The Role of Neurotrophins in Inflammation and Allergy.

Allergic inflammation is the result of a specific pattern of cellular and humoral responses leading to the activation of the innate and adaptive immune system, which, in turn, results in physiological and structural changes affecting target tissues such as the airways and the skin. Eosinophil activation and the production of soluble mediators such as IgE antibodies are pivotal features in the pathophysiology of allergic diseases. In the past few years, however, convincing evidence has shown that neurons and other neurosensory structures are not only a target of the inflammatory process but also participate in the regulation of immune responses by actively releasing soluble mediators. The main products of these activated sensory neurons are a family of protein growth factors called neurotrophins. They were first isolated in the central nervous system and identified as important factors for the survival and differentiation of neurons during fetal and postnatal development as well as neuronal maintenance later in life. Four members of this family have been identified and well defined: nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5. Neurotrophins play a critical role in the bidirectional signaling mechanisms between immune cells and the neurosensory network structures in the airways and the skin. Pruritus and airway hyperresponsiveness, two major features of atopic dermatitis and asthma, respectively, are associated with the disruption of the neurosensory network activities. In this chapter, we provide a comprehensive description of the neuroimmune interactions underlying the pathophysiological mechanisms of allergic and inflammatory diseases.

The relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus.

Our aim was to obtain knowledge of how meteorological conditions affect community epidemics of respiratory syncytial virus (RSV) infection. To this end we recorded year-round RSV activity in nine cities that differ markedly in geographic location and climate. We correlated local weather conditions with weekly or monthly RSV cases. We reviewed similar reports from other areas varying in climate. Weekly RSV activity was related to temperature in a bimodal fashion, with peaks of activity at temperatures above 24-30 degrees C and at 2-6 degrees C. RSV activity was also greatest at 45-65% relative humidity. RSV activity was inversely related to UVB radiance at three sites where this could be tested. At sites with persistently warm temperatures and high humidity, RSV activity was continuous throughout the year, peaking in summer and early autumn. In temperate climates, RSV activity was maximal during winter, correlating with lower temperatures. In areas where temperatures remained colder throughout the year, RSV activity again became nearly continuous. Community activity of RSV is substantial when both ambient temperatures and absolute humidity are very high, perhaps reflecting greater stability of RSV in aerosols. Transmission of RSV in cooler climates is inversely related to temperature possibly as a result of increased stability of the virus in secretions in the colder environment. UVB radiation may inactivate virus in the environment, or influence susceptibility to RSV by altering host resistance.

Administration of fludarabine-loaded autologous red blood cells in simian immunodeficiency virus-infected sooty mangabeys depletes pSTAT-1-expressing macrophages and delays the rebound of viremia after suspension of antiretroviral therapy.

A major limitation of highly active antiretroviral therapy is that it fails to eradicate human immunodeficiency virus (HIV) infection due to its limited effects on viral reservoirs carrying replication-competent HIV, including monocytes/macrophages (M/M). Therefore, therapeutic approaches aimed at targeting HIV-infected M/M may prove useful in the clinical management of HIV-infected patients. In previous studies, we have shown that administration of fludarabine-loaded red blood cells (RBC) in vitro selectively induces cell death in HIV-infected M/M via a pSTAT1-dependent pathway. To determine the in vivo efficacy of this novel therapeutic strategy, we treated six naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) with either 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) only, fludarabine-loaded RBC only, or PMPA in association with fludarabine-loaded RBC. The rationale of this treatment was to target infected M/M with fludarabine-loaded RBC at a time when PMPA is suppressing viral replication taking place in activated CD4+ T cells. In vivo administration of fludarabine-loaded RBC was well tolerated and did not induce any discernible side effect. Importantly, addition of fludarabine-loaded RBC to PMPA delayed the rebound of viral replication after suspension of therapy, thus suggesting a reduction in the size of SIV reservoirs. While administrations of fludarabine-loaded RBC did not induce any change in the CD4+ or CD8+ T-cell compartments, we observed, in chronically SIV-infected SMs, a selective depletion of M/M expressing pSTAT1. This study suggests that therapeutic strategies based on the administration of fludarabine-loaded RBC may be further explored as interventions aimed at reducing the size of the M/M reservoirs during chronic HIV infection.

