Agarose gel profiles of polymerase chain reaction products from cell supernatant of Campylobacter jejuni –induced lymphocytes from 4 representative patients with Guillain-Barré syndrome (GBS). A, β-Actin messenger RNA (mRNA) from GBS patients. B and C, Tumor necrosis factor mRNA from the progressive and recovery phases of GBS, respectively. Lane M, molecular weight marker (100 base pairs [bp]); lane 1, negative control; and lanes 2 through 5, the expression of mRNA from C jejuni –induced lymphocytes from GBS patients.
Comparative evaluation of mean (SD) cytokine expressions by reverse transcription–polymerase chain reaction during the progressive and recovery phases of Guillain-Barré syndrome. A, Interferon γ (IFN-γ); B, interleukin 1β (IL-1β); C, tumor necrosis factor (TNF); D, IL-6; E, transforming growth factor β1; F, IL-4; G, IL-10; and H, IFN-γ:IL-4 ratio (P < .001 for all).
Comparative evaluation of cytokine expressions by enzyme-linked immunosorbent assay during the progressive and recovery phases of Guillain-Barré syndrome. A, Interferon γ (IFN-γ); B, interleukin 1β (IL-1β) (P < .001); C, tumor necrosis factor (TNF) (P < .001); D, IL-6 (P < .001); E, transforming growth factor β1 (TGF-β1) (P < .001); F, IL-4 (P < .001); G, IL-10; and H IFN-γ:IL-4 ratio. Error bars indicate 95% confidence intervals.
Nyati KK, Prasad KN, Rizwan A, Verma A, Paliwal VK. TH1 and TH2 Response to Campylobacter jejuni Antigen in Guillain-Barré Syndrome. Arch Neurol. 2011;68(4):445-452. doi:10.1001/archneurol.2011.51
To determine the expression of proinflammatory and anti-inflammatory cytokines in lymphocytes from the progressive and recovery phases of Guillain-Barré syndrome (GBS) after stimulation with Campylobacter jejuni outer membrane proteins.
Sanjay Gandhi Postgraduate Institute of Medical Sciences.
Sixty-five patients with GBS, 60 age- and sex-matched disease control individuals, and 68 healthy control individuals were included in the study.
Main Outcome Measures
Lymphocytes from patients with GBS were stimulated with C jejuni outer membrane proteins, and the levels of different proinflammatory (TH1 [helper T cell, subtype 1]) and anti-inflammatory (TH2) cytokines were determined by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay.
In the progressive phase of the disease, the expressions of interferon γ (IFN-γ), interleukin 1β (IL-1β), tumor necrosis factor (TNF), IL-6, IL-10, and the IFN-γ:IL-4 ratio were significantly upregulated, but expressions of transforming growth factor (TGF)–β1 and IL-4 were lower in patients compared with disease and healthy controls. In contrast, the levels of IFN-γ, IL-1β, TNF, IL-6, IL-10, and the IFN-γ:IL-4 ratio were significantly lower, but TGF-β1 and IL-4 were upregulated in the recovery phase of GBS patients compared with controls.
Upregulation of TH1 cytokines in the early disease course may be associated with immune-mediated disease progression due to neuronal inflammation, but upregulation of TH2 immune response during the later phase aids recovery from the disease.
Guillain-Barré syndrome (GBS) is an immune-mediated inflammatory disease that affects the myelin cells and axons of peripheral nerves. It is generally believed that exogenous antigens may trigger an autoimmune peripheral demyelination by a molecular mimicry–induced loss of tolerance. Campylobacter jejuni is the most common microorganism implicated in the development of GBS.1- 3 It is hypothesized that the development of GBS after C jejuni infection is due to the triggering of humoral and cellular immune responses against antigenic epitopes on C jejuni that crossreact with shared epitopes in nerve myelin cells.1,4 Besides antibodies, complement system components and inflammatory cell infiltrates (lymphocytes and macrophages) also contribute to nerve damage in GBS5 by targeting Schwann cells, the principal myelinating glial cells of peripheral nerves.4 Furthermore, Schwann cells can potentially modulate multiple aspects of inflammatory cascade4 by producing cytokines and toxic substances.6 Because the inflammatory cell infiltrates exert most of their effects through immunomodulatory cytokines and Schwann cells can also produce cytokines, it is of central importance to explore cytokine orchestration in the development of GBS after C jejuni infection.
