Anti-IFNAR treatment does not reverse neuropsychiatric disease in MRL/lpr lupus mice.

OBJECTIVE: Many systemic lupus erythematosus patients display a type I interferon (IFN) signature, and IFNα levels positively correlate with disease severity. Previous studies blocking the type I IFN pathway systemically in lupus models showed some beneficial effects. However, its effects on neuropsychiatric manifestations have yet to be carefully assessed, even though IFNα has been associated with induction of depression. Our aim was to investigate whether disrupting the type I IFN pathway would attenuate the development of murine neuropsychiatric lupus. METHODS: Female MRL/lpr mice were administered an antitype I IFN receptor (IFNAR) antibody or a control antibody intraperitoneally three times weekly for 12 weeks starting at age 4-5 weeks. Behavior was assessed during and at the end of the treatment schedule. RESULTS: No significant differences were seen between the anti-IFNAR- and control-treated mice when assessing for depression-like behavior or cognitive dysfunction, although anti-IFNAR antibody-treated mice displayed significant decreases in levels of IFN-stimulated genes. Anti-IFNAR treatment also did not significantly improve brain histology, cellular infiltration, or blood-brain barrier integrity. CONCLUSIONS: Surprisingly, our results showed no improvement in neuropsychiatric disease and suggest that the role of IFNAR signaling in the pathogenesis of neuropsychiatric lupus continues to need to be carefully assessed.

Natural killer cell expression of Ki67 is associated with elevated serum IL-15, disease activity and nephritis in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder whose pathology involves multiple immune cell types, including B and T lymphocytes as well as myeloid cells. While it is clear that autoantibody-producing B cells, as well as CD4+ T cell help, are key contributors to disease, little is known regarding the role of innate lymphoid cells such as natural killer (NK) cells in the pathogenesis of SLE. We have characterized the phenotype of NK cells by multi-color flow cytometry in a large cohort of SLE patients. While the overall percentage of NK cells was similar or slightly decreased compared to healthy controls, a subset of patients displayed a high frequency of NK cells expressing the proliferation marker, Ki67, which was not found in healthy donors. Although expression of Ki67 on NK cells correlated with Ki67 on other immune cell subsets, the frequency of Ki67 on NK cells was considerably higher. Increased frequencies of Ki67+ NK cells correlated strongly with clinical severity and active nephritis and was also related to low NK cell numbers, but not overall leukopenia. Proteomic and functional data indicate that the cytokine interleukin-15 promotes the induction of Ki67 on NK cells. These results suggest a role for NK cells in regulating the immune-mediated pathology of SLE as well as reveal a possible target for therapeutic intervention.

IgG1+ B-cell immunity predates IgE responses in epicutaneous sensitization to foods.

BACKGROUND: The generation of IgE-mediated food allergy in humans is silent and only diagnosed upon manifestation of clinical symptoms. While experimental models have been used to investigate some mechanisms of allergic sensitization, the generation of humoral immunity and memory remains to be elucidated. Here, we defined the evolution of allergen-specific B-cell responses during epicutaneous sensitization to foods. METHODS: Wild-type and genetic knockout animals, and drug or antibody strategies for cell depletion and immunoglobulin signaling blockade were used to investigate epicutaneous sensitization and disease progression; we analyzed allergen-specific germinal centers and IgG1+ memory B cells by flow cytometry, evaluated humoral responses, and determined clinical reactivity (anaphylaxis). RESULTS: Epicutaneous sensitization caused microscopic skin damage, inflammation, and recruitment of activated dendritic cells to the draining lymph nodes. This process generated allergen-specific IgG1+ germinal center B cells, serum IgG1, and anaphylaxis that was mediated by the alternative pathway. Whether we used peanut and/or ovalbumin from the egg white for sensitization, the allergen-specific IgG1+ memory compartment predominantly exhibited an immature, pro-germinal center phenotype (PDL-2- CD80- CD35+ CD73+ ). Subsequent subclinical exposures to the allergen induced IgE+ germinal center B cells, serum IgE, and likely activated the classical pathway of anaphylaxis. CONCLUSIONS: Our data demonstrate that IgG1+ B-cell immunity against food allergens in epicutaneous sensitization precedes the generation of IgE responses. Therefore, the assessment of allergen-specific cellular and humoral IgG1+ immunity may help to identify individuals at risk of developing IgE-mediated food allergy and hence provide a window for therapeutic interventions.