Perturbations of cell cycle control in T cells contribute to the different outcomes of simian immunodeficiency virus infection in rhesus macaques and sooty mangabeys.

In contrast to human immunodeficiency virus (HIV) infection of humans and experimental simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs), SIV infection of sooty mangabeys (SMs), a natural host African monkey species, is typically nonpathogenic and associated with preservation of CD4+ T-cell counts despite chronic high levels of viral replication. In previous studies, we have shown that the lack of SIV disease progression in SMs is related to lower levels of immune activation and bystander T-cell apoptosis compared to those of pathogenic HIV/SIV infection (G. Silvestri, D. Sodora, R. Koup, M. Paiardini, S. O'Neil, H. M. McClure, S. I. Staprans, and M. B. Feinberg, Immunity 18:441-452, 2003; G. Silvestri, A. Fedanov, S. Germon, N. Kozyr, W. J. Kaiser, D. A. Garber, H. M. McClure, M. B. Feinberg, and S. I. Staprans, J. Virol. 79:4043-4054, 2005). In HIV-infected patients, increased T-cell susceptibility to apoptosis is associated with a complex cell cycle dysregulation (CCD) that involves increased activation of the cyclin B/p34-cdc2 complex and abnormal nucleolar structure with dysregulation of nucleolin turnover. Here we report that CCD is also present during pathogenic SIV infection of RMs, and its extent correlates with the level of immune activation and T-cell apoptosis. In marked contrast, naturally SIV-infected SMs show normal regulation of cell cycle control (i.e., normal intracellular levels of cyclin B and preserved nucleolin turnover) and a low propensity to apoptosis in both peripheral blood- and lymph node-derived T cells. The absence of significant CCD in the AIDS-free, non-immune-activated SMs despite high levels of viral replication indicates that CCD is a marker of disease progression during lentiviral infection and supports the hypothesis that the preservation of cell cycle control may help to confer the disease-resistant phenotype of SIV-infected SMs.

Origins of reactive airways disease in early life: do viral infections play a role?

UNLABELLED: There is mounting evidence suggesting that infection with respiratory syncytial virus (RSV) in early life increases the risk of developing reactive airway disease (RAD) later in childhood. A recent prospective study demonstrated that children hospitalized with RSV bronchiolitis in infancy face a significantly increased risk of recurrent wheezing and allergy at least until the age of 7 y that is independent of hereditary factors. Proposed mechanisms for this link include immune dysregulation, in which RSV-specific IgE or an imbalance between T-lymphocyte-dependent immune pathways may be involved, and abnormal neural control, in which the non-adrenergic, non-cholinergic pathways are altered by RSV infection. More recent studies suggest that immune and neural mechanisms may be linked and that post-RSV airway inflammation may be explained, at least in part, on the basis of these neuroimmune interactions. CONCLUSION: Passive immunoprophylaxis may protect against persistent viral-induced inflammation of the respiratory tract, long-term changes in pulmonary function and increased frequency of RAD episodes.

The association between respiratory syncytial virus infection and reactive airway disease.

Evidence has been accumulating that respiratory syncytial virus (RSV) lower respiratory tract infection (LRTI) in infants may be linked to subsequent development of reactive airway disease (RAD) in childhood, and therefore research into the prevention of RSV LRTI may have important implications for the prevention of RAD. This article reviews the epidemiological evidence linking RSVand RAD and some ofthe theories concerning cellular and molecular mechanisms of post-viral airway inflammation in order to understand how RSV prophylaxis may assist in reducing the occurrence of RSV LRTI and RAD.

Exogenous interleukin-2 administration corrects the cell cycle perturbation of lymphocytes from human immunodeficiency virus-infected individuals.

