Surgically Implanted Electrodes Enable Real-Time Finger and Grasp Pattern Recognition for Prosthetic Hands.

Currently available prosthetic hands are capable of actuating anywhere from five to 30 degrees of freedom (DOF). However, grasp control of these devices remains unintuitive and cumbersome. To address this issue, we propose directly extracting finger commands from the neuromuscular system via electrodes implanted in residual innervated muscles and regenerative peripheral nerve interfaces (RPNIs). Two persons with transradial amputations had RPNIs created by suturing autologous free muscle grafts to their transected median, ulnar, and dorsal radial sensory nerves. Bipolar electrodes were surgically implanted into their ulnar and median RPNIs and into their residual innervated muscles. The implanted electrodes recorded local electromyography (EMG) with Signal-to-Noise Ratios ranging from 23 to 350 measured across various movements. In a series of single-day experiments, participants used a high speed pattern recognition system to control a virtual prosthetic hand in real-time. Both participants were able to transition between 10 pseudo-randomly cued individual finger and wrist postures in the virtual environment with an average online accuracy of 86.5% and latency of 255 ms. When the set was reduced to five grasp postures, average metrics improved to 97.9% online accuracy and 135 ms latency. Virtual task performance remained stable across untrained static arm positions while supporting the weight of the prosthesis. Participants also used the high speed classifier to switch between robotic prosthetic grips and complete a functional performance assessment. These results demonstrate that pattern recognition systems can use the high-quality EMG afforded by intramuscular electrodes and RPNIs to provide users with fast and accurate grasp control. SUMMARY: Surgically implanted electrodes recorded finger-specific electromyography enabling reliable finger and grasp control of an upper limb prosthesis.

Correlation between liver cirrhosis and risk of death from oral cancer: Taiwan cohort study.

BACKGROUND: A nationwide population-based cohort was used to examine the severity of liver cirrhosis and risk of mortality from oral cancer. METHODS: The cohort consisted of 3583 patients with oral cancer treated by surgery between 2008 and 2011 in Taiwan. They were grouped on the basis of normal liver function (n = 3471), cirrhosis without decompensation (n = 72) and cirrhosis with decompensation (n = 40). The primary endpoint was mortality. Hazard ratios of death were also determined. RESULTS: The mortality rates in the respective groups were 14.8 per cent, 20.8 per cent and 37.5 per cent at one year (p < 0.001). The adjusted hazard ratios of death at one year for each group compared to the normal group were 2.01 (p = 0.021) for cirrhotic patients without decompensation, 4.84 (p < 0.001) for those with decompensation and 2.65 (p < 0.001) for those receiving chemotherapy. CONCLUSION: Liver cirrhosis can be used to predict one-year mortality in oral cancer patients. Chemotherapy should be used with caution and underlying co-morbidities should be managed in cirrhotic patients to reduce mortality risk.

Development of primer pairs from diverse chloroplast genomes for use in plant phylogenetic research.

Variation in the chloroplast DNA sequence is useful for plant phylogenetic studies. However, the number of variable sequences provided by chloroplast DNA for suggested genes or genomic regions in plant phylogenetic analyses is often inadequate. To identify conserved regions that can be used to design primers and amplify variable sequences for use in plant phylogenetic studies, the complete chloroplast genomic sequences of six plant species (including Oryza sativa, Arabidopsis thaliana, Glycine max, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris), searched from the taxonomy database of NCBI were investigated. A total of 93 conserved regions, 32 in large single copy and 61 in inverted repeat regions, were identified. A set of five primer pairs were designed according to the conserved sequences located in the psbA~trnK, psbB~psbH, rpl23~trnI, trnR~trnN, and trnY~trnD regions to amplify variable DNA fragments. An additional 18 plant accessions from 14 species were used to validate their utility. Each of the tested species could be distinguished by length polymorphisms of fragments amplified with the five primer pairs. trnR~trnN and rpl23~trnI amplified fragments specific to monocot and legume species, respectively. Three primer pairs located in the psbA~trnK, psbB~psbH, and trnR~trnN regions were applied to amplify variable DNA sequences for phylogenetic analysis using the maximum parsimony method. The consistent result between taxonomy and phylogenetic analysis on the variable sequences amplified with these three primer pairs was revealed. The five newly developed primer pairs are recommended as tools for use in the identification of plant species and in phylogenetic studies.