CD19-positive antibody-secreting cells provide immune memory.

Long-lived antibody-secreting cells (ASCs) are critical for the maintenance of humoral immunity through the continued production of antibodies specific for previously encountered pathogen or vaccine antigens. Recent reports describing humoral immune memory have suggested the importance of long-lived CD19- bone marrow (BM) ASCs, which secrete antibodies recognizing previously encountered vaccine antigens. However, these reports do not agree upon the unique contribution of the CD19+ BM ASC subset toward humoral immunity. Here, we found both CD19+ and negative ASCs from human BM were similar in functional capacity to react to a number of vaccine antigens via ELISpot assays. The CD19+ cells were the predominant ASC population found in lymphoid tissues, and unlike the CD19- ASCs, which were found only in spleen and BM, the CD19+ ASCs were found in tonsil and blood. CD19+ ASCs from the BM, spleen, and tonsil were capable of recognizing polio vaccine antigens, indicating the CD19+ ASC cells play a novel role in long-lasting immune defense. Comparative gene expression analysis indicated CD19+ and negative BM ASCs differed significantly by only 14 distinct messenger RNAs and exhibited similar gene expression for cell cycle, autophagy, and apoptosis control necessary for long life. In addition, we show identical CDR-H3 sequences found on both BM ASC subsets, indicating a shared developmental path. Together, these results provide novel insight for the distribution, function, genetic regulation, and development of long-lived ASCs and may not only impact improved cell therapies but also enhance strategies for vaccine development.

T helper cell IL-4 drives intestinal Th2 priming to oral peanut antigen, under the control of OX40L and independent of innate-like lymphocytes.

Intestinal T helper type 2 (Th2) immunity in food allergy results in IgG1 and IgE production, and antigen re-exposure elicits responses such as anaphylaxis and eosinophilic inflammation. Although interleukin-4 (IL-4) is critically required for allergic sensitization, the source and control of IL-4 during the initiation of Th2 immunity in vivo remains unclear. Non-intestinal and non-food allergy systems have suggested that natural killer-like T (NKT) or γδ T-cell innate lymphocytes can supply the IL-4 required to induce Th2 polarization. Group 2 innate lymphoid cells (ILCs) are a novel IL-4-competent population, but their contribution to initiating adaptive Th2 immunity is unclear. There are also reports of IL-4-independent Th2 responses. Here, we show that IL-4-dependent peanut allergic Th2 responses are completely intact in NKT-deficient, γδ T-deficient or ILC-deficient mice, including antigen-specific IgG1/IgE production, anaphylaxis, and cytokine production. Instead, IL-4 solely from CD4(+) Th cells induces full Th2 immunity. Further, CD4(+) Th cell production of IL-4 in vivo is dependent on OX40L, a costimulatory molecule on dendritic cells (DCs) required for intestinal allergic priming. However, both Th2 cells and ILCs orchestrated IL-13-dependent eosinophilic inflammation. Thus, intestinal Th2 priming is initiated by an autocrine/paracrine acting CD4(+) Th cell-intrinsic IL-4 program that is controlled by DC OX40L, and not by NKT, γδ T, or ILC cells.

Molecular requirements for inhibition of the chemokine receptor CCR8--probe-dependent allosteric interactions.

BACKGROUND AND PURPOSE: Here we present a novel series of CCR8 antagonists based on a naphthalene-sulfonamide structure. This structure differs from the predominant pharmacophore for most small-molecule CC-chemokine receptor antagonists, which in fact activate CCR8, suggesting that CCR8 inhibition requires alternative structural probes. EXPERIMENTAL APPROACH: The compounds were tested as inverse agonists and as antagonists against CCL1-induced activity in Gα(i) signalling and chemotaxis. Furthermore, they were assessed by heterologous competition binding against two radiolabelled receptor ligands: the endogenous agonist CCL1 and the virus-encoded antagonist MC148. KEY RESULTS: All compounds were highly potent inverse agonists with EC(50) values from 1.7 to 23 nM. Their potencies as antagonists were more widely spread (EC(50) values from 5.9 to 1572 nM). Some compounds were balanced antagonists/inverse agonists whereas others were predominantly inverse agonists with >100-fold lower potency as antagonists. A correspondingly broad range of affinities, which followed the antagonist potencies, was disclosed by competition with [(125)I]-CCL1 (K(i) 3.4-842 nM), whereas the affinities measured against [(125)I]-MC148 were less widely spread (K(i) 0.37-27 nM), and matched the inverse agonist potencies. CONCLUSION AND IMPLICATIONS: Despite highly potent and direct effects as inverse agonists, competition-binding experiments against radiolabelled agonist and tests for antagonism revealed a probe-dependent allosteric effect of these compounds. Thus, minor chemical changes affected the ability to modify chemokine binding and action, and divided the compounds into two groups: predominantly inverse agonists and balanced antagonists/inverse agonists. These studies have important implications for the design of new inverse agonists with or without antagonist properties.

