Role Of Cell-mediated Immunity Research Articles

Prolactin-Inducible Protein: From Breast Cancer Biomarker to Immune Modulator-Novel Insights from Knockout Mice.

The propensity for breast cancers to elicit immune responses in patients is well established. The accumulation of tumor infiltrating lymphocytes within the primary breast tumor has been linked to better prognosis and better response to therapy. The prolactin-inducible protein (PIP) is a 15 kD protein that is expressed under physiological conditions of the breast and is regarded as a marker of mammary differentiation. While highly expressed under pathological conditions of the mammary gland, including breast cancers, PIP is expressed in very few other cancers. Although the function of PIP is not well elucidated, numerous studies suggest that its primary role may be related to host defense and immune modulation. However, evidence to show a direct link between PIP and the immune response has been lacking. In this review, we discuss our recent work with Pip-deficient mice, linking PIP not only to a role in innate immunity but for the first time, providing evidence for a role in cell-mediated immunity. These functional studies in Pip null mice lend new insight into the role of PIP in immunity and suggest that PIP may play a similar immune-regulatory role in breast cancer.

Short communication: Inhibitory effects of dietary aflatoxin B1 on cytokines expression and T-cell subsets in the cecal tonsil of broiler chickens

Aflatoxin B1 (AFB1) is the most toxic form among the mycotoxins. Cytokines are important mediators of the immune system. T-cell subsets play a crucial role in cell-mediated immunity. The aim of present study was to evaluate the effects of dietary AFB1 on the cytokines expression and T-cell subsets in the cecal tonsil of broiler chickens throughout a 21-day experimental period. One hundred and fifty six one-day-old broiler chickens were randomly divided into control group (0 mg AFB1/kg feed) and AFB1 group (0.6 mg pure AFB1/kg feed). At 7, 14 and 21 days of age, the levels of seven cytokines (IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF-α) mRNA expression as well as the proportions of T-cell subsets (CD3+, CD3+CD4+, CD3+CD8+) by qRT-PCR and flow cytometry methods were assessed in the cecal tonsils. The levels of the seven cytokines mRNA expression and the percentages of T-cell subsets significantly decreased at 14 and 21 days of age in the AFB1 group compared with the control group. However, the CD4+/CD8+ ratio was not significantly changed. These results demonstrate that 0.6 mg/kg AFB1 dietary exposure reduced the levels of cytokines mRNA expression and the percentages of T-cell subsets in the cecal tonsils of broiler chickens, suggesting that the cell-mediated immunity of cecal tonsils might be impaired in broilers.

T Hücrelerin Eş Uyaran ve İnhibitörleri: CD28 Ailesi

T cells that play role in cell-mediated immunity must be activated in order to generate immune responses. The two signals are required for T cell activation. The first of these signals is provided by the antigen. The second signal is provided by the co-stimulatory molecules. For many years it was believed that T cells do not require co-stimulation for activation. However, in vitro and in vivo studies have shown that T cells need the co-stimulatory molecules for activation and expansion. Co-stimulation for T-cell activation and growth is provided by molecules of CD28 and the tumor necrosis factor (TNF) family members. Four co-stimulatory molecules belonging to the CD28 family have been identified. These family members are CD28, inducible co-stimulator (ICOS), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, CD152) and programmed cell death protein 1 (PD-1). Although CD28 and ICOS showed positive effects, CTLA- 4 and PD-1 molecule have a negative effect on T cell activation. In this review, we discussed co-stimulatory molecules belonging to the CD28 family, the ligands of these molecules, and the interactions of co-stimulatory molecules with ligands.

Expression analyses of interferon gamma, tumor necrosis factor alpha and inducible nitric oxide synthase in the hemocyte morphotypes of two commercially important Indian molluscs

Bellamya bengalensis and Lamellidens marginalis are the two common species of molluscs of Indian subcontinent which bear immense aquacultural prospect. These edible varieties of freshwater molluscs have nutritional, ecological, ethnomedicinal and industrial significance. Report of nonspecific immunological status of them is grossly inadequate in the current scientific literature. Hemocytes, the circulating blood cells of invertebrate molluscs play a pivotal role in cell mediated immunity. Information of existence of cytokines like IFNγ, TNFα and enzyme iNOS is very limited and controversial in molluscan hemocytes. We detected IFNγ, an immunomodulatory molecule and TNFα, a tumoricidal and apoptosis inducing cytokine in hemocyte morphotypes of molluscs B. bengalensis and L. marginalis by flow cytometry, iNOS had been detected in the hemocyte morphotypes of B. bengalensis and not in L. marginalis. We report the existence of IFNγ in molluscs for the first time. Comparative expression of IFNγ, TNFα and iNOS in hemocytes would provide a better information base to understand biology, evolution and immunological role of these molecules in molluscs and related metazoans.

