Platelets are specialized hemostatic cells that circulate in the blood as anucleate cytoplasts. We report that platelets unexpectedly possess a functional spliceosome, a complex that processes pre-mRNAs in the nuclei of other cell types. Spliceosome components are present in the cytoplasm of human megakaryocytes and in proplatelets that extend from megakaryocytes. Primary human platelets also contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. In response to integrin engagement and surface receptor activation, platelets precisely excise introns from interleukin-1beta pre-mRNA, yielding a mature message that is translated into protein. Signal-dependent splicing is a novel function of platelets that demonstrates remarkable specialization in the regulatory repertoire of this anucleate cell. While this mechanism may be unique to platelets, it also suggests previously unrecognized diversity regarding the functional roles of the spliceosome in eukaryotic cells.

After budding, the human immunodeficiency virus (HIV) must 'mature' into an infectious viral particle. Viral maturation requires proteolytic processing of the Gag polyprotein at the matrix-capsid junction, which liberates the capsid (CA) domain to condense from the spherical protein coat of the immature virus into the conical core of the mature virus. We propose that upon proteolysis, the amino-terminal end of the capsid refolds into a β-hairpin/helix structure that is stabilized by formation of a salt bridge between the processed amino-terminus (Pro1) and a highly conserved aspartate residue (Asp51). The refolded amino-terminus then creates a new CA-CA interface that is essential for assembling the condensed conical core. Consistent with this model, we found that recombinant capsid proteins with as few as four matrix residues fused to their aminotermini formed spheres in vitro, but that removing these residues refolded the capsid amino-terminus and redirected protein assembly from spheres to cylinders. Moreover, point mutations throughout the putative CA-CA interface blocked capsid assembly in vitro, core assembly in vivo and viral infectivity. Disruption of the conserved amino-terminal capsid salt bridge also abolished the infectivity of Moloney murine leukemia viral particles, suggesting that lenti-and oncoviruses mature via analogous pathways.

Neutrophils are highly specialized innate effector cells that have evolved for killing of pathogens. Human neonates have a common multifactorial syndrome of neutrophil dysfunction that is incompletely characterized and contributes to sepsis and other severe infectious complications. We identified a novel defect in the antibacterial defenses of neonates: inability to form neutrophil extracellular traps (NETs). NETs are lattices of extracellular DNA, chromatin, and antibacterial proteins that mediate extracellular killing of microorganisms and are thought to form via a unique death pathway signaled by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-generated reactive oxygen species (ROS). We found that neutrophils from term and preterm infants fail to form NETs when activated by inflammatory agonists-in contrast to leukocytes from healthy adults. IntroductionPolymorphonuclear leukocytes (PMNs, neutrophils) are highly specialized cellular effectors in host defense and immune surveillance. Mature human PMNs from healthy adults have a unique repertoire of activities, including phagocytosis, degranulation of antimicrobial enzymes and peptides, and generation of oxygen radicals with antimicrobial properties. 1-6 Synthesis of inflammatory and regulatory lipids and proteins complements these innate mechanisms. 1,4,5 PMNs have evolved for capture, containment, and destruction of bacteria and fungi and also have activity against intracellular pathogens and viruses. 2,3 PMNs have additional important roles in tissue repair and integration of innate and adaptive immune responses. 6 If, however, these specialized defensive mechanisms become dysregulated or unregulated, PMNs can paradoxically be mediators of inflammatory tissue injury. 1,6 Consistent with their requisite activities in host defense, defects in PMN functions cause immune deficiency syndromes. 2,7 Neutrophil defects can be hereditary, developmental, or acquired in nature. Specific genetic deficiencies in PMN function cause significant morbidity in subsets of children and adults and, in parallel, provide unique insights into molecular mechanisms that regulate leukocyte activities. 7,8 Nevertheless, these disorders are rare and arcane. In contrast, the developmental syndrome of neonatal neutrophil dysfunction, which is particularly important in premature infants, is common and contributes to infections in infants worldwide. As an example, neonatal PMN dysfunction is thought to be a pivotal feature of sepsis in the newborn. 9-11 The incidence of neonatal sepsis is estimated to be 1 to 5 cases per 1000 live births in the United States and to be even higher after very low birth weight premature deliveries (15-19/1000); in contrast, the incidence of sepsis is much lower in children older than 1 year of age and in young adults. 12-15 Furthermore, the incidence of neonatal sepsis is as high as 25% in some areas of the developing world. 16,17 Thus, neonatal PMN dysfunction is a contributor to a public health problem of significant proportions, and also may pr...