Molecular and clinical rationale for therapeutic targeting of interleukin-5 and its receptor.

Interleukin-5 is a Th2 homodimeric cytokine involved in the differentiation, maturation, migration, development, survival, trafficking and effector function of blood and local tissue eosinophils, in addition to basophils and mast cells. The IL-5 receptor (IL-5R) consists of an IL-5-specific α subunit that interacts in conformationally dynamic ways with the receptor's ßc subunit, an aggregate of domains it shares with binding sites of IL-3 and granulocyte-macrophage colony-stimulating factor. IL-5 and IL-5R drive allergic and inflammatory immune responses characterizing numerous diseases, such as asthma, atopic dermatitis, chronic obstructive pulmonary disease, eosinophilic gastrointestinal diseases, hyper-eosinophilic syndrome, Churg-Strauss syndrome and eosinophilic nasal polyposis. Although corticosteroid therapy is the primary treatment for these diseases, a substantial number of patients exhibit incomplete responses and suffer side-effects. Two monoclonal antibodies have been designed to neutralize IL-5 (mepolizumab and reslizumab). Both antibodies have demonstrated the ability to reduce blood and tissue eosinophil counts. One additional monoclonal antibody, benralizumab (MEDI-563), has been developed to target IL-5R and attenuate eosinophilia through antibody-dependent cellular cytotoxicity. All three monoclonal antibodies are being clinically evaluated. Antisense oligonucleotide technology targeting the common ßc IL-5R subunit is also being used therapeutically to inhibit IL-5-mediated effects (TPI ASM8). Small interfering RNA technology has also been used therapeutically to inhibit the expression of IL-5 in animal models. This review summarizes the structural interactions between IL-5 and IL-5R and the functional consequences of such interactions, and describes the pre-clinical and clinical evidence supporting IL-5R as a therapeutic target.

Reversed binding of a small molecule ligand in homologous chemokine receptors - differential role of extracellular loop 2.

BACKGROUND AND PURPOSE: The majority of small molecule compounds targeting chemokine receptors share a similar pharmacophore with a centrally located aliphatic positive charge and flanking aromatic moieties. Here we describe a novel piperidine-based compound with structural similarity to previously described CCR8-specific agonists, but containing a unique phenyl-tetrazol moiety which, in addition to activity at CCR8 was also active at CCR1. EXPERIMENTAL APPROACH: Single point mutations were introduced in CCR1 and CCR8, and their effect on small molecule ligand-induced receptor activation was examined through inositol trisphosphate (IP(3) ) accumulation. The molecular interaction profile of the agonist was verified by molecular modeling. KEY RESULTS: The chemokine receptor conserved glutamic acid in TM-VII served as a common anchor for the positively charged amine in the piperidine ring. However, whereas the phenyl-tetrazol group interacted with TyrIV:24 (Tyr(172) ) and TyrIII:09 (Tyr(114) ) in the major binding pocket (delimited by TM-III to VII) of CCR8, it also interacted with TrpII:20 (Trp(90) ) and LysII:24 (Lys(94) ) in the minor counterpart (delimited TM-I to III, plus TM-VII) in CCR1. A straightening of TM-II by Ala-substitution of ProII:18 confirmed its unique role in CCR1. The extracellular loop 2 (ECL-2) contributed directly to the small molecule binding site in CCR1, whereas it contributed to efficacy, but not potency in CCR8. CONCLUSION AND IMPLICATIONS: Despite high ligand potency and efficacy and receptor similarity, this dual-active and bitopic compound binds oppositely in CCR1 and CCR8 with different roles of ECL-2, thereby expanding and diversifying the influence of extracellular receptor regions in drug action.