Interleukin-10 as an indicator of chronic course of oral candidiasis in diabetics: an in vitro study

The importance of host defense against candidiasis and the role of cell-mediated immunity in host defense have been the subject of many studies. Increased expression of virulence factors in Candida isolates from diabetes mellitus (DM) patients with oropharyngeal candidiasis (OPC) has also been reported. To study the difference in T helper type 2 (Th2) and T helper type 1 (Th1) responses and neutrophil respiratory burst response, as a parameter of acute phase and adaptive immune responses, respectively, by using Candida isolates from lesions of oropharyngeal candidiasis (OPC) in DM patients compared to oral Candida isolates from healthy carriers. Analytical experimental laboratory-based study: (a) yeast identification and speciation was done. (b) Peripheral blood mononuclear cells (PBMCs) and neutrophils, obtained from healthy volunteers, were stimulated with oral Candida isolates of DM patients and healthy carriers (controls). Thereafter, cytokine analysis and neutrophil respiratory burst response analysis were done. IL-10 release was uniformly low at all concentrations of Candida albicans antigen isolated from healthy controls as compared to when C. albicans isolated from DM patients was used as antigenic stimulus. Mean INF-γ concentration was highest when C. albicans, isolated from healthy carriers was used as antigenic stimulus for PBMCs. There was no significant difference in neutrophil respiratory burst response among study and control groups. The observations highlight a significant IL-10 dominance, which may be an indicator of the inclination of host immune system toward a chronic progressive disease condition, indicating the pathogenic potential of select Candida strains.

NIEluter: Predicting peptides eluted from HLA class I molecules

The immune system has evolved to make a diverse repertoire of peptides processed from self and foreign proteomes, which are displayed in antigen-binding grooves of major histocompatibility complex (MHC) proteins at cell surface for surveillance by T cells. These antigenic peptides are termed Naturally Processed Peptides or Naturally Presented Peptides (NPPs), which play a major role in cell-mediated immunity and rational vaccine design. Therefore, it is intensely desirable to predict NPPs from a given protein antigen, or to foretell if an MHC-binding peptide can be eluted from a given MHC protein. In this paper, we describe NIEluter, an ensemble predictor based on support vector machine (SVM). It consists of a combination of five SVM models trained with position-specific amino acid composition, position-specific dipeptide composition, Hidden Markov Model, binary encoding, and BLOSUM62 feature. NIEluter can predict NPPs of length 8–11 from six HLA alleles (A0201, B0702, B3501, B4403, B5301, and B5701) at present. Evaluated with five-fold cross-validation and independent datasets if available, NIEluter shows good performance. It outperforms MHC-NP in 7 out of 24 types of situation and precedes NetMHC3.2 in most cases, indicating that it is a helpful complement to available tools. NIEluter has been implemented as a free web service, which can be accessed at http://immunet.cn/nie/cgi-bin/nieluter.pl.

Genetically modified T cells in cancer therapy: opportunities and challenges.

Tumours use many strategies to evade the host immune response, including downregulation or weak immunogenicity of target antigens and creation of an immune-suppressive tumour environment. T cells play a key role in cell-mediated immunity and, recently, strategies to genetically modify T cells either through altering the specificity of the T cell receptor (TCR) or through introducing antibody-like recognition in chimeric antigen receptors (CARs) have made substantial advances. The potential of these approaches has been demonstrated in particular by the successful use of genetically modified T cells to treat B cell haematological malignancies in clinical trials. This clinical success is reflected in the growing number of strategic partnerships in this area that have attracted a high level of investment and involve large pharmaceutical organisations. Although our understanding of the factors that influence the safety and efficacy of these therapies has increased, challenges for bringing genetically modified T-cell immunotherapy to many patients with different tumour types remain. These challenges range from the selection of antigen targets and dealing with regulatory and safety issues to successfully navigating the routes to commercial development. However, the encouraging clinical data, the progress in the scientific understanding of tumour immunology and the improvements in the manufacture of cell products are all advancing the clinical translation of these important cellular immunotherapies.