Plasma adiponectin in heart failure with and without cachexia: catabolic signal linking catabolism, symptomatic status, and prognosis.

BACKGROUND AND AIMS: Adiponectin (ADPN) as an adipose tissue hormone contributes to regulation of energy metabolism and body composition and is associated with cardiovascular risk profile parameters. Cardiac cachexia may develop as a result of severe catabolic derangement in chronic heart failure (CHF). We aimed to determinate an abnormal ADPN regulation as a link between catabolic signalling, symptomatic deterioration and poor prognosis. METHODS AND RESULTS: We measured plasma ADPN in 111 CHF patients (age 65 ± 11, 90% male, left ventricular ejection fraction (LVEF) 36 ± 11%, peak oxygen consumption (peakVO2) 18.1 ± 5.7 l/kg*min, body mass index (BMI) 27 ± 4 kg/m(2), all mean ± standard deviation) and 36 healthy controls of similar age and BMI. Body composition was assessed by dual energy X-ray absorptiometry, insulin sensitivity was evaluated by homoeostasis model assessment, exercise capacity by spiroergometry. Plasma ADPN did not differ between CHF vs. controls (13.5 ± 11.0 vs. 10.5 ± 5.3 mg/l, p > 0.4), but increased stepwise with NYHA functional class (I/II/III: 5.7 ± 1.4/10.7 ± 8.3/19.2 ± 14.0 mg/l, ANOVA p < 0.01). Furthermore, ADPN correlated with VO2 at anaerobic threshold (r = -0.34, p < 0.05). ADPN was highest in cachectic patients (cCHF, 16%) vs. non-cachectic (ncCHF) (18.7 ± 15.0 vs. 12.5 ± 9.9 mg/l; p < 0.05). ADPN indicated mortality risk independently of established prognosticators (HR: 1.04 95% CI: 1.02-1.07; p < 0.0001). ADPN above the mean (13.5 mg/l) was associated with a 3.4 times higher mortality risk in CHF vs. patients with ADPN levels below the mean. CONCLUSION: Circulating ADPN is abnormally regulated in CHF. ADPN may be involved in impaired metabolic signalling linking disease progression, tissue wasting, and poor outcome in CHF.

Tachykinin NK3-receptor deficiency does not inhibit pulmonary eosinophilia in allergic mice.

Tachykinins are important in the development of pulmonary inflammation in mice but the tachykinin receptor subtype mediating this response has not been defined. To elucidate the role of tachykinin NK3-receptors on allergen-induced pulmonary inflammation, studies were performed on ovalbumin (OVA) sensitized and challenged mice with genetic disruption of the tachykinin NK3-receptor (NK3-/-). Aerosol OVA (0.5%) challenge produced eosinophil influx into the bronchoalveolar lavage fluid and lung tissue, goblet cell hyperplasia and damage to the airway epithelium of both NK3-/- mice and in wild type control mice (NK3+/+). There was no difference in the magnitude of these allergic inflammatory pulmonary responses between NK3-/- and NK3+/+ mice. These results find no role for tachykinin NK3-receptors on the pulmonary eosinophilia and lung damage after antigen challenge in mice.

Inhibition of experimental acute pulmonary inflammation by pirfenidone.

Pirfenidone, a putative tumor necrosis factor-alpha (TNF-alpha) inhibitor, has recently gained recognition for its therapeutic use in the treatment of idiopathic pulmonary fibrosis. As pulmonary fibrosis may be the result of lung inflammatory processes, we examined the anti-inflammatory potential of pirfenidone in several models of acute pulmonary inflammation. In antigen-induced allergic paradigms, 24 h after antigen challenge, sensitized mice or guinea pigs develop a prominent pulmonary inflammation, reflected by a significant increase in the number of recoverable bronchoalveolar lavage (BAL) total cells and eosinophils. In both species, the pretreatment of animals with pirfenidone (10 and 30 mg/kg) resulted in a dose-dependent inhibition of the antigen-induced pulmonary inflammation, which was reflected by a significant decrease in the BAL eosinophils and total cells by the 30 mg/kg dose. In a non-allergic model of pulmonary inflammation, rats challenged with intratracheal LPS develop a significant increase in BAL neutrophils and total cells, along with significant increases in TNF-alpha and IL-6. Pretreatment with pirfenidone (3 and 30 mg/kg) showed a dose-dependent inhibition of the LPS-induced pulmonary inflammation, reflected by a significant decrease in the number of BAL total and neutrophilic cells at both the 3 and 30 mg/kg dose. However, pirfenidone had no effect on the peak BAL levels of TNF-alpha. In contrast, pirfenidone significantly inhibited BAL levels of IL-6. In summary, we have shown that pirfenidone can inhibit allergic and non-allergic inflammatory cell recruitment and that its pulmonary anti-inflammatory activity is independent of TNF-alpha inhibition.