Influenza A facilitates sensitization to house dust mite in infant mice leading to an asthma phenotype in adulthood.

The origins of allergic asthma, particularly in infancy, remain obscure. Respiratory viral infections and allergen sensitization in early life have been associated with asthma in young children. However, a causal link has not been established. We investigated whether an influenza A infection in early life alters immune responses to house dust mite (HDM) and promotes an asthmatic phenotype later in life. Neonatal (8-day-old) mice were infected with influenza virus and 7 days later, exposed to HDM for 3 weeks. Unlike adults, neonatal mice exposed to HDM exhibited negligible immune responsiveness to HDM, but not to influenza A. HDM responsiveness in adults was associated with distinct Ly6c+ CD11b+ inflammatory dendritic cell and CD8α+ plasmacytoid (pDC) populations that were absent in HDM-exposed infant mice, suggesting an important role in HDM-mediated inflammation. Remarkably, HDM hyporesponsiveness was overcome when exposure occurred concurrently with an acute influenza infection; young mice now displayed robust allergen-specific immunity, allergic inflammation, and lung remodeling. Remodeling persisted into early adulthood, even after prolonged discontinuation of allergen exposure and was associated with marked impairment of lung function. Our data demonstrate that allergen exposure coincident with acute viral infection in early life subverts constitutive allergen hyporesponsiveness and imprints an asthmatic phenotype in adulthood.

A mouse GM-CSF receptor antibody attenuates neutrophilia in mice exposed to cigarette smoke.

We investigated the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a subchronic exposure model of cigarette smoke (CS)-induced inflammation using antibodies directed against GM-CSF or the GM-CSF receptor (GM-CSFR) α-chain. CS-induced mononuclear and neutrophilic inflammation following 4 days of CS exposure in BALB/c mice was assessed in bronchoalveolar lavage (BAL) fluid. An increase in mature dendritic cells (DCs) (CD11c+ and major histocompatibility complex II+) and Gr-1-high neutrophils was also observed by flow cytometric analysis of whole-lung tissue. Daily i.p. injection of 400 µg GM-CSF or GM-CSFR antibody prior to daily smoke exposure attenuated the accumulation of neutrophils within the BAL by 60%. A reduction in mature DCs was also observed. Anti-GM-CSFR antibody administration did not have an effect on the percentage of lung T-cells; however, a significant decrease in activated CD69+ CD8+ T-cells was observed. Anti-GM-CSFR antibody administration decreased the mRNA and protein expression of interleukin-12 p40 and matrix metalloproteinase 12. Taken together, intervention with this receptor antibody implicates the GM-CSF pathway as an important mediator of smoke-induced inflammation.

Identification of a potent anti-IL-15 antibody with opposing mechanisms of action in vitro and in vivo.

BACKGROUND AND PURPOSE: Interleukin-15 (IL-15) is important in the activation and proliferation of lymphocytic cell populations and is implicated in inflammatory disease. We report the characterization of a novel monoclonal antibody DISC0280 which is specific for human IL-15. EXPERIMENTAL APPROACH: DISC0280 was characterized in a direct binding assay of IL-15 with IL-15 receptor α (IL-15Rα) and by its ability to alter IL-15 mediated proliferation of a range of cell lines (cytotoxic T lymphocyte line-2, M-07e, KIT225). A pharmacodynamic model injecting male C57/BL6 mice with IL-15 or IL-15/IL-15Rα, with or without DISC0280, and assessing changes in lymphocytic cell populations and serum cytokines was utilized. KEY RESULTS: DISC0280 inhibited the binding of IL-15 to IL-15Rα and also potently inhibits IL-15 dependent proliferation of cells expressing IL-15Rα, shared interleukin 2/ interleukin 15 receptor ß chain (IL-15Rß) and common gamma chain (γ(c) ). DISC0280 also inhibited the IL-15 dependent proliferation of M-07e cells that only express IL-15Rß/γ(c) subunits. Human IL-15 injected into mice caused an increase in NK1.1(+) and CD3(+) cells in the spleen and peripheral blood and these effects were unexpectedly potentiated by giving DISC0280 with human IL-15. This increase in cells caused by DISC0280/IL-15 co-administration was greater than that observed when IL-15 was administered complexed with soluble IL-15Rα. CONCLUSIONS AND IMPLICATIONS: The ability of DISC0280 to bind to the IL-15Rα-binding site on IL-15 allows trans-presentation of IL-15 by DISC0280 in vivo, similar to the trans-presentation by soluble IL-15Rα. DISC0280 may be therefore suitable as a clinical substitute for IL-15.