Low Serum Zinc and Increased Acid Phosphatase Activity in Type 2 Diabetes Mellitus with Periodontitis Subjects

Micronutrient zinc plays a major role in influencing the periodontal conditions in Type 2 diabetes mellitus (T2DM) subjects. In the developed countries nearly 40% of the people are zinc deficient among the T2DM. Now it is estimated that nearly 2 billion subjects in the developing world may be zinc deficient. The periodontal diseases are highly prevalent and can affect up to 90% of the world wide population. Many chronic diseases have been associated with periodontal disease which results in adverse pregnancy outcomes, cardiovascular disease, stroke, pulmonary disease, and diabetes, but the causal relations have not been established. Zinc in human play an important role in cell mediated immunity and was also an antioxidant and anti-inflammatory agent. Zinc helps in the stabilization of lysosomal membranes. The increased acid phosphatase activity might be a result of destructive processes in alveolar bone in advanced stages of periodontal disease. In light of the available data, the study aimed to show how low serum zinc and increased level of lysosomal enzyme, acid phosphatase affect the subjects of Type 2 diabetes mellitus with periodontitis.

Proprotein convertase FURIN constrains Th2 differentiation and is critical for host resistance against Toxoplasma gondii.

The proprotein convertase subtilisin/kexin enzymes proteolytically convert immature proproteins into bioactive molecules, and thereby they serve as key regulators of cellular homeostasis. The archetype proprotein convertase subtilisin/kexin, FURIN, is a direct target gene of the IL-12/STAT4 pathway and it is upregulated in Th1 cells. We have previously demonstrated that FURIN expression in T cells critically regulates the maintenance of peripheral immune tolerance and the functional maturation of pro-TGF-β1 in vivo, but FURIN's role in cell-mediated immunity and Th polarization has remained elusive. In this article, we show that T cell-expressed FURIN is essential for host resistance against a prototypic Th1 pathogen, Toxoplasma gondii, and for the generation of pathogen-specific Th1 lymphocytes, including Th1-IL-10 cells. FURIN-deficient Th cells instead show elevated expression of IL-4R subunit α on cell surface, sensitized IL-4/STAT6 signaling, and a propensity to polarize toward the Th2 phenotype. By exploring FURIN-interacting proteins in Jurkat T cells with Strep-Tag purification and mass spectrometry, we further identify an association with a cytoskeleton modifying Ras-related C3 botulinum toxin substrate/dedicator of cytokinesis 2 protein complex and unravel that FURIN promotes F-actin polymerization, which has previously been shown to downregulate IL-4R subunit α cell surface expression and promote Th1 responses. In conclusion, our results demonstrate that in addition to peripheral immune tolerance, T cell-expressed FURIN is also a central regulator of cell-mediated immunity and Th1/2 cell balance.

Effect of Age on Nitric Oxide Level in Murrah Buffalo (Bubalus bubalis) Neonates

Immune system of calf is more susceptible to oxidative stress during neonatal period due to immature defense system against superoxide radicals. Nitric oxide (NO), a component of bactericidal mechanisms of phagocytic leukocytes, plays a pivotal role in cell-mediated immunity. Present research has addressed many NO-related aspects of neonatal adaptation in the postpartum period. The experiment was conducted on twenty Murrah buffaloes selected from NDRI herd. Approximately 15 ml blood was drawn in sterile heparinised vacutainer tubes from each calf born from these buffaloes, by jugular veni-puncture on day 0 (Before colostrum feeding), followed by day 1, 3, 7, 14, 21, 28, 42, 56, 70, 84, 98, 112 and 126 post birth at 6.00 AM in the morning. Plasma was aliquoted and analysed for total NO by modified Griess Reaction as described by Shoker et al. (1997). Plasma nitrate and nitrite levels were significantly elevated (P<0.01) at birth in buffalo calves and decreased rapidly within first week of life with mean values decreasing more than 80%. The mean values declined from 797.08±43.62 μM/L (Precolostral levels) to 78.97±68.97 μM/L on day 126 post- birth. The levels afterwards though less than day 7 but were not comparable to mature buffaloes (1.09±0.41 μM/L to 7.63±2.75 μM/L) even after 126 days post birth exhibiting fluctuations in between. Therefore, we can say that buffalo neonates have an immature immune system as compared to the adult. Leukocytes from neonates produce unusually high concentrations of NO when compared with those produced by adult buffalo, thus, buffalo neonates have an immature immune system. Therefore, further research is needed in this aspect to explore the possibility of nitric oxide involvement in the extra-uterine adjustment of buffalo neonate.

