Treatment of thrombocytopenia in chimpanzees infected with human immunodeficiency virus by pegylated recombinant human megakaryocyte growth and development factor

Three chimpanzees experimentally infected with human immunodeficiency virus (HIV) developed significant chronic thrombocytopenia after 5, 4, and 2 years, with peripheral platelet counts averaging 64 +/- 19 x 10(3)/microL (P = .004 compared with 228 +/- 92 x 10(3)/microL in 44 normal control animals), mean platelet volumes of 11.2 +/- 1.8 fL (P > .5 compared with 10.9 +/- 0. 7 fL in normal controls), endogenous thrombopoietin (TPO) levels of 926 +/- 364 pg/mL (P < .001 compared with 324 +/- 256 pg/mL in normal controls), uniformly elevated platelet anti-glycoprotein (GP) IIIa49-66 antibodies, and corresponding viral loads of 534, 260, and 15 x 10(3) RNA viral copies/mL. Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) was administered subcutaneously (25 microg/kg twice weekly for 3 doses) to determine the effects of stimulating platelet production on peripheral platelet concentrations in this cohort of thrombocytopenic HIV-infected chimpanzees. PEG-rHuMGDF therapy increased (1) peripheral platelet counts 10-fold (from 64 +/- 19 to 599 +/- 260 x 10(3) platelets/microL; P = .02); (2) marrow megakaryocyte numbers 30-fold (from 11.7 +/- 6.5 x 10(6)/kg to 353 +/- 255 x 10(6)/kg; P = .04); (3) marrow megakaryocyte progenitor cells fourfold (from a mean of 3.6 +/- 0.6 to 14.1 x 10(3) CFU-Meg/1, 000 CD34(+) marrow cells); and (4) serum levels of Mpl ligand from 926 +/- 364 pg/mL (endogenous TPO) to predosing trough levels of 1, 840 +/- 353 pg/mL PEG-rHuMGDF (P = .02). The peripheral neutrophil counts were also transiently increased from 5.2 +/- 2.6 x 10(3)/microL to 9.9 +/- 5.0 x 10(3)/microL (P = .01), but neither the erythrocyte counts nor the reticulocyte counts were altered significantly (P > .1). The serum levels of antiplatelet GPIIIa49-66 antibodies exhibited reciprocal reductions during periods of thrombocytosis (P < .07). PEG-rHuMGDF therapy did not increase viral loads significantly (395, 189, and 53 x 10(3) RNA viral copies/mL; P > .5 compared with baseline values). The striking increase in peripheral platelet counts produced by PEG-rHuMGDF therapy implies that thrombocytopenia in HIV-infected chimpanzees is attributable to insufficient compensatory expansion in platelet production resulting from HIV-impaired delivery of platelets despite stimulated megakaryocytopoiesis. These data suggest that PEG-rHuMGDF therapy may similarly correct peripheral platelet counts in thrombocytopenic HIV-infected patients.     

Cytologic evaluation of bronchoalveolar lavage fluid from horses with recurrent airway obstruction after aerosol and parenteral administration of beclomethasone dipropionate and dexamethasone, respectively

OBJECTIVE: To determine cytologic changes in horses with recurrent airway obstruction (heaves) after administration of aerosolized beclomethasone dipropionate and dexamethasone parenterally. ANIMALS: 6 horses with inducible and reversible heaves. PROCEDURE: Episodes of heaves were induced by exposure to moldy hay and straw for 7 days. Horses were assigned to treatment groups (aerosolized beclomethasone, parenterally administered dexamethasone, aerosolized propellant), and pulmonary inflammation was evaluated by serial cytologic examination of bronchoalveolar lavage (BAL) fluid samples obtained on days 0, 7, 10, 14, and 21. Total and differential cell counting and phenotypic analysis of lymphocyte subpopulations in BAL fluid were performed. RESULTS: 7 days of natural challenge induced neutrophilic inflammation. Neutrophil counts in BAL fluid were reduced in beclomethasone- and dexamethasone-treated horses on days 10 and 14 but rebounded to pretreatment values on day 21. The proportion of proinflammatory lymphocyte subpopulations (CD4+ and B+) and MHC class-II antigen expression were increased on days 14 and 21 in propellant-treated horses, compared with beclomethasone- and dexamethasone-treated horses. CONCLUSIONS: Aerosolized beclomethasone attenuated neutrophilic pulmonary inflammation and prevented alteration in lymphocyte subpopulations in horses with heaves. Results were similar to the response associated with parenterally administered dexamethasone. Short-term administration of aerosolized beclomethasone without minimizing environmental allergen exposure is not expected to provide prolonged anti-inflammatory benefit for horses with heaves.     