Human immunodeficiency virus (HIV)-induced immunodeficiency is characterized by progressive loss of CD4(+) T cells associated with functional abnormalities of the surviving lymphocytes. Increased susceptibility to apoptosis and loss of proper cell cycle control can be observed in lymphocytes from HIV-infected individuals and may contribute to the lymphocyte dysfunction of AIDS patients. To better understand the relation between T-cell activation, apoptosis, and cell cycle perturbation, we studied the effect of exogenous interleukin-2 (IL-2) administration on the intracellular turnover of phase-dependent proteins. Circulating T cells from HIV-infected patients display a marked discrepancy between a metabolic profile typical of G(0) and a pattern of expression of phase-dependent proteins that indicates a more-advanced position within the cell cycle. This discrepancy is enhanced by in vitro activation with ConA and ultimately results in a marked increase of apoptotic events. Conversely, treatment of lymphocytes with IL-2 alone restores the phase-specific pattern of expression of cell cycle-dependent proteins and is associated with low levels of apoptosis. Interestingly, exogenous IL-2 administration normalizes the overall intracellular protein turnover, as measured by protein synthesis, half-life of newly synthesised proteins, and total protein ubiquitination, thus providing a possible explanation for the effect of IL-2 on the intracellular kinetics of cell cycle-dependent proteins. The beneficial effect of IL-2 administration is consistent with the possibility of defective IL-2 function in vivo, which is confirmed by the observation that lymphocytes from HIV-infected patients show abnormal endogenous IL-2 paracrine/autocrine function upon in vitro mitogen stimulation. Overall these results confirm that perturbation of cell cycle control contributes to HIV-related lymphocyte dysfunction and, by showing that IL-2 administration can revert this perturbation, suggest a new mechanism of action of IL-2 therapy in HIV-infected patients.

Abnormal intracellular kinetics of cell-cycle-dependent proteins in lymphocytes from patients infected with human immunodeficiency virus: a novel biologic link between immune activation, accelerated T-cell turnover, and high levels of apoptosis.

Human immunodeficiency virus (HIV)-infection is characterized by loss of CD4+ T cells associated with high levels of immune activation, T-cell proliferation, and lymphocyte apoptosis. To investigate the role of intrinsic perturbations of cell-cycle control in the immunopathogenesis of acquired immunodeficiency syndrome (AIDS), we studied the expression of cell-cycle-dependent proteins in lymphocytes from HIV-infected patients. Cyclin B1 expression, Nucleolar Organizer Regions (NORs) number, and NORs area of distribution were all consistently increased in HIV-infected patients, but returned to normal after effective antiretroviral therapy, suggesting that viral replication is directly implicated in the genesis of the observed changes. Analysis of cyclin B1 intracellular turnover showed that the increased cyclin B1 expression is (1) caused by defective degradation in the presence of normal rates of synthesis, and (2) is temporally associated with decreased levels of ubiquitination. After in vitro activation of lymphocytes from healthy individuals, cyclin B1 and cdc25 expression and ubiquitination, p34 cdc2 activity, NORs morphology, and C23/nucleolin localization showed a 72- to 96-hour cyclic pattern that led to a biologic state similar to baseline. On the contrary, complex but consistent changes of the same indices followed activation of T lymphocytes from HIV-infected patients, resulting in a 5-fold increase in apoptosis. Overall, our data indicate that a profound dysregulation of cell-cycle control is present in lymphocytes from HIV-infected patients. This finding may provide a novel biologic link between immune activation, accelerated lymphocyte turnover, and increased apoptosis during HIV infection.

Exaggerated neurogenic inflammation and substance P receptor upregulation in RSV-infected weanling rats.