Cytokines are a group of polypeptides involved in host defense and repair. The balance between the proinflammatory (TH1 [helper T cell, subtype 1]) and anti-inflammatory (TH2) cytokines is necessary for a successful immune response. It is possible that systemically and locally released cytokines are important in the pathogenesis of GBS. Evidence suggests that tumor necrosis factor (TNF) is critically involved in immune-mediated demyelination and axonal damage of peripheral nerves7 by affecting myelin protein and glycolipid synthesis, but transforming growth factor β (TGF-β) can downmodulate activated glial, inflammatory, and endothelial cells.6 Studies6 regarding experimental autoimmune encephalomyelitis and experimental autoimmune neuritis (EAN) revealed differential upregulation and expression of various cytokines. However, the data published so far are conflicting, and comprehensive analysis has not yet been reported, to our knowledge. Experimental autoimmune neuritis is the only animal model of GBS in which differential upregulation of cytokines during various phases of EAN has been documented.8,9 Most of the data in existence regarding the role of immune responses relate to the entire spectrum of GBS patients, but studies regarding immune response to patients with C jejuni –associated GBS are still elusive. Because of the scarcity of nerve biopsy specimens from GBS patients, the role of cytokines in the development of GBS after C jejuni infection is not completely understood. Earlier, the lymphocyte transformation test was evaluated for the detection of a response to C jejuni antigens in the lymphocytes from GBS patients.3 Furthermore, in the present study, we investigated the cytokine profile expressed by the lymphocytes of GBS patients after stimulation by C jejuni outer membrane proteins (OMPs) and compared the results with those from the progressive and recovery phases of the disease.
Sixty-five patients with GBS admitted to the Neurology Ward, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India, who fulfilled the diagnostic criteria described by Asbury and Cornblath10 were enrolled in the study. The GBS patients were admitted a mean (SD) of 4.5 (3.2) days after the onset of motor symptoms. According to Hughes et al,11 severity of disease was scored at the time of each blood sample using the following scale: (1) able to walk more than 5 m without assistance, (2) able to walk more than 5 m with assistance, (3) bedridden or requiring the full-time use of a wheelchair, or (4) requiring assisted ventilation for at least part of the day. The first blood sample was collected from patients within 48 hours of admission (progressive phase of the disease) before treatment with intravenous (IV) immunoglobulins, and the second blood sample was collected during the fifth week of the hospital stay (recovery phase of the disease) after a 5-day course of IV immunoglobulin treatment (0.4 g/kg/d). Patients with GBS had not received any steroid treatment during the study. Sixty age- and sex-matched disease control individuals and 68 healthy controls were also included in the study. Disease controls were patients with transverse myelitis, diabetic neuropathy, stroke, and chronic inflammatory neuropathy, and healthy controls were individuals without any history of apparent infectious illness within the past 4 weeks. The study was approved by the ethics committee of the Sanjay Gandhi Postgraduate Institute of Medical Sciences, and informed consent was obtained from all study participants.
Lymphocytes were isolated from all study participants from freshly collected peripheral blood drawn in heparinized tubes (Becton Dickinson Diagnostic Systems, Franklin Lakes, New Jersey) by Ficoll-Hypaque density gradient centrifugation (research grade; Pharmacia Biotech, Uppsala, Sweden).3 Cell viability was determined by trypan blue staining (Gibco BRL, Auckland, New Zealand) and counted on a hemocytometer per the standard procedure. Cells were then suspended in T-cell medium consisting of RPMI-1640 medium (Gibco BRL) supplemented with 10% fetal calf serum, 2mM L-glutamine (Gibco BRL), and antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin).
The lymphocyte transformation test was performed following the standard protocol, as previously described.3 In brief, lymphocytes (1 × 106 cells per well) in 200 μL of RPMI-1640 with supplements were cultured in triplicate in 96-well flat-bottom tissue culture plates in the presence of 20 μg/mL of OMPs (test) or 5.0 μg/mL of phytohemagglutinin (positive control) per well. Cells cultured in the medium without OMPs and phytohemagglutinin antigen served as negative controls. The reaction was stopped 24 hours after stimulation. Cells and cell supernatants were collected separately and stored at −80°C for reverse transcription–polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively.