Expression of CCR8 is increased in asthma.

BACKGROUND: Chemokines and their receptors could play key roles in the recruitment of T cells to the asthmatic lung. CCR8 is preferentially expressed on T-helper type 2 cells, and is thought to play a role in the pathogenesis of human asthma. OBJECTIVE: Determine the expression of CCR8 on T cells in blood, bronchoalveolar lavage (BAL) and bronchial mucosa from asthmatics and normal subjects. METHODS: CCR8 expression in blood and BAL from asthma and normal subjects was studied using flow cytometry. CCR8 expression on IFN-gamma+ and IL-4+/IL-13+ blood and BAL T cells was studied following stimulation with Phorbol-Myristate-Acetate and Calcium Ionophore. Paraffin-embedded bronchial biopsies were used to study CCR8 in bronchial epithelium. RESULTS: The percentage of CD3+ cells expressing CCR8 in the blood was higher in asthmatics (4.7+/-0.4%) compared with normal subjects (3.0+/-0.4%; P<0.01). There was an approximately sixfold enrichment of CCR8 on IL-4+/IL-13+ cells compared with IFN-gamma+ T cells (P<0.001) in both asthmatic and normal subjects in both blood and BAL. Significantly more BAL T cells expressed CCR8 in asthmatic (8.6+/-0.8%) compared with normal subjects (3.9+/-0.7%) (P<0.01). In paired blood-BAL samples from asthmatics, significantly more CCR8+CD3+ T cells were present in BAL (9.0+/-0.9%) than in blood (5.6+/-0.9%; P<0.05). There were more CCR8-positive cells in bronchial biopsies from asthmatic (93+/-11 cells/mm2) compared with normal subjects (30+/-16 cells/mm2) (P<0.05). The ligand CCL1 was increased in the BAL of asthmatics compared with normal subjects (35+/-6 vs. 12.9+/-7 pg/mL; P<0.05). CONCLUSION: There may be a role for CCR8 in the recruitment of T cells to the lung in asthmatics.

Comparison of the timing of acute blood-brain barrier breakdown to rabbit immunoglobulin G in the cerebellum and spinal cord of mice with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for human multiple sclerosis (MS). Similar to MS patients, EAE animals can exhibit chronic or relapsing, remitting paralysis; leukocyte infiltration of the central nervous system (CNS); and breakdown of the blood-brain barrier (BBB), allowing access to serum components. EAE pathology in rodents is generally thought to progress from the spinal cord to the more rostral brain. This common notion is based on numerous reports on the severity and progression of cellular infiltration and BBB breakdown during the course of disease. We studied opening of the BBB in EAE mice immunized to the proteolipid protein (PLP) peptide, PLP 139-151, with or without the use of pertussis toxin. Peripherally injected rabbit immunoglobulin G showed significant penetration through a compromised BBB in EAE mice and was observed throughout the parenchyma as well as intracellularly in multiple neuronal types. Results demonstrate the novel finding that the cerebellar BBB is dramatically and briefly comprised, even before breakdown of the BBB in the thoracolumbar spinal cord and prior to symptomatic disease. The demonstration of susceptibility in the cerebellum provides an important target for studying the factors predisposing certain CNS regions to autoimmune-related compromise of the BBB, such as MS.

Functional properties of skeletal muscle from transgenic animals with upregulated heat shock protein 70.