Although intravenous Escherichia coli endotoxin has been used extensively in experimental studies to increase lung endothelial permeability, the effect of E. coli endotoxin on lung epithelial permeability has not been well studied. To examine this issue in sheep, bidirectional movement ofprotein across the lung epithelial barrier was studied by labeling the vascular space with 13"I-albumin and-by instilling 3 ml/kg of an isosmolar protein solution with`-I-albumin into the alveoli. E. coli endotoxin was administered according to one of three protocols: intravenous alone (5-500 ug/kg), intravenous (5 pg/kg) plus low-dose alveolar endotoxin (10 pg/kg), and high-dose alveolar endotoxin alone (50-100 pg/kg). Alveolar liquid clearance was estimated based on the concentration of the instilled native protein.Sheep were studied for either 4 or 24 h. Although intravenous E. coli endotoxin produced a marked increase in transvascular protein flux and interstitial pulmonary edema, there was no effect on the clearance of either the vascular ("3'I-albumin) or the alveolar (1251-albumin) protein tracer across the epithelial barrier. High-dose alveolar E. coil endotoxin caused a 10-fold increase in the number of leukocytes, particularly neutrophils, that accumulated in the air spaces. In spite of the marked chemotactic effect of alveolar endotoxin, there was no change in the permeability of the epithelial barrier to the vascular or alveolar protein tracers. Also, alveolar epithelial liquid clearance was normal. Morphologic studies confirmed that the alveolar epithelial barrier was not injured by either intravenous or alveolar E. coli endotoxin. Thus, the alveolar epithelium in sheep is significantly more resistant than the lung endothelium to the injurious effects of E. coli endotoxin. (J. Clin. Invest. 1991. 88:864-875.) Key words: E. coli endotoxin * alveolar epithelium -lung endothelium * alveolar liquid clearance * acute lung injury

Apoptosis mediated by Fas/Fas ligand (FasL) interaction has been implicated in human disease processes, including pulmonary disorders. However, the role of the Fas/FasL system in acute lung injury (ALI) and in the acute respiratory distress syndrome (ARDS) is poorly defined. Accordingly, we investigated both the soluble and cellular expression of the Fas/FasL system in patients with ALI or ARDS. The major findings are summarized as follows. First, the soluble expression of the Fas/FasL system was assessed in undiluted pulmonary edema fluid and simultaneous plasma. Pulmonary edema fluid obtained from patients with ALI or ARDS (n = 51) had significantly higher concentrations of both soluble Fas (27 ng/ml; median; P < 0.05) and soluble FasL (0.125 ng/ml; P < 0.05) compared to control patients with hydrostatic pulmonary edema (n = 40; soluble Fas, 12 ng/ml; soluble FasL, 0.080 ng/ml). In addition, the concentrations of both soluble Fas and soluble FasL were significantly higher in the pulmonary edema fluid of the patients with ALI or ARDS compared to simultaneous plasma samples (soluble Fas, 16 ng/ml; soluble FasL, 0.058 ng/ml; P < 0.05), indicating local release in the lung. Higher soluble Fas concentrations were associated with worse clinical outcomes. Second, cellular expression of the Fas/FasL system was assessed by semiquantitative immunofluorescence microscopy in lung tissue obtained at autopsy from a different set of patients. Both Fas and FasL were immunolocalized to a greater extent in the patients who died with ALI or ARDS (n = 10) than in the patients who died without pulmonary disease (n = 10). Both proteins were co-expressed by epithelial cells that lined the alveolar walls, as well as by inflammatory cells and sloughed epithelial cells that were located in the air spaces. Semiquantitative immunohistochemistry showed that markers of apoptosis (terminal dUTP nick-end labeling, caspase-3, Bax, and p53) were more prevalent in alveolar wall cells from the patients who died with ALI or ARDS compared to the patients who died without pulmonary disease. These findings indicate that alveolar epithelial injury in humans with ALI or ARDS is in part associated with local up-regulation of the Fas/FasL system and activation of the apoptotic cascade in the epithelial cells that line the alveolar air spaces.