Respiratory syncytial virus (RSV) infection in adult rats causes exaggerated inflammation after sensory nerve stimulation in the extrapulmonary, but not in the intrapulmonary airways. The goal of this study was to analyze neurogenic inflammation in weanling F-344 rats infected with RSV 18 +/- 2 d after birth. Five days after RSV inoculation, the extravasation of Evans blue-labeled albumin after nerve stimulation was significantly greater in the intrapulmonary airways of RSV-infected weanling rats than in pathogen-free control rats. In contrast, no difference was found in the extrapulmonary airways. The level of messenger RNA (mRNA) encoding the substance P (SP) receptor (neurokinin 1 [NK1]) increased fourfold in RSV-infected lungs, whereas mRNA encoding the VIPR1 receptor for the antiinflammatory vasoactive intestinal peptide (VIP) increased to a much lesser degree. mRNAs encoding the other neurokinin (NK2) and VIP (VIPR2) receptors were not affected by the virus. Selective inhibition of the NK1 receptor abolished neurogenic inflammation in RSV-infected intrapulmonary airways. Also, neurogenic inflammation and NK1 receptor upregulation in infected lungs were inhibited by prophylaxis with a monoclonal antibody against RSV. These data suggest that RSV lower respiratory tract infection makes the intrapulmonary airways of young rats abnormally susceptible to the proinflammatory effects of SP by selectively upregulating the expression of NK1 receptors.

A humanized monoclonal antibody against respiratory syncytial virus (palivizumab) inhibits RSV-induced neurogenic-mediated inflammation in rat airways.

Respiratory syncytial virus (RSV) is the most important respiratory pathogen in infancy and early childhood and may predispose to subsequent lower respiratory tract illness. Recent data indicate that RSV up-regulates the substance P receptor, making the airways abnormally susceptible to the proinflammatory effects of this peptide released from sensory nerves. The present study was designed to determine whether the administration of RSV antibodies prevents the potentiation of neurogenic inflammation in rat airways. Five days after inoculation, sensory nerve-mediated extravasation of Evans blue-labeled albumin was significantly greater in the airways of RSV-infected rats than in pathogen-free controls. Polyclonal immune globulin enriched for RSV-neutralizing antibodies (RSVIG) reduced neurogenic extravasation when injected 24 h before intranasal inoculation of the virus but not when injected before endotracheal inoculation. A humanized MAb against RSV fusion protein (palivizumab) was twice as potent as RSVIG when given before intranasal inoculation and also caused significant inhibition after endotracheal inoculation. Furthermore, palivizumab inhibited neurogenic inflammation in RSV-infected rats when given 72 h after virus inoculation. These data suggest that palivizumab protects the respiratory tract from RSV-induced inflammation when given before or in the early phase of the viral infection. The administration of palivizumab to high-risk infants may limit the severity of the acute airway inflammation and may protect against subsequent lower respiratory tract illness.

Respiratory syncytial virus upregulates expression of the substance P receptor in rat lungs.

Respiratory syncytial virus (RSV) is a major respiratory pathogen in infants. The first goal of this study was to determine whether the infection following endotracheal inoculation of RSV in Fischer 344 rats results in increased inflammatory responses to substance P (SP) either released by capsaicin from sensory nerves or injected into the circulation. Five days after inoculation, the extravasation of Evans blue-labeled albumin after capsaicin or SP was significantly greater in RSV-infected airways than in pathogen-free controls. The peptide-degrading activity of the regulatory enzyme neutral endopeptidase was unaffected by RSV. However, SP(NK(1)) receptor mRNA levels increased fivefold in RSV-infected lungs, and the density of SP binding sites in the bronchial mucosa increased threefold. These data suggest that RSV makes the airways abnormally susceptible to the proinflammatory effects of SP by upregulating SP(NK(1)) receptor gene expression, thereby increasing the density of these receptors on target cells. This effect may contribute to the inflammatory reaction to the virus and could be a target for the therapy of RSV disease and its sequelae.

Unscheduled cyclin B expression and p34 cdc2 activation in T lymphocytes from HIV-infected patients.