Effect of anti-mIL-9 antibody on the development of pulmonary inflammation and airway hyperresponsiveness in allergic mice.

Interleukin (IL)-9 is a T-cell-derived cytokine with pleiotropic activities on T helper 2 cells, B cells, and mast cells. IL-9 may therefore play an important role in the development of allergic pulmonary inflammatory diseases. In this study, an antimouse IL-9 (anti-mIL-9) antibody (Ab) was evaluated against pulmonary eosinophilia, histopathologic changes in lung tissues, serum immunoglobulin (Ig) E levels, and airway hyperresponsiveness (AHR) to methacholine in mice sensitized and challenged with ovalbumin (OVA). Additionally, steady-state levels of IL-4, IL-5, IL-13, and interferon-gamma messenger RNA (mRNA) in the lungs were measured. The anti-mIL-9 Ab (200 microg/mouse, intraperitoneally) was given as either four doses during the sensitization period or as a single dose before OVA challenge. Sensitized mice challenged with OVA displayed marked pulmonary eosinophilia, epithelial damage, and goblet cell hyperplasia. OVA challenge also increased mRNA levels of IL-4, IL-5, and IL-13 in the lungs. AHR was also increased twofold in sensitized, challenged mice. Treatment of sensitized, challenged mice with four doses of anti-mIL-9 Ab significantly reduced pulmonary eosinophilia, serum IgE levels, goblet cell hyperplasia, airway epithelial damage, and AHR, but had no effect on IL-4, IL-5, and IL-13 mRNA levels in the lungs. A single dose of the antibody was ineffective on all measures. These results indicate that an antibody to mIL-9 inhibits the development of allergic pulmonary inflammation and AHR in mice.

Comparison of PDE 4 inhibitors, rolipram and SB 207499 (ariflo), in a rat model of pulmonary neutrophilia.

Using a rat model of lipopolysaccharide (LPS)-induced pulmonary inflammation, the antiinflammatory activity of SB 207499 was evaluated and compared to that of the prototypic type-4 phosphodiesterase (PDE4) inhibitor, rolipram. In dose-response experiments, we found that rats exposed to 10 microg or 100 microg of intratracheal (it) LPS developed a prominent pulmonary inflammation, due to a significant increase in the number of recoverable bronchoalveolar lavage neutrophils. The pulmonary neutrophilia, provoked by the challenge of 10 microg LPS/rat, was significant at 2 h, peaked by 16 h, declined thereafter but remained elevated for up to 48 h. Additionally, the exposure of rats to 10 microg LPS caused the local pulmonary production of TNF- alpha. In contrast to the cellular influx, TNF- alpha production peaked at 2 h and rapidly declined to negligible levels by 8 h. While low levels were detected, the levels of IL-1 beta in bronchoalveolar lavage did not significantly differ from saline challenged animals. Rats pretreated with rolipram or SB 207499, displayed dose-dependent inhibition of the LPS-induced pulmonary inflammation. Nevertheless, the pulmonary production of TNF- alpha and IL-1 beta was unaffected by either SB 207499 or rolipram. When provoked with the 10 microg dose of LPS, adrenalectomized rats produced a similar 24 h induction of pulmonary neutrophilia. Pretreatment of adrenalectomized rats with the PDE4 inhibitors showed similar inhibitory results to those obtained in normal rats. In summary, we have shown, using a rat model of LPS-induced pulmonary neutrophilic inflammation, that the inhibitory activities of rolipram or SB207499 are not linked to the production of TNF- alpha or the inhibition of IL-1 beta, and occur independently of endogenous catecholamine or corticosteroid release.

Aberrant in vivo T helper type 2 cell response and impaired eosinophil recruitment in CC chemokine receptor 8 knockout mice.

Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (-/-) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8(-/)- mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.

Inhibition of pulmonary eosinophilia and airway hyperresponsiveness in allergic mice by rolipram: involvement of endogenously released corticosterone and catecholamines.

This study investigates the role of adrenal-derived catecholamines and corticosterone on the inhibition by rolipram, a phosphodiesterase (PDE)-4 inhibitor, of pulmonary eosinophilia and airway hyperresponsiveness (AHR) in allergic mice. The following experimental groups were studied in mice sensitized and challenged with ovalbumin (OVA): normal, adrenalectomized, propranolol (beta-adrenoceptor antagonist) and metyrapone (corticosterone synthesis inhibitor) treated. These interventions were studied both in the absence and in the presence of rolipram. Eosinophil numbers in the bronchoalveolar lavage (BAL) and AHR to methacholine were measured 24 h after OVA challenge. Treatment of sensitized mice with rolipram (0.3 - 10 mg kg(-1), p.o.), inhibited pulmonary eosinophilia and the AHR to methacholine in OVA-challenged mice. Adrenalectomy increased the number of eosinophils in the BAL of OVA-challenged mice but had no effect on AHR to methacholine. Adrenalectomy attenuated both the rolipram-induced inhibition of BAL eosinophilia and AHR to methacholine in OVA challenged mice. Propranolol (10 mg kg(-1), p.o.) had no effect on the inhibition of eosinophilia by rolipram but attenuated the inhibition of AHR to methacholine in OVA challenged mice. On the other hand, metyrapone (10 mg kg(-1), p.o.) attenuated the inhibition of eosinophilia by rolipram but had no effect on the inhibition of AHR to methacholine in OVA challenged mice. Metyrapone-treatment alone increased the number of eosinophils in the BAL of OVA-challenged mice. These results identify an important role for adrenal-derived catecholamines and corticosterone on the inhibition of pulmonary eosinophilia and AHR by rolipram in allergic mice.

Effect of Sch 55700, a humanized monoclonal antibody to human interleukin-5, on eosinophilic responses and bronchial hyperreactivity.

This report describes the development and the biology of Sch 55700, a humanized monoclonal antibody to human IL-5 (hIL-5). Sch 55700 was synthesized using CDR (complementarity determining regions) grafting technology by incorporating the antigen recognition sites for hIL-5 onto consensus regions of a human IgG4 framework. In vitro, Sch 55700 displays high affinity (Kd = 20 pmol/l) binding to hIL-5, inhibits the binding of hIL-5 to Ba/F3 cells (IC50 = 0.5 nmol/l) and blocks IL-5 mediated proliferation of human erythroleukemic TF-1 cells. In allergic mice, Sch 55700 (0.1-10 mg/kg, i.p. or i.m.) inhibits the influx of eosinophils in the lungs, demonstrates long duration of activity and the anti-inflammatory activity of this compound is additive with oral prednisolone. In allergic guinea pigs, Sch 55700 (0.03-30 mg/kg i.p.) inhibits both the pulmonary eosinophilia and airway hyperresponsiveness and at 30 mg/kg, i.p. inhibited allergic, but not histamine-induced bronchoconstriction. In allergic rabbits, Sch 55700 blocks cutaneous eosinophilia. Sch 55700 (0.1-1 mg/kg i.p.) also blocks the pulmonary eosinophilia and neutrophilia caused by tracheal injection of hIL-5 in guinea pigs. In allergic cynomolgus monkeys, a single dose of Sch 55700 (0.3 mg/kg i.v.) blocks the pulmonary eosinophilia caused by antigen challenge for up to six months. Sch 55700 is, therefore, a potent antibody against IL-5 in vitro and in a variety of species in vivo that could be used to establish the role of IL-5 in human eosinophilic diseases such as asthma.

Effects of cyclosporin A and dinactin on T-cell proliferation, interleukin-5 production, and murine pulmonary inflammation.