Cytokine RT-PCR was performed on RNA extracted from the stimulated and unstimulated cells using the Qiagen RNeasy kit (Qiagen Inc, Valencia, California) according to the manufacturer's instructions. Complementary DNA was synthesized by the RevertAid H minus first strand complementary DNA synthesis kit (Fermantas Life Sciences, Glen Burnie, Maryland) taking 100 ng of RNA for each sample. After reverse transcription, primer-specific amplification of interferon γ (IFN-γ), interleukin 1β (IL-1β), TNF, IL-6, TGF-β1, IL-4, and IL-10 was performed (Table 1); β-actin was used as the housekeeping gene. The PCR products were examined via 1.8% agarose gel electrophoresis and photographed under UV illumination. Band intensities were quantified by densitometry scanning software ImageQuant TL (GE Pharmacia Biotech, Newark, New Jersey). To normalize messenger RNA (mRNA) levels, densities of IFN-γ, IL-1β, TNF, IL-6, TGF-β1, IL-4, IL-10, and β-actin bands from the same samples were measured, and data were calculated as the ratios of band intensity values relative to the band intensity value of β-actin.
Quantitative analysis by ELISAs for IFN-γ, IL-1β, TNF, IL-6, TGF-β1, IL-4, and IL-10 in cell supernatants was performed using commercially available kits per the manufacturer's instructions (R&D Systems, Minneapolis, Minnesota). ELISAs were performed in triplicate independently for each sample. The results were expressed as picograms of cytokines per milliliter based on the standard provided with the kits.
Data were analyzed with SPSS statistical software, version 17.0 (SPSS Inc, Chicago, Illinois), and P ≤ .05 was considered significant. Values from densitometry were expressed as mean (SD) of triplicate experiments performed independently for each sample for semiquantitative RT-PCR and ELISA analysis. Differences in cytokine concentrations between the different groups (GBS vs disease controls and/or GBS vs healthy controls) were analyzed using the 1-way analysis of variance test. Comparison of 2 diseased groups (progressive vs recovery) was analyzed by the paired-sample t test.
Sixty-five patients with GBS (40 men and 25 women; mean [SD] age, 40.29 [15.25] years), 60 disease controls (38 men and 22 women; mean [SD] age, 38.65 [18.27] years), and 68 healthy volunteers (44 men and 24 women; mean [SD], 41.90 [14.47] years) were included in the study. All the GBS patients were in the progressive and recovery phases during the first and second blood samplings, respectively. The GBS patients were admitted a mean (SD) of 4.5 (3.2) days after onset of motor symptoms. Fifty-nine patients had a satisfactory recovery in a median (SD) duration of 33 (4.2) days without any relapses during the study period. The remaining 6 patients (9.2%) were excluded from the study because of difficulty walking without support during the second sampling. All demographic and clinical details of the patients are summarized in Table 2.
A representative agarose gel photograph for the TNF cytokine and β-actin during the progressive and recovery phases for a GBS patient is shown in Figure 1 (parts A-C).
The mean (SD) mRNA levels of TH1 cytokine IFN-γ and proinflammatory cytokines IL-1β, TNF, IL-6, and immunomodulatory IL-10 were significantly upregulated in GBS patients in the progressive phase of the disease compared with disease controls (0.29 [0.10] vs 0.24 [0.08], 0.72 [0.22] vs 0.45 [0.10], 1.45 [0.64] vs 0.55 [0.18], 0.56 [0.12] vs 0.43 [0.15], and 0.47 [0.14] vs 0.27 [0.07], respectively) and healthy controls (0.29 [0.10] vs 0.20 [0.10], 0.72 [0.22] vs 0.42 [0.12], 1.45 [0.64] vs 0.53 [0.13], 0.56 [0.12] vs 0.31 [0.11], and 0.47 [0.14] vs 0.22 [0.08], respectively). In contrast, significantly decreased expression of immunomodulatory TGF-β1 and TH2 cytokine IL-4 was found in GBS patients in the progressive phase of the disease compared with disease controls (0.13 [0.06] vs 0.16 [0.07] and 0.07 [0.04] vs 0.09 [0.07], respectively) and healthy controls (0.13 [0.06] vs 0.24 [0.08] and 0.07 [0.04] vs 0.10 [0.08], respectively). The IFN-γ:IL-4 ratio was significantly higher in patients compared with healthy controls (5.99 [5.84] vs 3.63 [3.66]) (Table 3).