OBJECTIVE: To study the role of cell cycle regulation during HIV infection by investigating in vivo and in vitro cyclin B and p34 cdc kinase expression. METHODS: Cyclin B expression was analysed by Western blot in CD4 and CD8 cells from 25 HIV-infected patients and 24 uninfected individuals. In eight patients, a sequential analysis was performed after initiation of antiretroviral therapy (ART), and correlations with CD4 cell count and HIV viremia were studied. Sequential changes in cyclin B expression and p34 cdc kinase expression and activity were also studied in lymphocytes activated in vitro with phytohaemagglutinin (PHA). RESULTS: Lymphocytes from untreated HIV-infected patients demonstrate persistent in vivo overexpression of cyclin B in both CD4 and CD8 cell subpopulations. When cells are stimulated to proliferate in vitro, biochemical events that characterize the entrance into the cell cycle [ornithine decarboxylase (ODC) activity, interleukin 2 production, interleukin 2 alpha-chain receptor (IL-2R, CD25) expression, total protein synthesis, total DNA synthesis] show similar timing and sequence in lymphocytes from HIV-infected and uninfected individuals. However, in peripheral blood lymphocytes (PBL) from HIV-infected patients, cyclin B and p34 cdc kinase show premature expression during the cell cycle. Both in vivo cyclin B overexpression and in vitro unscheduled cyclin B expression were almost completely reversed 2-4 weeks after initiation of effective ART. CONCLUSION: Increased and unscheduled expression of cyclin B and p34 cdc kinase is consistently observed in CD4 and CD8 cells from HIV-infected patients, both in vivo and after in vitro mitogenic stimulation. These alterations correlate with the level of viremia and may provide a link between the perturbation of lymphocyte proliferative homeostasis and the exaggerated propensity towards apoptosis.

Protein degradation and apoptotic death in lymphocytes during Fiv infection: activation of the ubiquitin-proteasome proteolytic system.

The movement of a cell through the sequential phases of apoptosis is accompanied by a progressive decrease in cell size with loss in protein mass. In lymphocytes from Hiv-infected persons, protein loss during apoptosis is due to increased protein degradation rather than decreased synthesis. To identify and characterize the proteolytic enzymes or enzyme systems involved in this process, we studied several features of protein turnover in lymphocytes from peripheral blood and lymph nodes during the natural and experimental infection by feline immunodeficiency virus (Fiv). This animal model allowed us to integrate in vivo results with in vitro observations of protein damage. Here we report that protein breakdown in apoptotic cells is concomitant with the activation of the ATP and ubiquitin-dependent multicatalytic system (proteasome). We suggest that proteasome activation is part of the proteolytic cascade in the execution phases of apoptosis in AIDS.

Apoptotic fraction in lymphoid tissue of FIV-infected SPF cats.

In the present study the apoptotic fraction has been investigated in peripheral blood mononuclear cells (PBMCs) and in lymphoid tissue of six clinically asymptomatic serologically positive specific pathogen free (SPF) FIV-infected cats with a decline in peripheral blood CD4+ lymphocytes, compared to five FIV- SPF controls. Apoptosis in PBMCs was scored in relation to cell cycle phases judged by the integrating cytometric measure of DNA content with 3H-thymidine and 3H-leucine incorporation. Apoptosis in lymphoid tissue was revealed with the ApopTag-peroxidase kit, quantified by image analysis and expressed as apoptotic index (number of apoptosis per 100 cells). The high percentage of apoptotic death in lymphocytes from FIV+ cats was chronologically related to the entrance of cells in the S phase of the cell cycle (p < 0.0001). No difference in the apoptotic index was revealed comparing the follicular, cortical + paracortical and medullary compartments in lymph nodes of FIV+ and FIV- cats. In each group of cats a similar pattern of apoptosis expression was revealed in lymph nodes: significantly higher in follicular vs. both cortical + paracortical and medullary compartments (p < 0.001). In the thymus a significant increase in apoptotic index was revealed in the cortical compartment of the FIV+ cats compared to FIV- (p < 0.001), while in the spleen both the red and white pulp expressed a higher value in FIV+ cats compared to FIV-(p < 0.05) and the former showed a pattern of expression as follows: in the red pulp significantly higher than in the white pulp (p < 0.001). This investigation suggests that the priming signals for apoptosis in FIV infection parallels the S phase of the cell cycle and peripheral blood changes could follow both thymic and splenic modifications in apoptotic expression.