We compared the effects of cyclosporin A (CSA) and a macrotetrolide antibiotic, dinactin, on human T-cell proliferation and cytokine production induced by stimulation of the T-cell receptor alone (monoclonal antibody [mAb] directed against CD3) or in combination with costimulatory signals (mAbs directed against CD3 and CD28). These agents were also examined in a murine model of interleukin (IL)-5-mediated pulmonary inflammation. Dinactin inhibited T-cell proliferation induced by IL-2, by mAb to CD3, and by mAbs to CD3 plus alpha-CD28 with identical dose-response curves (IC50 = 10-20 ng/ml). Dinactin inhibited cytokine production with IC50 values of 10 ng/ml for IL-4 and IL-5 and 30 or 60 ng/ml for interferon-gamma or IL-2, respectively. Unlike CSA, exogenous IL-2 did not alter the dinactin-mediated effects on T cells, and nuclear run-on and steady-state messenger RNA (mRNA) analysis showed that dinactin inhibited cytokine production through a post-transcriptional mechanism. CSA selectively blocked T-cell receptor-induced T-cell proliferation and cytokine production (IC50 = 10 ng/ml). Under costimulatory conditions, IL-5 synthesis was only minimally inhibited by high concentrations of CSA, and at CSA concentrations of less than 125 ng/ml, IL-5 was significantly increased above control values. Dinactin and CSA reduced pulmonary eosinophilia when administered within 1 d of airway antigen challenge. Of the cytokine mRNAs examined in the lungs of CSA-pretreated, antigen-challenged mice, IL-5 mRNA levels were the least reduced, paralleling the resistance of IL-5 to CSA observed in vitro and suggesting a role for CD28 in the in vivo induction of IL-5.

Effects of chronic anti-interleukin-5 monoclonal antibody treatment in a murine model of pulmonary inflammation.

The maturation of eosinophils in bone marrow, their migration to pulmonary tissue, and their subsequent degranulation and release of toxic granule proteins contributes to the pathophysiology observed in asthma. Interleukin-5 (IL-5) is essential for these processes to occur. Therefore, much emphasis has been placed on attempts to inhibit the production or activity of IL-5 in order to attenuate the inflammatory aspect of asthma. In this report, the immunological consequences of long-term exposure to an antibody recognizing IL-5 (TRFK-5) were studied in a murine pulmonary inflammation model. A single dose of TRFK-5 (1 mg/ kg, intraperitoneally) reversibly inhibited antigen-dependent lung eosinophilia in mice for at least 12 wk and inhibited the release of eosinophils from bone marrow for at least 8 wk. Normal responses to aerosol challenge were attained after 24 wk. In mice treated acutely with antibody (2 h before challenge), 50% inhibition of pulmonary eosinophilia occurred when 0. 06 mg/kg TRFK-5 was administered (intraperitoneally; ED50), resulting in 230 ng/ml (IC50) in serum. In mice treated with one dose of TRFK-5 (1 mg/kg) and rested before challenge, the antibody exhibited a half-life of 2.4 wk. After 18 to 19 wk, antigen challenge-induced eosinophilia was inhibited by 50% and serum levels of TRFK-5 were 25 ng/ml. TRFK-5 remaining in mice 8 wk after a single injection of TRFK-5 was sufficient to inhibit at least 50% of the eosinophilia induced in blood 3 h after injection of recombinant murine IL-5 (10 microg/kg, intravenously). To assess the biologic effect of long-term exposure of mice to antibody, several parameters of immune-cell function were measured. Throughout the extended period of activity of TRFK-5 (>/= 12 wk) there were no gross effects on antigen-dependent increases in T-cell recruitment into bronchoalveolar fluid (BALF), in IL-4 and IL-5 steady-state mRNA levels in lung tissue, or in immunoglobulin E (IgE) and IgG levels in serum. There was a small increase in IL-5 steady-state mRNA production in TRFK-5-treated mice after 2 h or 2 wk, but this was not observed at other times examined. In untreated mice, IL-5 steady-state mRNA production in response to antigen challenge decreased > 6-fold with age, although at all time points there was an increase in mRNA levels following challenge. Therefore, at later times, 25 ng/ml rather than 230 ng/ml of TRFK-5 inhibited BALF eosinophilia, probably because of reduced IL-5 levels. Twenty-four weeks after treatment with TRFK-5, when challenge-induced eosinophilia was restored, there was an excess of CD4(+) T cells in BALF from challenged mice. However, these T cells had no measurable effects on other responses to challenge, including cytokine production, B-cell accumulation, and immunoglobulin production in serum. Thus, the biologic duration of TRFK-5 was several months, and its activity was due to the presence of antibody above a therapeutic threshold rather than to any profound effect on the immune system.