Furthermore, in patients in the recovery phase of the disease, the mRNA levels of TH1 cytokine IFN-γ and proinflammatory cytokines IL-1β, TNF, and IL-6 were significantly downregulated compared with disease controls (0.07 [0.04] vs 0.20 [0.06], 0.26 [0.11] vs 0.38 [0.14], 0.43 [0.21] vs 0.53 [0.16], and 0.09 [0.05] vs 0.35 [0.08], respectively) and healthy controls (0.07 [0.04] vs 0.23 [0.11], 0.26 [0.11] vs 0.41 [0.11], 0.43 [0.21] vs 0.50 [0.12], and 0.09 [0.05] vs 0.31 [0.11], respectively), but upregulation of the TH2 cytokine IL-4 and immunomodulatory TGF-β1 was found in these patients compared with disease controls (0.16 [0.07] vs 0.10 [0.05] and 0.49 [0.20] vs 0.32 [0.21], respectively) and healthy controls (0.16 [0.07] vs 0.11 [0.08] and 0.49 [0.20] vs 0.22 [0.07], respectively). However, the expression of IL-10 was comparable at the mRNA level with disease and healthy controls. The IFN-γ:IL-4 ratio was significantly lower in patients compared with disease and healthy controls (Table 4).
The concentrations of the TH1 cytokine IFN-γ; proinflammatory cytokines IL-1β, TNF, and IL-6; and immunomodulatory IL-10 were significantly higher in GBS patients in the progressive phase of the disease compared with disease controls (87.98 [35.32] vs 74.23 [39.41], 151.81 [74.28] vs 79.33 [24.36], 76.23 [35.99] vs 23.93 [9.77], 146.96 [81.66] vs 79.63 [31.18], and 4.92 [1.68] vs 4.01 [1.94] pg/mL, respectively) and healthy controls (87.98 [35.32] vs 38.91 [24.51], 151.81 [74.28] vs 68.95 [26.90], 76.23 [35.99] vs 18.70 [6.37], 146.96 [81.66] vs 66.47 [31.77], and 4.92 [1.68] vs 2.82 [1.28] pg/mL, respectively). Beside the proinflammatory cytokines, the immunomodulatory cytokine TGF-β1 was downregulated in GBS patients in the progressive phase of the disease compared with disease controls (22.72 [10.81] vs 26.18 [10.18] pg/mL) and healthy controls (22.72 [10.81] vs 27.91 [13.84] pg/mL), whereas IL-4 showed decreased expression in GBS patients compared with healthy controls (0.60 [0.37] vs 0.87 [1.04] pg/mL). The IFN-γ:IL-4 ratio was significantly higher in GBS patients compared with healthy controls (205.66 [157.27] vs 135.87 [102.80] pg/mL) (Table 3).
Decreased expression of proinflammatory cytokines (IL-1β, TNF, and IL-6) was observed in patients in the recovery phase of the disease compared with disease controls (61.76 [18.84] vs 71.63 [27.88], 13.89 [6.25] vs 20.38 [7.78], and 56.83 [20.74] vs 65.85 [29.88] pg/mL, respectively) and healthy controls (61.76 [18.84] vs 71.57 [34.27], 13.89 [6.25] vs 17.16 [6.19], and 56.83 [20.74] vs 67.17 [33.15] pg/mL, respectively). In contrast, levels of immunomodulatory TGF-β1 and the TH2 cytokine IL-4 were significantly higher in patients in the recovery phase of GBS compared with disease controls (41.03 [11.97] vs 28.10 [9.69] and 3.67 [2.87] vs 0.97 [0.71] pg/mL, respectively) and healthy controls (41.03 [11.97] vs 29.76 [11.61] and 3.67 [2.87] vs 0.85 [0.99] pg/mL, respectively). Interestingly, the levels of IFN-γ, a signature cytokine of TH1 cells and IL-10, were not detectable in the recovery phase for GBS patients (Table 4).