The influence of inducible heat stress proteins on protecting contracting skeletal muscle against fatigue-induced injury was investigated. A line of transgenic mice overexpressing the inducible form of the 72-kDa heat shock protein (HSP72) in skeletal muscles was used. We examined the relationship between muscle contractility and levels of the constitutive (HSC73) and inducible (HSP72) forms of the 72-kDa heat shock protein in intact, mouse extensor digitorum longus (EDL), soleus (SOL), and the diaphragm (DPH). In all transgenic muscles, HSP72 was expressed at higher levels compared with transgene-negative controls, where HSP72 was below the level of detection. At the same time, HSC73 levels were downregulated in all transgenic muscle types. Shipment-related stress caused an elevation in the levels of HSP72 in all muscles for 1 wk after arrival of the animals. We also found that, although no statistical differences in response to intermittent fatiguing stimulation in the contractile properties of intact transgene-positive muscles compared with their transgene-negative counterparts were observed, the response of intact transgene-positive EDL muscles to caffeine was enhanced. These findings demonstrate that elevated HSP72 does not protect EDL, SOL, or DPH muscles from the effects of intermittent fatiguing stimulation. However, HSP72 may influence the excitation-contraction coupling (ECC) process, either directly or indirectly, in EDL muscle. If the effects on ECC were indirect, then these results would suggest that manipulation of a specific gene might cause functional effects that seem independent of the manipulated gene/protein.

Relative contribution of endogenous neurotrophins in hippocampal long-term potentiation.

Recent evidence has shown that brain-derived neurotrophic factor (BDNF) is involved in hippocampal long-term potentiation (LTP). Because the reagents used in acute experiments react not only with BDNF but also with neurotrophin-4/5 (NT4/5) and neurotrophin-3 (NT3), we examined the involvement of these neurotrophins in LTP using two highly specific, function-blocking monoclonal antibodies against BDNF and NT3, as well as a TrkB-IgG fusion protein. Our results show that NT3 antibodies did not have any effects on LTP. However, both TrkB-IgG fusion proteins and BDNF antibody similarly reduced LTP, suggesting that only BDNF but no other ligands of the TrkB-receptor are likely to be involved in LTP induction. The reduction in LTP depended on the inducing stimuli and was only observed with theta-burst stimulation (TBS) but not with tetanic stimulation. We further observed that LTP was only reduced if BDNF was blocked before and during TBS stimulation, and BDNF antibodies did not affect early or late stages of LTP if they were applied 10, 30, or 60 min after TBS stimulation. These results point toward a specific and unique role of endogenous BDNF but not of other neurotrophins in the process of TBS-induced hippocampal LTP. Additionally, they suggest that endogenous BDNF is required for a limited time period only shortly before or around LTP induction but not during the whole process of LTP.

Are there differences between the secretion characteristics of NGF and BDNF? Implications for the modulatory role of neurotrophins in activity-dependent neuronal plasticity.

In previous experiments the activity-dependent secretion of nerve growth factor (NGF) from native hippocampal slices and from NGF-cDNA transfected hippocampal neurons showed unusual characteristics [Blochl and Thoenen (1995) Eur J Neurosci 7:1220-1228; (1996) Mol Cell Neurosci 7:173-190]. In both hippocampal slices and cultured hippocampal neurons the activity-dependent secretion proved to be independent of extracellular calcium, but dependent on the release of calcium from intracellular stores. Under different experimental conditions, Goodman et al. [(1996) Mol Cell Neurosci 7:222-238] reported that the high potassium-mediated secretion of brain-derived neurotrophic factor (BDNF) from hippocampal cultures was dependent on extracellular calcium. Mowla et al. [(1997) Proc 27th Annu Meet Soc Neurosci New Orleans 875.10] reported on even further-reaching differences between NGF and BDNF secretion, namely, that in hippocampal neurons and in pituitary cell lines NGF was secreted exclusively according to the constitutive pathway, whereas BDNF was exclusively sorted according to the activity-dependent regulated pathway. In view of the crucial importance of such potential differences between the processing, sorting, and secretory mechanisms of different neurotrophins for their modulatory roles in activity-dependent neuronal plasticity, a thorough analysis under comparable experimental conditions was mandatory. We demonstrate that in native hippocampal slices and adenoviral-transduced hippocampal neurons there are no differences between NGF and BDNF with respect to the subcellular distribution and mechanism of secretion; that the activity-dependent secretion of both NGF and BDNF is dependent on intact intracellular calcium stores; and that the differences between our own observations and those of Goodman et al. (ibid.) regarding the dependence on extracellular calcium do not reflect differences between NGF and BDNF sorting and secretion, but reflect the differing experimental conditions used.