Platelet activating factor (PAF) in memory formation: role as a retrograde messenger in long-term potentiation

A model of experimental infection with EV1, a British isolate of maedi-visna virus (MVV), has been developed. Twelve male Texel sheep were allocated to three groups and inoculated by the respiratory route with different inocula. Six of the animals received 10(7.2) tissue culture infective dose (TCID50) of MVV EV1 strain. Two sheep were inoculated with the same dose of heat inactivated MVV EV1 strain. An additional group of four sheep was sham-inoculated with identically prepared virus-free culture media. Experimental infection was followed for 16 weeks. Prior to inoculation, routine haematology, bronchoalveolar lavage (BAL) and flow cytometric analysis of bronchoalveolar lavage fluid (BALF) lymphocytes were performed in all animals to provide baseline parameters. Flow cytometric analysis of BALF lymphocytes and differential BALF cell counts were performed. Precipitating antibodies to MVV developed in all MVV-inoculated animals during the first 4 weeks post-inoculation, while the rest remained seronegative to MVV. MVV-infected animals had significantly decreased (P < 0.05) percentages of macrophages and significantly increased (P < 0.05) percentages of lymphocytes in BALF 4 weeks post-inoculation. Phenotypic changes in BALF T lymphocytes from MVV-inoculated animals, compared with the other two groups, showed significantly decreased (P < 0.05) percentages of CD4+ and gamma delta + T lymphocytes, significantly increased (P < 0.05) percentages of CD8+ lymphocytes and significant inversion (P < 0.05) of the CD4+/CD8+ ratio at different sampling times, but between 2 and 12 weeks post-inoculation. These findings indicate that during experimental MVV-infection an early, short-term cellular reaction occurs in the lung, that is characterised by T lymphocyte phenotypic changes that are very similar, if not identical, to those observed in natural MVV infection.     

The phylogenetic position of alpha- and beta-tubulins from the Chlorarachnion host and Cercomonas (Cercozoa)

The purpose of this study was to investigate the effect of acid conditioning of root surfaces during recombinant human bone morphogenetic protein-2 (rhBMP-2) induced periodontal regeneration in vivo. The buccal aspect of molar roots were denuded of their periodontal ligament through a bony window created in the mandible of 34 Wistar rats under general anesthesia. Three groups of 11 or 12 animals received either 10 microL of 50 g/mL rhBMP-2 in a collagen gel over the surgical defect (BMP) or 10 microL of collagen gel only (COL) or were left untreated (UN). Each of the 3 groups were further subdivided into those that received prior root acid conditioning with 35% phosphoric acid gel and those without acid conditioning. Animals were sacrificed 10 days after surgery and the tissues processed for histological examination. The BMP groups with and without acid conditioning developed significantly more bone over the second molar (3.89+/-0.86% and 7.62+/-0.93%, respectively; mean+/-SE), compared with the respective COL (1.24+/-0.26% and 2.77+/-0.52%) and UN groups (1.34+/-0.35% and 3.69+/-0.37%) (P <0.05). Furthermore, significantly more bone was found in the BMP non-acid conditioned group compared with all other groups (P <0.05). Acid conditioning promoted significantly more ankylosis (50%) compared with non-acid conditioning (6.3%) (P=0.007). New cementum formation was greatest in the BMP acid conditioned group (628.4+/-253.8 microm2) and lowest in the non-acid conditioned UN group (207.6+/-36.4 microm2) (P <0.05). This is the first known report evaluating the effects of root acid conditioning after a single application of rhBMP-2 in vivo. Results suggest that root conditioning agents operating at low pH administered into the periodontal wound impairs early BMP-induced osteogenesis while simultaneously promoting BMP-induced cementogenesis.     

Effects of FK506 on experimental membranous glomerulonephritis induced by cationized bovine serum albumin in rats

BACKGROUND: There have been no reports on the effect of FK5 06, a new immunosuppressive agent, on experimental membranous glomerulonephritis (MN) induced by an exogenous antigen. Therefore we investigated the effects of FK506 on MN induced by cationized bovine serum albumin (C-BSA) in rats. METHODS: Two weeks after the rats were immunized with 1 mg of C-BSA and Freund's complete adjuvant, they received intravenous injections of 3 mg/kg of C-BSA 3 times weekly for 4 weeks. Rats were divided into four groups: group A (n = 5) received intramuscular injections of 3 mg/kg of FK506 daily for 5 days beginning 2 days before the first immunization; group B (n =4) received 1 mg/kg of FK506 daily for 2 weeks beginning 2 weeks after the first immunization; and group C (n =4) received 1 mg/kg of FK506 daily for 2 weeks beginning 4 weeks after the first immunization. Group D (n = 5) received no FK506 and served as the control group. Rats were sacrificed 8 weeks after the first immunization. RESULTS: Administration of FK506 in the preimmunization stage almost completely suppressed the development of MN in group A. Histological findings in groups B and C were similar to those in group D, the control group. However, urinary protein excretion was significantly lower in group B (24+/-46 mg/day) and C (220+/-44 mg/day) than in group D (330+/-61 mg/day). Expression of intracellular adhesion molecule-1 in glomeruli and the number of leukocyte functioning-associated molecules-1 were significantly decreased in groups A, B, and C compared with the control group. Administration of FK506 was not associated with any significant side-effects or histological abnormalities. The whole-blood trough levels of FK506 in groups B and C were 9.1 ng/ml and 9.2 ng/ml respectively. CONCLUSIONS: The efficacy of FK506 was significantly increased when the drug was administered in the early phase of immunization in this model. The present study suggests that FK506 may be useful in patients with intractable nephrotic syndrome such as MN.     