Anti-inflammatory activity of inhaled mometasone furoate in allergic mice.

Mometasone furoate (CAS 83919-23-7, Sch 32088) is a new inhaled corticosteroid that is being developed to treat allergic inflammatory airway disorders such as rhinitis and asthma. In this study, we investigated the effects of inhaled mometasone furoate in allergic mice that, after antigen challenge, develop an influx of eosinophils and T cells and display an increased mRNA expression of proinflammatory cytokines in the lungs. Mometasone furoate aerosol was generated from metered dose inhalers and delivered into an animal exposure chamber. The mice were exposed to mometasone furoate by nose-only inhalation at respired doses ranging from 0.5-33 micrograms/kg given 24, 18 and 2 h before aeroallergen challenge. The elevated eosinophil numbers in the bronchoalveolar lavage fluid and lung tissues of sensitized, ovalbumin challenged mice were dose-dependently inhibited by inhaled mometasone furoate. Increased numbers of Thy1+ T cells and CD4+ (T-helper) and CD8+ (T-cytotoxic) T cell subsets were seen in the bronchoalveolar lavage fluid of ovalbumin-challenged mice. Pretreatment of these animals with mometasone furoate (33 micrograms/kg) reduced the number of Thy1+ T cells and the T-helper subset. Furthermore, mometasone furoate (33 micrograms/kg) reduced the percentage of CD44+ T-helper cells (activated/memory cells) to the levels observed in non-sensitized, ovalbumin-challenged mice. There were increased levels of steady-state mRNA for interleukin-4, interleukin-5, and to a lesser extent, gamma-interferon in the lungs of sensitized mice after ovalbumin challenge and pretreatment with mometasone furoate reduced the steady-state mRNA levels of these cytokines. Our results demonstrate a potent lung anti-inflammatory effect of inhaled mometasone furoate and identify that inhibition of T cell influx, eosinophil accumulation and modulation of cytokine activity are important components of this response.

Eosinophils retain their granule major basic protein in a murine model of allergic pulmonary inflammation.

Accumulation of eosinophils in the lung with concomitant tissue damage are defining histopathologic features of human asthma. Through degranulation and the release of proinflammatory proteins such as major basic protein (MBP), eosinophils may perpetuate this inflammatory response. We investigated the extent of eosinophil degranulation in a murine model of allergic pulmonary inflammation. In this paradigm, the mice develop pulmonary eosinophilia, mucus hypersecretion, tissue damage, and airway edema and hyperreactivity. To evaluate the degree of eosinophil degranulation, we used a polyclonal antibody to murine MBP (mMBP) to perform dot blot analysis of bronchoalveolar lavage (BAL) cells and fluids, and immunohistochemical fluorescent analysis of lung tissue sections. After ovalbumin antigen challenge, we were unable to detect immunoreactive mMBP in the BAL fluids from either nonsensitized or sensitized mice. However, after lysis of the recoverable BAL cells, we were able to detect mMBP by immunoblot analysis, with the levels of immunoreactive mMBP directly related to the number of recoverable eosinophils. We also examined paraffin-embedded, lung tissue sections for patterns of mMBP deposition. Whereas lung sections from allergic mice revealed prominent peribronchial eosinophilia after antigen challenge, tissue sections from nonsensitized animals rarely displayed eosinophils. Despite the presence of numerous eosinophils, no immunohistologic evidence of extracellular mMBP could be found in antigen-challenged allergic mice. Furthermore, rechallenged allergic mice displayed a significant increase in the number of recruited pulmonary eosinophils but all immunoreactive mMBP was still intracellular. We conclude that the recruited pulmonary eosinophils have not substantially degranulated. These results suggest that, in this murine model of allergic inflammation, eosinophil degranulation and release of mMBP does not contribute to the observed pulmonary inflammation and airway hyperreactivity.