Elastin is a major component of the mammalian lung, predominantly found in the alveoli. Destruction of alveolar elastic fibers is implicated in the pathogenic mechanism of emphysema in adults. These data define a role for elastin in the structure and function of the mature lung, and suggest that elastin is important for alveogenesis. To investigate the role of elastin in lung development, we examined mice lacking elastin (Eln-/-). At birth, the distal air sacs of Eln-/- lungs dilate to form abnormally large cavities. This phenotype appears before the synthesis and deposition of alveolar elastin, a process mediated by myofibroblasts and initiated after postnatal Day 4. Morphometric analyses demonstrate that the perinatal development of terminal airway branches is arrested in Eln-/- mice. The branching defect is accompanied by fewer distal air sacs that are dilated with attenuated tissue septae, a condition reminiscent of emphysema. Elastin expression in the lung parenchyma before alveogenesis is localized to the mesenchyme surrounding the developing airways, supporting a role for elastin in airway branching. Thus, in addition to its role in the structure and function of the mature lung, elastin is essential for pulmonary development and is important for terminal airway branching.

Nitric oxide (NO) serves multiple functions in the developing lung, and pulmonary NO production is decreased in a baboon model of chronic lung disease (CLD) after premature birth at 125 days (d) gestation (term = 185d). To determine whether postnatal NO administration alters the genesis of CLD, the effects of inhaled NO (iNO, 5 ppm) were assessed in the baboon model over 14d. iNO caused a decrease in pulmonary artery pressure in the first 2d and a greater rate of spontaneous closure of the ductus arteriosus, and lung compliance was greater and expiratory resistance was improved during the first week. With iNO, postmortem pressure-volume curves were shifted upward, lung DNA content and cell proliferation were increased, and lung growth was preserved to equal that which occurs during the same period in utero. In addition, the excessive elastin deposition characteristic of CLD was normalized by iNO, and there was evidence of stimulation of secondary crest development. Thus, in the baboon model of CLD, iNO improves early pulmonary function and alters lung growth and extracellular matrix deposition. As such, NO biosynthetic pathway dysfunction may contribute to the pathogenesis of CLD.

The cardioprotective effects of an mAb to P-selectin designated mAb PB1.3 was examined in a feline model of myocardial ischemia (MI) and reperfusion. PB1.3 (1 mg/kg), administered after 80 min of ischemia (i.e., 10 min before reperfusion), significantly attenuated myocardial necrosis compared to a nonblocking mAb (NBP1.6) for P-selectin (15±3 vs 35±3% of area at risk, P < 0.01). Moreover, endothelial release of endothelium derived relaxing factor, as assessed by relaxation to acetylcholine, was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with mAb PB1.3 compared to mAb NBP1.6 (67±6 vs 11±3, P < 0.01). This endothelial preservation was directly related to reduced endothelial adherence of PMNs in ischemic-reperfused coronary arteries. Immunohistochemical localization of P-selectin was significantly upregulated in the cytoplasm of endothelial cells that lined coronary arteries and veins after 90 min of ischemia and 20 min of reperfusion. The principal site of intracytoplasmic expression was in venous vessels. mAb PB1.3 significantly decreased (P < 0.01 ) adherence of unstimulated PMNs to thrombin and histamine stimulated endothelial cells in a concentration-dependent manner in vitro. These results demonstrate that PMN adherence to endothelium by P-selectin is an important early consequence of reperfusion injury, and a specific monoclonal antibody to P-selectin exerts significant endothelial preservation and cardioprotection in myocardial ischemia and reperfusion. (J. Clin. Invest. 1993. 91:2620-2629