Alkalinizing water-soluble local anesthetic solutions by addition of cyclodextrin

OBJECTIVE: To correlate indices of airway reactivity to bronchoalveolar lavage (BAL) fluid cytologic features in horses with a recent decline in exercise tolerance. ANIMALS: 20 actively working horses from 2 to 24 years old. PROCEDURE: Bronchoalveolar lavage fluid samples were obtained and analyzed. Forced oscillatory mechanics (1-7 Hz) technique was used for measurements of total respiratory system resistance (RRS), compliance (CRS), and resonant frequency (fres). Changes in RRS (1 Hz) during histamine challenge were used to generate histamine dose-response curves, from which the provocative concentrations that evoked a 75 or 100% increase in baseline RRS (PCRRS75 and PCRRS 100, respectively) were determined. Age, sex, baseline lung mechanics, and BAL cytologic findings were correlated with PCRRS75 and PCRRS100. RESULTS: No horse of the study had clinical signs or history of obstructive pulmonary disease or increased percentage (> 7%) of neutrophils in bronchoalveolar lavage fluid samples. Mean (+/- SEM) RRS, CRS, and fres were 0.67 +/- 0.06 cm of H2O/L/s, 0.52 +/- 0.04 L/cm H2O, and 2.46 +/- 0.02 Hz, respectively. There was no correlation between age or sex, and RRS, CRS, fres, PCRRS75, or PCRRS100. There was a significant correlation (rs = -0.78, P < 0.001) between percentage of BAL fluid mast cells and PCRRS75 or PCRRS100, but correlation with other cell types and indices of airway reactivity were not observed. CONCLUSION: The strong association between mast cell percentage in BAL fluid and airway reactivity in this group suggests that mast cell products may contribute to bronchospasm, airway wall thickening, and/or loss of elastic recoil, which underlie airway hyperreactivity. Alternatively, mast cells may contribute to nonspecific airway reactivity in horses through unknown mechanisms.     

Costing methods in the Canadian literature on the economic evaluation of health care. A survey and assessment

Administration of interleukin 2 (IL-2) has been associated with potent in vitro antitumor effects. However, systemic in vivo toxicity has been problematic. Because local delivery and liposomal formulations of IL-2 may improve the therapeutic index, we used dogs to evaluate and compare immunological activation of inhaled free IL-2 and IL-2 liposomes. Twelve normal dogs were treated with nebulized IL-2 formulations and controls for 2 to 7 weeks. Cellular immune activation of peripheral blood mononuclear cells and bronchoalveolar lavage (BAL) effector leukocytes against tumor cell lines, changes in effector leukocyte populations, and toxicity were monitored. No toxicity was seen with either aerosolized free IL-2 or IL-2 liposomes. Free IL-2 given at 0.5 x 10(6) Biologic Response Modifier Program (BRMP) units twice daily to dogs resulted in increased peripheral blood mononuclear cell activation compared with saline control-treated dogs. IL-2 liposomes given at 0.5 x 10(6) BRMP units twice daily to dogs resulted in significantly increased BAL effector activation compared with IL-2 liposomes given at 1.0 x 10(6) BRMP units once daily (P = 0.018) and empty liposome controls (P = 0.016). The BAL leukocyte cell count was increased significantly after inhalation of IL-2 liposomes versus inhalation of free IL-2 (P = 0.011). BAL effector populations included a greater proportion and total number of lymphocytes and eosinophils after treatment with IL-2 liposomes. Nontoxic activation of pulmonary immune effectors for the treatment of cancer in the lung may be possible using nebulized IL-2 liposomes.     

Effects of 0.05% chlorhexidine lavage on the tarsocrural joints of horses

In six horses, a 0.05% solution of chlorhexidine diacetate was used to lavage one tarsocrural joint; the contralateral control joint was lavaged with lactated Ringer's solution. Horses were evaluated daily for lameness. Synovial fluid samples were collected on days 1, 4, and 8 for determination of protein concentration, total and differential leukocyte counts, and mucin clot formation. After death on day 8, synovium and osteochondral samples were collected from the tarsocrural joints for examination of morphology and proteoglycan staining. Lavage with chlorhexidine solution caused lameness that was reduced but still evident at day 8. Synovial protein concentration was significantly increased by chlorhexidine lavage; the greatest increase occurred on day 1. Joint lavage increased synovial leukocyte counts on day 1, primarily by increasing polymorphonuclear (PMN) cell counts. Although total synovial leukocyte counts returned to normal by day 4, PMN cell counts remained elevated through day 8; PMN cell counts for chlorhexidine-lavaged joints were typically twice that of control joints. Chlorhexidine lavage caused synovial ulceration, inflammation, and abundant fibrin accumulation. Consistent differences in proteoglycan staining were not detected between control and chlorhexidine-lavaged joints. Joint lavage with 0.05% chlorhexidine diacetate, the lowest known bactericidal concentration, is not recommended for equine joints.     