Complex adaptive immunity to enteric fevers in humans: lessons learned and the path forward.

Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, and S. Paratyphi A and B, causative agents of paratyphoid fever, are major public health threats throughout the world. Although two licensed typhoid vaccines are currently available, they are only moderately protective and immunogenic necessitating the development of novel vaccines. A major obstacle in the development of improved typhoid, as well as paratyphoid vaccines is the lack of known immunological correlates of protection in humans. Considerable progress has been made in recent years in understanding the complex adaptive host responses against S. Typhi. Although the induction of S. Typhi-specific antibodies (including their functional properties) and memory B cells, as well as their cross-reactivity with S. Paratyphi A and S. Paratyphi B has been shown, the role of humoral immunity in protection remains undefined. Cell mediated immunity (CMI) is likely to play a dominant role in protection against enteric fever pathogens. Detailed measurements of CMI performed in volunteers immunized with attenuated strains of S. Typhi have shown, among others, the induction of lymphoproliferation, multifunctional type 1 cytokine production, and CD8+ cytotoxic T-cell responses. In addition to systemic responses, the local microenvironment of the gut is likely to be of paramount importance in protection from these infections. In this review, we will critically assess current knowledge regarding the role of CMI and humoral immunity following natural S. Typhi and S. Paratyphi infections, experimental challenge, and immunization in humans. We will also address recent advances regarding cross-talk between the host’s gut microbiota and immunization with attenuated S. Typhi, mechanisms of systemic immune responses, and the homing potential of S. Typhi-specific B- and T-cells to the gut and other tissues.

Host Defense Against Cryptococcal Disease: Is There a Role for B Cells and Antibody-Mediated Immunity?

The role of B cells and antibody-mediated immunity (AMI) is poorly understood regarding infections with the encapsulated yeast species, Cryptococcus. Human cryptococcal disease, or cryptococcosis, generally occurs in the setting of immune suppression, including deficits of T cells and other components of cell-mediated immunity (CMI), as observed in HIV/AIDS, cancer, solid-organ transplant, and similar conditions. The protective role of CMI is, therefore, well-described in the literature. However, CMI deficiencies alone cannot adequately explain the quantum of cryptococcal disease noted in human and animal populations, and a wealth of clinical and experimental data, mostly spanning the past several decades, has shed light upon a significant role of AMI in anticryptococcal immunity. Recent evidence suggests that rather than functioning discretely, these two host immune compartments work synergistically, with the AMI modulating CMI functions in order to provide a critical balance for host benefit. We describe what is currently known.

A TB Antigen-Stimulated CXCR3 Ligand Assay for the Diagnosis of Active Pulmonary TB

The ligands for CXC chemokine receptor 3 (CXCR3) recruit T-helper type 1 cells, which play a major role in cell-mediated immunity in TB. A total of 409 subjects were enrolled. The study population comprised 186 patients with active TB, 58 patients with non-TB pulmonary diseases, 50 control subjects with a positive interferon (IFN)-γ release assay (IGRA) result, and 115 control subjects with a negative IGRA result. Whole-blood samples were collected using IGRA methodology. After incubation, plasma IFN-γ levels and two CXCR3 ligands, IFN-inducible T-cell α-chemoattractant (I-TAC, CXCL11) and monokine induced by IFN-γ (MIG, CXCL9), were measured by enzyme-linked immunosorbent assay. Receiver operating characteristic (ROC) analysis was performed. Sensitivity and specificity were based on cutoff values selected to maximize the Youden index. The TB antigen-stimulated levels of IFN-γ, I-TAC, and MIG were significantly increased in the active pulmonary TB group compared with all other groups. From ROC analysis, for the diagnosis of active TB, I-TAC and MIG outperformed IFN-γ in all comparisons with the IGRA-positive and -negative control groups and the non-TB pulmonary disease group. The areas under the curve (95% CI) for differentiating active pulmonary TB from all other groups were 0.893 (0.864-0.924) for IFN-γ, 0.962 (0.946-0.978) for I-TAC, and 0.944 (0.922-0.965) for MIG. Sensitivity and specificity were 90.3% and 90.7%, respectively, for I-TAC; 92.5% and 85.2% for MIG; and 84.9% and 79.8% for IFN-γ. TB antigen-stimulated assays of I-TAC and MIG may be useful surrogate markers in the diagnosis of active pulmonary TB.