Pulmonary biology of anti-interleukin 5 antibodies

Interleukin 5 (IL-5) is a critical cytokine for the maturation of eosinophil precursors to eosinophils in the bone marrow and those eosinophils then accumulate in the lungs during asthma. We have studied anti-bodies on allergic responses in mice, guinea pigs anf monkeys and are extending this experiment into humans with a humanized antibody. In a monkey model of pulmonary inflammation and airway hyperreactivity, we found that the TRFK-5 antibody blocked both responses for three months following a single dose of 0.3 mg/kg i.v. This antibody also blocked lung eosinophilia in mice by inhibiting release from the bone marrow. To facilitate multiple dosing and to reduce immunogenicity in humans, we prepared Sch 55700, humanized antibody against IL-5. Sch 55700 was also active against lung eosinophilia in allergic monkeys and mice and against pulmonary eosinophilia and airway hyperresponsiveness in guinea pigs. Furthermore, as opposed to steroids, Sch 55700 dis not cause immunosuppression in guinea pigs. Studies with antibody in humans will be critical to establishing the therapeutic potential of IL-5 inhibition.

Role of nitric oxide on eosinophilic lung inflammation in allergic mice.

Nitric oxide (NO) is an important mediator of inflammatory reactions and may contribute to the lung inflammation in allergic pulmonary diseases. To assess the role of NO in pulmonary inflammation, we studied the effect of four nitric oxide synthase (NOS) inhibitors, N-nitro-L-arginine methyl ester (L-NAME), aminoguanidine, N(G)-monomethyl-L-arginine (NMMA) and L-N6-(1-Iminoethyl) lysine (L-NIL), on the influx of eosinophils into the bronchoalveolar lavage (BAL) fluid and lung tissue of antigen-challenged allergic mice. We also analyzed lung tissues for the presence of steady state mRNA for inducible nitric oxide synthase (iNOS) and iNOS protein. Furthermore, BAL fluid and serum were analyzed for their nitrite content. B6D2F1/J mice were sensitized to ovalbumin (OVA) and challenged with aerosolized OVA. The NOS inhibitors were given 0.5 h before and 4 h after the antigen challenge. OVA challenge induced a marked eosinophilia in the BAL fluid and lung tissue 24 h after challenge. The OVA-induced pulmonary eosinophilia was significantly reduced by L-NAME (10 and 50 mg/kg, intraperitoneally [i.p.]). The inactive isomer, D-NAME (50 mg/kg, i.p.) had no effect. When mice were treated with L-NAME (20 mg/kg, i.p.) and an excess of NOS substrate, L-arginine (200 mg/kg, i.p.), the OVA-induced pulmonary eosinophilia was restored. Treatment with aminoguanidine (0.4-50 mg/kg, i.p.) also reduced the pulmonary eosinophilia. Treatment with NMMA (2-50 mg/kg, i.p.) partially reduced the eosinophilia, but L-NIL (10-50 mg/kg, i.p.), a selective iNOS inhibitor, had no effect. L-NAME had no effect on the reduction of eosinophils in the bone marrow following OVA challenge to sensitized mice. OVA challenge to sensitized mice had no effect on iNOS protein expression or iNOS mRNA in the lungs or on the levels of nitrite in the BAL fluid. These results suggest that NO is involved in the development of pulmonary eosinophilia in allergic mice. The NO contributing to the eosinophilia is not generated through the activity of iNOS nor does NO contribute to the efflux of eosinophils from the bone marrow in response to antigen challenge. It is speculated that after antigen challenge, the localized production of NO, possibly from pulmonary vascular endothelial cells, is involved in the extravasation of eosinophils from the circulation into the lung tissue.

Pulmonary biology of anti-interleukin 5 antibodies.

Interleukin-5 (IL-5) is a critical cytokine for the maturation of eosinophil precursors to eosinophils in the bone marrow and those eosinophils then accumulated in the lungs during asthma. We have studied anti IL-5 antibodies on allergic responses in mice, guinea pigs and monkeys and are extending this experiment into humans with a humanized antibody. In a monkey model of pulmonary inflammation and airway hyperreactivity, we found that the TRFK-5 antibody blocked both responses for three months following a single does of 0.3 mg/kg, i.v. This antibody also blocked lung eosinophilia in mice by inhibiting release from the bone marrow. To facilitate multiple dosing and to reduce immunogenicity in humans, we prepared Sch 55700, a humanized antibody against IL-5. Sch 55700 was also active against lung eosinophilia in allergic monkeys and mice and against pulmonary eosinophilia and airway hyperresponsiveness in guinea pigs. Furthermore, as opposed to steroids, Sch 55700 did not cause immunosuppression in guinea pigs. Studies with this antibody in humans will be critical to establishing the therapeutic potential of IL-5 inhibition.