Leukocyte counts in patients with Kawasaki disease: from the results of nationwide surveys of Kawasaki disease in Japan

In the latest survey of Kawasaki disease in Japan, questionnaires on maximal leukocyte counts of the patients were included to clarify whether leukocyte counts could be of value for the diagnosis and prediction of outcome. A questionnaire form and diagnostic guidelines for Kawasaki disease were sent to all pediatric departments of hospitals with > or = 100 beds throughout Japan, and information including maximal leukocyte counts was obtained on patients with Kawasaki disease diagnosed during the 2-y period from January 1993 to December 1994. Of the 11,458 patients diagnosed during the 2-y period, maximal leukocyte counts were reported in 11,062 patients (96.5%). The mean value and the distribution of maximal leukocyte counts were lower in the age group under 1 y. The mean values and the distribution of leukocyte counts were lowest in suspected cases among three diagnostic categories: typical cases of Kawasaki disease, atypical cases, and suspected cases. The mean values of maximal leukocyte counts of the patients with cardiac sequelae were significantly higher than those without cardiac sequelae in each age group. The proportion of patients with cardiac sequelae increased with leukocyte counts in each age group. The Receiver/Response Operating Characteristic (ROC) curve for maximal leukocyte counts in Kawasaki disease revealed that the accuracy of maximal leukocyte counts for prediction of cardiac sequelae was highest in the age group < 6 months, and the most accurate cut-off point was 16 x 10(9)/l. The strongest association between higher leukocyte counts (> or = 16 x 10(9)/l) and cardiac sequelae was observed in the age group < 6 M. A large-scale analysis of leukocyte counts in patients with Kawasaki disease revealed age-dependent relationship between maximal leukocyte counts, diagnostic categories and outcome. Maximal leukocyte counts may be helpful for the prediction of outcome with the consideration of age.     

Challenges posed by health care restructuring in Ontario

Gene therapy is becoming one of the most promising modalities for the treatment of acquired immunodeficiency syndrome. The purpose of this study was to investigate the mobilization and collection of peripheral blood progenitor cells from human immunodeficiency virus (HIV)-infected individuals using granulocyte colony-stimulating factor (G-CSF). A total of 10 patients (9 male, 1 female; median age 36.5 years) with varying circulating CD4+ cell counts (13.9-1467/microL) were administered 10 microg/kg G-CSF daily for 6 days. Peripheral white blood cells (WBCs), CD34+ cell counts, lymphocyte subsets, and plasma viremia were monitored before each G-CSF injection. An average sixfold increase in WBCs was observed, which stabilized on day 4 or thereafter. The level of CD34+ cells was increased by 20-fold, and did not differ between days 5 and 6. Smaller increases in CD4+, CD8+, and CD4+CD8+ cells were observed. HIV viral load, as measured by RNA copy number in plasma, was not significantly altered by G-CSF administration. The leukapheresis product (LP), collected on day 7, contained an average of 6.25+/-4.52 (mean +/- standard deviation) x 10(10) WBCs and 3.08+/-2.98 x 10(6) CD34+ cells/kg. The levels of different CD34+ cell subsets were similar to those in the LPs of G-CSF-mobilized healthy individuals from an earlier study. Primitive hematopoietic cells (CD38- and CD38-HLA-DR+ cells) were detected in LPs (1.19+/-0.46% and 0.87+/-0.23%, respectively, of CD34+ cells). All parameters (WBC counts, lymphocyte populations, CD34+ cells, and HIV-1 RNA copies) measured 3 weeks after leukapheresis returned to baseline values. The administration of G-CSF was well tolerated by the HIV patients; side effects included bone pain, headache, flulike symptoms, and fatigue. There were no correlations between baseline CD4+ cell count and the WBCs, mononuclear cells, or CD34+ cells collected in the LP. Similarly, no correlation existed between baseline CD4+ and CD34+ cells, peak CD34+ cells, or days to achieve peak CD34+ cell counts after G-CSF mobilization. Our results showed that: (1) maximal mobilization can be achieved after 4 days of G-CSF administration; (2) therapeutic quantities of hematopoietic cells can be collected and used for gene therapy; and (3) G-CSF administration is well tolerated and does not cause a clinically significant increase in viremia.     