Development of a simplified and convenient assay for cell-mediated immunity to the mumps virus

Because methods for measuring cell-mediated immunity (CMI) to the mumps virus are expensive, time-consuming, and technically demanding, the role of CMI in mumps virus infection remains unclear. To address this issue, we report here the development of a simplified method for measuring mumps virus-specific CMI that is suitable for use in diverse laboratory and clinical settings. A mumps vaccine was cultured with whole blood, and interferon (IFN)-γ released into the culture supernatant was measured using an enzyme-linked immunosorbent assay. IFN-γ production in blood from vaccinated subjects markedly increased in response to the vaccine and decreased before the antibody titer decreased in some cases, suggesting that this assay may be used as a simple surrogate method for measuring CMI specific for the mumps virus.

The highest mountain: T-cell technology

T-lymphocytes (T-cell) therapy offers a treatment for cancers. Developing this technology in the future provides the opportunity to revolutionise treatment and to make cancer a chronic condition. T-cells in themselves are a type of lymphocytes (itself a type of white blood cell) that play a central role in cell mediated immunity. They can be distinguished from other lymphocytes, such as B-cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. T-cells have the capacity to destroy diseased cells, but tumours present a considerable challenge that reduces their impact. As cancer cells are frequently ‘invisible’ to the immune system, and they create an environment that suppresses T-cell activity., genetic engineering of T-cells can be used therapeutically to overcome these challenges. T-cells can be taken from the blood of cancer patients and then modified to recognise and destroy cancer-specific antigens.

P57 regulates T-cell development and prevents lymphomagenesis by balancing p53 activity and pre-TCR signaling

AbstractT cells are key components of the immune system, playing a central role in cell-mediated immunity. The sequential differentiation of T cells is associated with strict regulation of the cell cycle at each developmental stage. A balance between p53 activity and pre–T cell receptor (TCR) signaling regulates proliferation and differentiation decisions made by these cells. The relation between maintenance of this balance and the function of cell cycle regulators has remained largely unknown, however. We now show that mice with T cell–specific deficiency of the cyclin-dependent kinase inhibitor p57 manifest a differentiation block at the early stage of T cell maturation. Further genetic analysis showed that this defect is attributable to an imbalance between p53 activity and pre-TCR signaling caused by hyperactivation of the E2F-p53 pathway. Moreover, ablation of both p57 and p53 in T cells led to the development of aggressive thymic lymphomas with a reduced latency compared with that apparent for p53-deficient mice, whereas ablation of p57 alone did not confer susceptibility to this hematologic malignancy. Our results thus show that the p57-E2F-p53 axis plays a pivotal role in the proper development of T cells as well as in the prevention of lymphomagenesis.

The Efficacy of Glucocorticoid on Macrolide Resistant Mycoplasma Pneumonia in Children

Mycoplasma pneumoniae (MP) is a common etiology of pediatric and adult community-acquired pneumonia that represents 10%-40% of cases;1-3 however, the immunopathogenesis of MP in humans is poorly understood. The role of cell-mediated immunity in MP infections is controversial. Both human infection tissue and animal model lung tissue are consistent in demonstrating mononuclear perilumenal infiltrates. Animal model studies have found hyperaccentuated reactions after repeated infection that is also mimicked by repeated exposure to non-viable microbe.4

Chinese goose (Anser cygnoides) CD8a: Cloning, tissue distribution and immunobiological in splenic mononuclear cells