The levels of IFN-γ, IL-1β, TNF, and IL-6 were significantly increased at the time of disease progression compared with patients in the recovery phase of the disease. Expression of TGF-β1 and IL-4 was significantly upregulated in the recovery phase compared with the progressive phase of the patients at the mRNA (Figure 2) and protein levels (Table 5 and Figure 3).
Furthermore, induction of lymphocytes with phytohemagglutinin revealed at least a 2.5-fold increase in the secretion of different cytokines in patients in the progressive and recovery phases compared with negative controls. In addition, the concentrations of all the cytokines in the cell supernatant of the negative controls and in the GBS patients in the progressive and recovery phases of disease were comparable (Table 5).
The present study provides evidence of the polarized TH1-like response in patients in the progressive phase of GBS; the recovery phase of GBS was associated with a TH2-like cytokine profile. During the study, TH1 and TH2 immune response in lymphocytes of GBS patients was determined after stimulation with C jejuni OMPs. In the progressive phase of the disease, the expression of the TH1 cytokine (IFN-γ) and proinflammatory cytokines (IL-1β, TNF, and IL-6), including immunomodulatory IL-10, was significantly higher, but TGF-β1 and IL-4 were found to be downregulated in GBS patients compared with disease controls (P = .006, P < .001, P < .001, P < .001, P = .014 and P = .04, respectively) and healthy controls (P = .001, P < .001, P = .001, P = .001, P < .001, and P = .04, respectively). In contrast, the levels of the proinflammatory cytokines (IL-1β, TNF, and IL-6) in GBS patients in the recovery phase were significantly lower compared with healthy controls (P < .001, P = .03, and P < .001, respectively) and disease controls (P < .001, P = .005, and P < .001, respectively), but increased expressions of immunomodulatory TGF-β1 and the TH2 cytokine IL-4 were determined in the late or recovery phase of the disease when compared with both groups of controls (P < .001 for both). An increased IFN-γ:IL4 ratio in the progressive phase compared with the recovery phase of GBS patients was also determined, which further supports the hypothesis of TH1 and TH2 cytokine–dominated immune responses.
Several studies12- 14 suggest that GBS associated with C jejuni as an antecedent pathogen may be more severe than GBS caused by other infectious agents. The host immune response against C jejuni has been assumed to be responsible for the pathogenesis of GBS15 by inducing crossreactive antibodies against host gangliosides. As a result, a cascade of immune-mediated inflammatory responses can be generated by specific immune recognition involving T lymphocytes, monocytes, and various cytokines responsible for causing demyelination in the host peripheral nervous system. These cytokines may assist in the disruption of the blood-nerve barrier; as a result, immune cells can infiltrate across the barrier and obtain direct access to myelin and Schwann cells, thus affecting the peripheral nerve conduction. Anatomically, the blood-nerve barrier is deficient in the distal nerve terminals and nerve roots, and these regions are preferentially affected by an immune attack. The resolution of physiologic nerve conduction failure at the nodes of Ranvier leads to rapid recovery in some patients; however, axonal degeneration is associated with slow and incomplete recovery in other patients.
Often, GBS is a transitory disease that may end in a full recovery. Various studies from different parts of the world suggest that the balance between TH1 and TH2 cytokines determines the course and the end result of the disease. In the earlier studies, TH1 cytokines are considered to play an important role in inducing cell-mediated autoimmune diseases, although TH2 cytokines suppress such diseases,8,16,17 which supports our present findings. In multiple sclerosis (MS), the cytokine effect may vary, with progression of disease being sometimes autoaggressive and sometimes protective. Interferon γ has been shown to induce clinical exacerbation in patients with MS. Increased IFN-γ and IL-10 production precedes relapse in MS patients.18 A study19 has shown that levels of proinflammatory cytokines, such as IFN-γ, TNF, IL-2, and IL-6, were elevated, which might lead to demyelination during progressive MS, but the TH2 cytokine (IL-4) was upregulated and proinflammatory cytokines were downregulated after treatment with IV methylprednisolone during recovery from MS. In the present study, we found significantly higher concentrations of IFN-γ, IL-1β, TNF, and IL-6 during the progressive phase of the disease. It is likely that autoimmune attack on gangliosides, neurons, or axons results from overexpression of TH1 cytokines by activated T cells, leading to severe neurophysiologic abnormalities.