Lead exposure causes generation of reactive oxygen species and functional impairment in rat sperm

The relationships between blood lead, sperm lead, sperm reactive oxygen species (ROS) level, and sperm fertile capability were investigated to understand the effects of lead exposure on sperm function and the mechanism of these effects. Male Sprague-Dawley rats, 7 weeks old, were randomly divided into control group and lead-treated group. The controls and lead-treated animals received intraperitoneal injection of 10 mg sodium acetate and 10 mg lead acetate/kg body weight, respectively, weekly for 6 or 9 weeks. The blood lead and epididymal sperm lead were analyzed by graphite furnace atomic absorption spectrophotometer. Chemiluminescence was measured to evaluate the generation of sperm ROS. Sperm-oocyte penetration rate (SOPR) was measured to evaluate sperm function. After 6 weeks of lead exposure, the rats had average blood lead levels of 32 microg/dl, sperm lead levels of 0.67 +/- 0.11 microg/10(9) sperm, unchanged epididymal sperm counts, percent of motile sperms, and motile epididymal sperm counts compared with control animals. However, after 9 weeks of lead exposure, the rats had average blood lead levels of 48.0 +/- 4.3 microg/dl, sperm lead levels of 0.88 +/- 0.16 microg/10(9) sperm, statistically lower epididymal sperm counts, and lower motile epididymal sperm counts. There was a good correlation between the blood lead and sperm lead(r2 = 0.946, P < 0.001). The sperms of lead-exposed rats produced significantly higher counts ofchemiluminescence than did those from the control rats (P < 0.001). The chemiluminescence counts were positively associated with sperm lead level (r2 = 0.613, P < 0.001). Epididymal sperm counts, motility and motile epididymal sperm counts were negatively associated with sperm chemiluminescence (r2 = 0.255, 0.152, and 0.299; P < 0.01, 0.05, and 0.01, respectively). The SOPR were positively associated with epididymal sperm counts, motility and motile epididymal sperm counts (r2 = 0.136, 0.285, and 0.264; P < 0.05, 0.01, and 0.001, respectively). The sperm chemiluminescence was negatively associated with SOPR (r2 = 0.519, P < 0.001). It is concluded that lead exposure probably affected the sperm function by activating one of the pathways of ROS generation.     

Bronchoalveolar lavage neutrophilia is associated with obliterative bronchiolitis after lung transplantation: role of IL-8

Obliterative bronchiolitis (OB) is a devastating complication in lung transplantation. We postulated that the pathogenesis of OB is mediated, in part, by neutrophils. We serially collected bronchoalveolar lavage (BAL) fluid from lung transplant recipients. Patients were divided into two groups depending on the presence or absence of OB. Samples from patients who never developed OB were further divided according to whether rejection was present. These samples were labeled healthy or rejection. Samples from patients who developed OB were divided according to whether the sample was obtained before (future OB) or at the time of diagnosis of OB (OB). The OB group, as compared with the healthy and rejection group, had significantly elevated neutrophil counts (3.9 x 10(5) +/- 1.8 x 10(5) vs 0.3 x 10(5) +/- 0.07 x 10(5) and 0.4 x 10(5) +/- 0.1 x 10(5), respectively, p < 0.01 for both) and levels of IL-8 (3131 +/- 1468 pg/ml vs 240 +/- 62 pg/ml and 172 +/- 47 pg/ml, p < 0.01 for both). Furthermore, we demonstrated immunolocalization of IL-8 associated with alpha smooth muscle actin-positive cells in the peribronchial region of OB. To confirm that the IL-8 present in BAL fluid from patients with OB was bioactive, we performed neutrophil chemotaxis experiments that showed that IL-8 accounted for a significant amount of the neutrophil chemotactic activity. We also found a trend toward higher levels of neutrophils and IL-8 in BALs from the future OB as compared with the healthy group (7.1 x 10(4) +/- 4.2 x 10(4) vs 3.4 x 10(4) +/- 0.7 x 10(4) and 500 +/- 306 pg/ml vs 240 +/- 62 pg/ml). In conclusion, we have provided the novel observation that in lung transplant recipients with OB, neutrophilia is present and highly correlated with the presence of IL-8.     

The role of neutrophils in the pathogenesis of idiopathic pulmonary fibrosis

STUDY OBJECTIVES: The prognostic value of the neutrophil count in BAL fluid (BALF) has been controversial. The role of neutrophils in this inflammatory lung disease, therefore, was evaluated in this study by additional measures. MATERIALS AND METHODS: We performed BAL in 22 patients with idiopathic pulmonary fibrosis (IPF) diagnosed by open lung biopsy specimen. Percent polymorphonuclear leukocyte (PMN) in BALF and absolute neutrophil counts were compared with those of normal nonsmokers. Elastase complexed to alpha-1-proteinase inhibitor (alpha1-PI) in plasma and BALF was measured as a marker of elastase burden, and neutrophil distribution in 22 lung tissues was observed by immunohistochemistry using antineutrophil elastase antibody. RESULTS: Percent PMN and absolute neutrophil counts in BALF did not increase in patients with IPF as compared with normal nonsmokers (n=15); the plasma elastase-alpha1-PI complex value (mean+/-SE) of patients with IPF (668.5+/-112.4 ng/mL) was significantly high as compared with that of normal nonsmokers (130.3+/-21.3, p<0.001). In addition, the BALF elastase-alpha1-PI complex value (mean+/-SE) of patients with IPF was also significantly high (333.1+/-87.0 ng/mg albumin) as compared with that of normal nonsmokers (83.1+/-29.3 ng/mg albumin, p<0.05). Immunohistochemistry demonstrated considerable numbers of neutrophils infiltrating the lung parenchyma in biopsy specimens obtained by open lung biopsy. CONCLUSIONS: These results suggested that although the neutrophil count in BALF could not represent the distribution of neutrophil in the lung, high levels of neutrophil elastase were demonstrated in lung parenchyma and also in both BALF and sera. Therefore, neutrophils might indeed play an important role in the pathogenesis of IPF.     