CD8 molecule is a cell membrane glycoprotein, which plays an important role in cell-mediated immunity. Here, we identified Chinese goose CD8α (goCD8α) gene for the first time. The full-length cDNA of goCD8α is 1459bp in length and contains a 711bp open reading frame. Phylogenetic analysis shows that the waterfowl CD8α formed a monophyletic group. Semi-quantitative RT-PCR analysis showed that transcripts of goCD8α mRNA were high in the immune-related organs and mucosal immune system in gosling, and high in thymus and spleen comparing to other immune-related tissues in goose. The obvious increase of CD8α expression was observed in spleen of acute new type gosling viral enteritis virus (NGVEV) infected bird, while the increase of CD8α were observed in the thymus, bursa of fabricius, and cecum of chronic infected bird. The CD8α mRNA transcription level in spleen mononuclear cells was significantly up-regulated when stimulated by phytohemagglutinin, but not by lipopolysaccharide in vitro.

NetMHCIIpan-3.0, a common pan-specific MHC class II prediction method including all three human MHC class II isotypes, HLA-DR, HLA-DP and HLA-DQ

Major histocompatibility complex class II (MHCII) molecules play an important role in cell-mediated immunity. They present specific peptides derived from endosomal proteins for recognition by T helper cells. The identification of peptides that bind to MHCII molecules is therefore of great importance for understanding the nature of immune responses and identifying T cell epitopes for the design of new vaccines and immunotherapies. Given the large number of MHC variants, and the costly experimental procedures needed to evaluate individual peptide-MHC interactions, computational predictions have become particularly attractive as first-line methods in epitope discovery. However, only a few so-called pan-specific prediction methods capable of predicting binding to any MHC molecule with known protein sequence are currently available, and all of them are limited to HLA-DR. Here, we present the first pan-specific method capable of predicting peptide binding to any HLA class II molecule with a defined protein sequence. The method employs a strategy common for HLA-DR, HLA-DP and HLA-DQ molecules to define the peptide-binding MHC environment in terms of a pseudo sequence. This strategy allows the inclusion of new molecules even from other species. The method was evaluated in several benchmarks and demonstrates a significant improvement over molecule-specific methods as well as the ability to predict peptide binding of previously uncharacterised MHCII molecules. To the best of our knowledge, the NetMHCIIpan-3.0 method is the first pan-specific predictor covering all HLA class II molecules with known sequences including HLA-DR, HLA-DP, and HLA-DQ. The NetMHCpan-3.0 method is available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.0 .

Anti-nociceptive effect of IL-12p40 in a rat model of neuropathic pain

IL-12p70 is a proinflammatory cytokine secreted by dendritic cells, monocytes and macrophages. It plays a crucial role in cell-mediated immunity by inducing proliferation of T cell and natural killer cells, and enhancing their cytotoxic activity. In adaptive immune response, it acts on naive T cells to differentiate into Th1-type cells. It is composed of two subunits, p35 and p40. The latter can be secreted in the form of monodimer or heterodimer, which is also referred as IL-12p80. Recently IL-12p70 has been proven to locally provoke nociceptive effect in naïve rats. This study investigated pain response following systemic administration of IL-12p70 and IL-12p40 homodimer in chronic neuropathic pain model, induced by chronic constriction injury. The doses tested were IL-12p40 homodimer or IL12p70 at 15, 150 and 1500ng/kg, respectively. Pain was assessed at 1, 4, 7 and 24h after injection, in the form of tactile allodynia and mechanical hyperalgesia. The side effect of sensory motor disability was measured by rotarod performance. By all behavioral measures, IL-12p70 of any dosage, at any time point, had no significant effect on tactile allodynia and mechanical hyperalgesia. A high dose of IL-12p40 homodimer induced significant analgesic effect by the measure of hind paw tactile allodynia from 1h to 4h after injection. Medium and low doses of IL-12p40 homodimer exerted their analgesic effect 4h post injection. Mechanical hyperalgesia, following high and medium doses of IL-12p40 administration, was significantly reduced at 4h after application. Also, no significant sensory motor dysfunction was detected for all dosage for both homodimers. These findings suggest that systemic application of IL-12p40 homodimer induces time-dependent analgesia to mechanical stimulation in rats exposed to neuropathic pain.