Increments in TNF levels have been associated with disease severity and may induce demyelination, axonal damage, and toxic effect in several autoimmune diseases, including GBS, and the decrease in TNF levels inhibits the development of experimental autoimmune encephalomyelitis, EAN, and GBS.6 Studies20- 22 suggest that TNF may act in the acute or afferent phase of an immune reaction and likely contributes to the development of inflammatory and immunopathologic lesions. Increased expression of TGF-β1 may be important during the recovery phase of GBS as found in the present study. Earlier, Créange et al23 found that TGF-β1 might help in the termination of inflammatory disease and in recovery. The higher levels of TGF-β1 induce migration, differentiation, and proliferation of Schwann cells, which may contribute to the recovery of the peripheral nervous system from severe damage. Similarly, the IL-4 responses are beneficial in GBS and may have a role in terminating the process of this self-limiting inflammatory disease.24,25 In contrast, the IFN-γ production is probably related to the severity of the GBS, with clinical improvement associated with its decreased production, as we observed in the present study. However, the data regarding IFN-γ in GBS are conflicting, which may be owing to use of different techniques, time of sample collection, the collection of samples from different stages of the diseases, and the methods of cytokine detection. Therefore, Press et al26 did not observe upregulation in IFN-γ levels in their study and concluded that IFN-γ was not involved in the pathogenesis of GBS. In a subsequent study, Sharief et al27 found high levels of IL-6 and IL-10, but TNF was not significantly altered. They reported that non-TH1 cytokines augmented in the early phase of the disease might describe the self-limited and benign course of the disease. They also reported that IV immunoglobulin cells used for the treatment of GBS suppress the levels of proinflammatory cytokines, such as TNF and IL-1β, during recovery; however, these levels remained relatively high in untreated patients. Large postphagocytic macrophages observed at later stages of demyelination in EAN were TNF negative, suggesting an early pathogenic role of TNF in EAN.28
Increased IL-10 expression in the early phase of GBS downregulates TH1 cytokine synthesis and may act as a physiologic countermeasure of immunologic mediators of neuroinflammation.29,30 Our results are in accordance with those of an earlier study30 in which higher numbers of IL-10–secreting cells are observed in the acute phase compared with the recovery phase of GBS. In the present study, IL-10, a regulator of the TH1 immune response, was expressed only in the progressive phase of the patients at mRNA and protein levels compared with both group of controls, although IL-10 mRNA was comparable, but IL-10 protein was not detectable in the recovery phase of the patients compared with both control groups. This finding may be explained by the fact that the entire mRNA does not necessarily synthesize protein, which may be governed by posttranscriptional factors.
In conclusion, the present study may explain the pathophysiologic role of TH1 cytokines in the progressive phase of C jejuni –associated GBS resulting from the immune-mediated nerve tissue damage and neuronal inflammation. A subsequent TH2 immune response likely aids recovery from the disease. However, further studies are needed, especially regarding nerve biopsy specimens from the peripheral nervous system, to provide direct insights into the putative immunopathogenesis of the disease associated with C jejuni infection.
Correspondence: Kashi N. Prasad, MD, Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Rae-bareli Rd, Lucknow, Uttar Pradesh 226 014 India (firstname.lastname@example.org).
Accepted for Publication: April 19, 2010.
Author Contributions: Drs Nyati and Prasad had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Nyati and Prasad. Acquisition of data: Nyati, Rizwan, and Verma. Analysis and interpretation of data: Nyati, Prasad, Verma, and Paliwal. Drafting of the manuscript: Nyati, Rizwan, and Verma. Critical revision of the manuscript for important intellectual content: Prasad and Paliwal. Statistical analysis: Nyati, Prasad, Rizwan, and Verma. Obtained funding: Prasad. Study supervision: Prasad and Paliwal.
Financial Disclosure: None reported.
Funding/Support: The study was supported by grants 5/3/3/24/2006-ECD-I and 80/569/2007-ECD-I (Mr Nyati) from the Indian Council of Medical Research and grant 09/590 (0138)/2007-EMR-I (Ms Verma) from the Council of Scientific and Industrial Research, Government of India, New Delhi.