Early increase of CD4+ CD45RA+ and CD4+ CD95- cells with conserved repertoire induced by anti-retroviral therapy in HIV-infected patients.

Administration of anti-retroviral drugs induces a decrease of viral load associated with increase of CD4+ cell count in most HIV-infected patients. To investigate the early changes in CD4+ cell phenotype induced by anti-retroviral therapy, six patients with CD4+ cell count > 100/mm3 and never treated with anti-HIV therapy were enrolled and blood samples collected several times within 14 days from the initiation of therapy with Zidovudine plus Didanosine. CD4+ cell count and HIV viraemia were investigated at each time point, as well as the expression of CD45RA, CD45RO and CD95/Fas molecules on CD4+ cells, and the T cell receptor (TCR) Vbeta repertoire of CD4+ cells. All patients showed a rapid and dramatic decrease in viral load with a corresponding increase of CD4+ cell count. The main remodelling of CD4+ cell subpopulations took place in the first 14 days of therapy, and consisted of: (i) increased CD4+CD45RA+/CD4+CD45RO+ ratio; (ii) decrease of CD95/Fas expression. The rise in absolute number of CD4+CD45RA+ cells was paralleled by an increase of CD4+CD95/Fas- cells and accounted for most of the early increment of CD4+ cell count. The TCR Vbeta repertoire of CD4+ cells was conserved after anti-HIV therapy, with the exception of two patients with expanded CD4+Vbeta12+ cells, which also tested CD45RA+ and CD95/Fas-. These experiments show that newcomer CD4+ lymphocytes are CD45RA+CD95/Fas- cells, suggesting that blocking HIV replication causes an early and antigen-independent proliferation of possibly 'naive' cells unprimed for CD95/Fas-mediated apoptosis. These cells expressed a conserved and widespread TCR repertoire, suggesting that their capability for antigenic recognition is intact.

Effect of beta-endorphin on cell growth and cell death in human peripheral blood lymphocytes.

Beta-endorphin (beta-end) was investigated for its ability to influence sequential metabolic events that accompany the movements of T-lymphocytes into the cell cycle. When cultured lymphocytes are exposed to this endogenous opioid peptide an increase in polyamine transport across cell membrane is observed. This membrane modification is an early cell cycle event, whose enhancement leads to the intracellular polyamine accumulation. It is shown that beta-end is able to enhance spermidine transport and that the exposition of cells to this peptide is perceived as an apoptotic signal. The possible relationship between induction of apoptotic death and enhancement of polyamine uptake is discussed.

Mediastinal lymphadenopathy caused by Mycobacterium avium-intracellulare complex in a child with normal immunity: successful treatment with anti-mycobacterial drugs and laser bronchoscopy.

We report on the case of a 9-month-old Caucasian girl referred to our institution with a history of fever of unknown origin and wheezing, unresponsive to bronchodilator and anti-inflammatory therapy. Subsequent investigation led to a diagnosis of mediastinal lymphadenopathy caused by Mycobacterium avium-intracellulare (MAI). The infected lymph tissue infiltrated and obstructed the right bronchus and significantly compressed the left bronchus to the point of near closure. Given the high degree of morbidity and potential mortality from thoracic surgery in this patient, we treated her with a combination of anti-mycobacterial drugs (rifabutin, clarithromycin, ciprofloxacin, clofazimine, amikacin, ethambutol) and glucocorticoids to relieve airway compression. The endobronchial granulation tissue was resected by laser bronchoscopy. This combined approach led to eventual normalization of radiologic and endoscopic findings, and the anti-mycobacterial chemotherapy was discontinued 12 months after the first bronchoalveolar lavage culture was negative for MAI. The patient remains asymptomatic 1 year after completion of this course of therapy. We suggest that mediastinal lymphadenopathy with bronchial infiltration and extrinsic airway compression caused by MAI in otherwise healthy children can be successfully treated with aggressive chemotherapy, glucocorticoids, and laser bronchoscopy.