The effect of salmeterol on nocturnal symptoms, airway function, and inflammation in asthma

STUDY OBJECTIVE: To determine the efficacy of salmeterol alone in a group of patients with moderate asthma with nocturnal worsening of symptoms. DESIGN: Double-blind, randomized, placebo-controlled crossover study. SETTING: Tertiary care hospital specializing in respiratory diseases. PARTICIPANTS: Ten patients with nocturnal asthma. INTERVENTIONS: Subjects were randomized to salmeterol, 100 micrograms twice daily, or placebo for 6 weeks with a 1-week washout between treatment periods. Symptoms, nocturnal awakenings, and beta 2-agonist use were recorded daily. Spirometry was performed at weeks 1 and 6 of each period at bedtime and at 4 AM, and methacholine challenge was performed at 4 AM followed by bronchoscopy with BAL. BAL fluid analysis included cell count and differential count, eosinophil cationic protein, Charcot-Leyden crystal protein, leukotriene B4, and thromboxane B2. RESULTS: The percentage of nights with awakenings decreased significantly with salmeterol (69.8 +/- 8.7% vs 30.6 +/- 10.8% for placebo and salmeterol, respectively; p = 0.02). The percentage of 24-h days with supplemental inhaled beta 2-agonist use significantly decreased with salmeterol (85.9 +/- 9.4% vs 70.4 +/- 10.1% for placebo and salmeterol, respectively; p = 0.04). There were no significant differences in bronchial reactivity, 4 AM FEV1, overnight percentage change in FEV1, or indexes of airway inflammation. CONCLUSIONS: Salmeterol alone improves the number of nocturnal awakenings and supplemental 24-h beta 2-agonist use in nocturnal asthma without significantly altering lung function and airway inflammation.     

Extreme leukoreduction of major histocompatibility complex class II positive B cells enhances allogeneic platelet immunity

In a murine model of platelet alloimmunization, we examined the definitive role that mononuclear cells (MC) have in modulating platelet immunity by using platelets from severe combined immunodeficient (SCID) mice. CB.17 (H-2(d)) SCID or BALB/c (H-2(d)) mouse platelets were transfused weekly into fully allogeneic CBA (H-2(k)) mice and antidonor antibodies measured by flow cytometry. MC levels in BALB/c platelets were 1.1 +/- 0.6/microL and SCID mouse platelets could be prepared to have significantly lower (<0. 05/microL) MC numbers. Transfusions with 10(8) BALB/c platelets (containing approximately 100 MC/transfusion) stimulated IgG antidonor antibodies in 100% of the recipients by the fifth transfusion, whereas 10(8) SCID mouse platelets (containing approximately 5 MC/transfusion) stimulated higher-titered IgG alloantibodies by the second transfusion. When titrations of BALB/c peripheral blood MC were added to the SCID mouse platelets, levels approaching 1 MC/microL reduced SCID platelet immunity to levels similar to BALB/c platelets. Characterization of the alloantibodies showed that the low levels of MC significantly influenced the isotype of the antidonor IgG; the presence of 1 MC/microL was associated with induction of noncomplement fixing IgG1 antidonor antibodies, whereas platelet transfusions, devoid of MC (<0. 05/microL), were responsible for complement-fixing IgG2a production. When magnetically sorted defined subpopulations of MC were added to the SCID platelets, major histocompatability complex (MHC) class II positive populations, particularly B cells, were found to be primarily responsible for the reduced SCID mouse platelet immunity. The presence of low numbers of MC within the platelets was also associated with an age-dependent reduction in platelet immunogenicity; this relationship however, was not observed with SCID mouse platelets devoid of MC. The results suggest that a residual number of MHC class II positive B cells within allogeneic platelets are required for maximally reducing alloimmunization.     