Brain-derived neurotrophic factor levels in the nervous system of wild-type and neurotrophin gene mutant mice.

Although brain-derived neurotrophic factor is the most abundant and widely distributed neurotrophin in the nervous system, reproducible determinations of its levels have been hampered by difficulties in raising suitable monoclonal antibodies. Following immunization of mice with recombinant fish and mammalian brain-derived neurotrophic factor, monoclonal antibodies were generated and used in an immunoassay based on the recognition of two different epitopes. Neither antibody crossreacts with neurotrophin homodimers other than brain-derived neurotrophic factor, although reactivity was detected with brain-derived neurotrophic factor/neurotrophin-3 heterodimers. As both nerve growth factor and neurotrophin-3 are known to affect the development of a variety of neurons expressing the brain-derived neurotrophic factor (bdnf) gene, this assay was used to determine levels in tissues isolated from newborn mice carrying a null mutation in the nerve growth factor (ngf) or the neurotrophin-3 (nt3) gene. Marked differences were observed between mutants and wild-type littermates in the PNS, but not in the CNS, suggesting that neither nerve growth factor nor neurotrophin-3 is a unique regulator of brain-derived neurotrophic factor levels in the newborn mouse CNS.

Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation?

Brain-derived neurotrophic factor (BDNF) has potent effects on neuronal survival and plasticity during development and after injury. In the nervous system, neurons are considered the major cellular source of BDNF. We demonstrate here that in addition, activated human T cells, B cells, and monocytes secrete bioactive BDNF in vitro. Notably, in T helper (Th)1- and Th2-type CD4(+) T cell lines specific for myelin autoantigens such as myelin basic protein or myelin oligodendrocyte glycoprotein, BDNF production is increased upon antigen stimulation. The BDNF secreted by immune cells is bioactive, as it supports neuronal survival in vitro. Using anti-BDNF monoclonal antibody and polyclonal antiserum, BDNF immunoreactivity is demonstrable in inflammatory infiltrates in the brain of patients with acute disseminated encephalitis and multiple sclerosis. The results raise the possibility that in the nervous system, inflammatory infiltrates have a neuroprotective effect, which may limit the success of nonselective immunotherapies.

Neurotrophin release by neurotrophins: implications for activity-dependent neuronal plasticity.

Neurotrophins, secreted in an activity-dependent manner, are thought to be involved in the activity-dependent refinement of synaptic connections. Here we demonstrate that in hippocampal neurons and the rat pheochromocytoma cell line PC12 application of exogenous neurotrophins induces secretion of neurotrophins, an effect that is mediated by the activation of tyrosine kinase neurotrophin receptors (Trks). Like activity-dependent secretion of neurotrophins, neurotrophin-induced neurotrophin secretion requires mobilization of calcium from intracellular stores. Because neurotrophins are likely to be released from both dendrites and axons, neurotrophin-induced neurotrophin release represents a potential positive feedback mechanism, contributing to the reinforcement and stabilization of synaptic connections.

Increased superoxide production during fatigue in the perfused rat diaphragm.

This study test the hypothesis that a temporal relationship exists between the production of superoxide anion (O2-) and the contractile activity of perfused rat diaphragm. O2- levels were determined minute to minute by measuring the reduction of cytochrome c in the perfusate as the diaphragms were subjected to various levels of contractile activity. After equilibrating at low contractile rates (one 500 ms 80 Hz train/min), diaphragms were fatigued by increasing their contractile activity for 5 min (one 500 ms 80 Hz train/s) and then allowed to recover for 30 min (one 500 ms 80 Hz train/min). During equilibration, diaphragms did not produce O2- above the background level measured in the presence of superoxide dismutase (SOD). Within the first minute of fatigue-inducing stimulation, however, the rate of O2- production increased to 0.70 +/- 0.17 nmol/min and remained elevated until the recovery period when production returned towards baseline. SOD blocked this stimulation-related increase of O2-. Tension (+/-SOD) fell to 12% of the control value during the fatigue-inducing stimulation. During recovery the contractile response returned to 51% of control, indicating long-lasting effects on the contractile machinery. SOD did not limit fatigue or improve recovery, probably because it is a large protein that cannot cross cell membranes and protect the cells by scavenging O2- at its site of production.