indian population. Both the heterozygous (GT) and homozygous (TT) genotypes at rs3091250 SNP were found to be significantly more frequent in AMD patients as compared to the GG genotypes. There was no significant difference between AMD patients in wet and dry. A significant association between AMD and CCR3 (rs3091250) polymorphism localized on chromosome 3p21.3 was detected. The results suggested the possible contribution of rs3091250 allele in AMD. From the study it can be postulated that for early detection of CNV, CCR3 could be a novel marker which can be exploited as a new therapeutic entity through future studies.

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Sharma N K, Gupta A , Prabhakar S , Singh R, Bhatt A K, Anand A. CC chemokine receptor-3 as new target for age-related macular degeneration. Gene 523 (2013) 106–111

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