Overexpression of leukotriene C4 synthase in bronchial biopsies from patients with aspirin-intolerant asthma

Aspirin causes bronchoconstriction in aspirin-intolerant asthma (AIA) patients by triggering cysteinyl-leukotriene (cys-LT) production, probably by removing PGE2-dependent inhibition. To investigate why aspirin does not cause bronchoconstriction in all individuals, we immunostained enzymes of the leukotriene and prostanoid pathways in bronchial biopsies from AIA patients, aspirin-tolerant asthma (ATA) patients, and normal (N) subjects. Counts of cells expressing the terminal enzyme for cys-LT synthesis, LTC4 synthase, were fivefold higher in AIA biopsies (11.5+/-2.2 cells/mm2, n = 10) than in ATA biopsies (2.2+/-0.7, n = 10; P = 0. 0006) and 18-fold higher than in N biopsies (0.6+/-0.4, n = 9; P = 0. 0002). Immunostaining for 5-lipoxygenase, its activating protein (FLAP), LTA4 hydrolase, cyclooxygenase (COX)-1, and COX-2 did not differ. Enhanced baseline cys-LT levels in bronchoalveolar lavage (BAL) fluid of AIA patients correlated uniquely with bronchial counts of LTC4 synthase+ cells (rho = 0.83, P = 0.01). Lysine-aspirin challenge released additional cys-LTs into BAL fluid in AIA patients (200+/-120 pg/ml, n = 8) but not in ATA patients (0. 7+/-5.1, n = 5; P = 0.007). Bronchial responsiveness to lysine-aspirin correlated exclusively with LTC4 synthase+ cell counts (rho = -0.63, P = 0.049, n = 10). Aspirin may remove PGE2-dependent suppression in all subjects, but only in AIA patients does increased bronchial expression of LTC4 synthase allow marked overproduction of cys-LTs leading to bronchoconstriction.     

Effect of exposure of guinea pigs to cigarette smoke on elastolytic activity of pulmonary macrophages

STUDY OBJECTIVE: To determine the effect of exposure to cigarette smoke on the elastolytic activity of guinea pigs' alveolar macrophages (AMs), and to compare elastolytic activity of AMs obtained by BAL with that of lung macrophages (LMs) obtained from minced lung tissue. METHODS: AMs were obtained by BAL from seven adult guinea pigs exposed to cigarette smoke for 5 d/wk during 6 weeks, as well as from age-matched control guinea pigs. From each animal, one lung was used to obtain LMs by mincing and teasing the lung, followed by enzymatic digestion and isolation of mononuclear cells by Hypaque-Ficoll separation. The other lung was inflated and fixed to quantitate emphysema by the destructive index (DI). Elastolytic activity (microgram of elastin degraded by 10(6) macrophages) was determined at 24, 48, and 72 h, by culturing AMs and LMs (1 x 10(6) cells in 1 mL of medium) in 3H-elastin-coated wells. RESULTS: In animals exposed to cigarette smoke, the total number of BAL cells (8.6+/-2.1 x 10(6)) and DI (21.8+/-8.1) were significantly higher than in nonexposed animals (6.4+/-1.8 x 10(6), p<0.05 for cells, and 12.1+/-4.1, p<0.01 for DI). Elastolytic activity of AMs from smoke-exposed guinea pigs was significantly higher at 24, 48, and 72 h than elastolytic activity of AMs from control animals (19.0+/-9.4 vs 10.0+/-5.3, p<0.05 at 72 h). Likewise, elastolytic activity of LMs was significantly higher in exposed than nonexposed guinea pigs (11.8+/-7.7 vs 7.4+/-5.0 at 72 h, p<0.05). Elastolytic activity of LMs was not significantly different from elastolytic activity of AMs, both in exposed guinea pigs (11.8+/-7.7 vs 19.0+/-9.4 at 72 h) and nonexposed animals (7.4+/-5.0 vs 10.0+/-5.3 at 72 h). CONCLUSIONS: These results indicate that elastolytic activity of both AMs and LMs of guinea pigs increases significantly after exposure to cigarette smoke and that AMs and LMs have similar elastolytic activities.     

Impact of Rantes and MCP-1 chemokines on in vivo basophilic cell recruitment in rat skin injection model and their role in modifying the protein and mRNA levels for histidine decarboxylase

RANTES and related molecules, constitute the C-C class of chemokine supergene family and a group of cytokines produced by hematopoietic cells constitute the MCP-1 or C-X-C class. The roles of most of these chemokines are not well known, although members of the C-X-C family are inflammatory agents. Here, we report that intradermal injection of RANTES 10 ng/50 microL subcutaneously in the abdominal skin produced a strong inflammatory reaction, as evidenced by Evans blue dye, greater than FMLP (10(-6) mol/L) (approximately 57%); while MCP-1, 10 ng/50 microL was less effective than FMLP (10(-6) mol/L) (approximately 54%). Moreover, the histologic analysis of the cells stained with Toluidine blue (0.1%) were analyzed at a magnification of x40). RANTES 10 ng/50 microL and LPS produced higher numbers (142 +/- 11 and 193 +/- 21 of cells/200 mm2, respectively) of basophilic cell accumulation in the skin injection sites compared with FMLP (10(-6) mol/L) (127 +/- 14/200 mm2), while MCP-1 10 ng/50 microL was less effective (88 +/- 10/200 mm2). Electron microscopy (x13,800) studies of skin injection sites revealed that RANTES was chemoattractant for mast cells. In a Northern blot analysis from homogeneous tissue biopsy from the intradermal injection sites, RANTES was more potent than MCP-1 in increasing histidine decarboxylase (HDC) mRNA, the sole enzyme responsible for the production of histamine from histidine. Since PGD2 is formed by mast cells on cell activation, we also studied the effect of RANTES and MCP-1 on PGD2 production in inflamed tissue in vivo. RANTES (20, 10, and 5 ng) and MCP-1 (20, 10, and 5 ng) strongly stimulated PGD2, in a dose-dependent manner, with a potency rank order of RANTES (10 ng/mL) approximately two times greater than MCP-1 (10 ng/mL).     

Child feeding and care behaviors are associated with xerophthalmia in rural Nepalese households

OBJECTIVE: To compare the blood-pressure-lowering effects of an angiotensin converting enzyme inhibitor, perindopril, with those of an angiotensin II type 1 receptor antagonist, L-158,809, for adult spontaneously hypertensive rats. DESIGN: A cross-over design was used, to treat adult spontaneously hypertensive rats with one drug for 10 weeks, and then with the other for 5 weeks. METHODS: Adult, male spontaneously hypertensive rats (aged 15 weeks) were treated daily by gavage for 10 weeks with perindopril (P group) or L-158,809 (L group), then treatment was crossed over so that rats in the P group were treated with L-158,809 (P/L group) and rats in the L group were treated with perindopril (L/P group) for 5 weeks. Blood pressure was measured weekly. Plasma angiotensin converting enzyme activity, renal angiotensin receptor density, and arterial structure and functioning were measured after the single and crossover treatment periods. RESULTS: Treatment lowered the blood pressure from 206 +/- 2 mmHg in rats in the control group, to 126 +/- 2 in rats in the P group and 150 +/- 2 in rats in the L group. After the cross-over period, blood pressure decreased further from 150 +/- 2 to 129 +/- 3 mmHg in rats in the L/P group, whereas blood pressure of spontaneously hypertensive rats in the P/L group increased from 126 +/- 2 to 148 +/- 2 mmHg. Perindopril treatment almost abolished plasma angiotensin converting enzyme activity, whereas L-158,809 treatment had no effect. Renal angiotensin II receptor density was decreased versus baseline in rats in the P and L groups. The affinity of binding was decreased versus baseline in rats in the L group. A positive correlation to blood pressure was found for mesenteric artery wall thickness and wall: lumen ratio. Concentration for half-maximal effect for the response of mesenteric arteries from rats in the P group to norepinephrine was lower than that of the control group rats. Angiotensin II potentiated the norepinephrine-stimulated contraction of arteries from rats in the control and P groups, but not that of arteries from rats in the groups treated with L-158,809. CONCLUSION: Perindopril was more effective than was L-158,809 at lowering the blood pressure of adult spontaneously hypertensive rats, and at altering the structure and functioning of the arteries.     

Poststorage diastolic abnormalities of heart transplants: is vascular dysfunction or myocardial contracture the culprit?

We tested the hypothesis that mast cells contribute to platelet-activating factor (PAF)-induced airways hyperreactivity and hyperpermeability in mice. Airways reactivity to acetylcholine (ACh) and lung permeability to Evans blue (EB) dye were measured before and after PAF challenge in genetically mast cell-deficient (WBB6F1 W/Wv) and normal congenic (WBB6F1 +/+) mice, as well as mast cell-reconstituted (BMT W/Wv) mice. In addition, prostaglandin D2 (PGD2), a mast cell-specific mediator, was measured in the bronchoalveolar lavage (BAL) from +/+ and W/Wv mice to determine if lung mast cell activation was a consequence of PAF challenge. Genetically PAF-sensitive AKR/J mice were also treated with the mast cell stabilizer nedocromil prior to assessment of PAF effects on ACh reactivity. Intravenous PAF (10 micrograms/kg) induced a significant (P < 0.05) increase in airways reactivity to ACh (25 micrograms/kg) in both +/+ (371 +/- 52%) and W/Wv (122 +/- 24%) mice. There was a significantly greater increase in +/+ compared with W/Wv mice. PAF-induced hyperreactivity to ACh in BMT W/Wv mice (191 +/- 44%) was significantly (P < 0.05) greater than age-matched W/Wv mice (80 +/- 16%), but not significantly different from age-matched +/+ mice (153 +/- 44%). PAF (10 micrograms/kg) also significantly (P < 0.5) increased lung permeability in +/+ and W/Wv mice, but there was no significant difference between groups. BAL PGD2 increased significantly in +/+ mice following PAF challenge (559 +/- 24 ng/ml) compared with vehicle controls (152 +/- 8 pg/ml). There was no significant increase in BAL PGD2 from W/Wv mice. Nedocromil pretreatment significantly (P < 0.05) decreased PAF-induced hyperreactivity in AKR/J mice but not in W/Wv mice (P > 0.05). We conclude that mast cells contribute significantly to PAF-induced hyperreactivity but not hyperpermeability in mice.