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1. The alveolocapillary membrane faces an extraordinary task in partitioning the plasma and lung hypophase proteins, with a surface area approximately 50-fold that of the body and only 0.1–0.2 μm thick. 2. Lung permeability is compromised under a variety of circumstances and the delineation between physiological and pathological changes in permeability is not always clear. Although the tight junctions of the epithelium, rather than the endothelium, are regarded as the major barrier to fluid and protein flux, it is becoming apparent that the permeability of both are dynamically regulated. 3. Whereas increased permeability and the flux of plasma proteins into the alveolar compartment has dire consequences, fortuitously the flux of surfactant proteins from the airspaces into the circulation may provide a sensitive means of non- invasively monitoring the lung, with important implications for treatment modalities. 4. Surfactant proteins are unique in that they are present in the alveolar hypophase in high concentrations. They diffuse down their vast concentration gradients (approximately 1:1500–7000) into the circulation in a manner that reflects lung function and injury score. Surfactant proteins vary markedly in size (approximately 20–650 kDa) and changes in the relative amounts appear particularly diagnostic with regard to disease severity. Alveolar levels of surfactant proteins remain remarkably constant despite respiratory disease and, unlike the flux of plasma proteins into the alveolus, which may reach equilibrium in acute lung injury, the flux of surfactant proteins is unidirectional because of the concentration gradient and because they are rapidly cleared from the circulation. 5. Ultimately, the diagnostic usefulness of surfactant proteins as markers of alveolocapillary permeability will demand a sound understanding of their kinetics through the vascular compartment. [ABSTRACT FROM AUTHOR]

Cell stretch stimulates both surfactant and cytokine production. The authors proposed that stretch, through these effects, modifies the pathogenesis of lipopolysaccharide-induced acute lung injury (ALI), and that this is CO(2) dependent. Rat alveolar type II cells and macrophages were co-cultured with lipopolysaccharide in 5%, 10%, or 20% CO(2) +/- stretch (30%, 60 cycles/min) for 6 hours. Intracellular TNF-alpha and IL-6 increased whereas secreted cytokine and surfactant decreased with increasing CO(2). Stretch independently increased intracellular TNF-alpha and decreased IL-6 secretion. Elevated CO(2) may therefore diminish secretion of proinflammatory cytokines by alveolar cells, contributing to an explanation for protective hypercapnia in ALI.

Objective: Low tidal volume (V(T)) ventilation strategies may be associated with permissive hypercapnia, which has been shown by ex vivo and in vivo studies to have protective effects. We hypothesized that hypercapnic acidosis may be synergistic with low V(T) ventilation; therefore, we studied the effects of hypercapnia and V(T) on unstimulated and lipopolysaccharide-stimulated isolated perfused lungs., Materials and Methods: Isolated perfused rat lungs were ventilated for 2 hours with low (7 mL/kg) or moderately high (20 mL/kg) V(T) and 5% or 20% CO(2), with lipopolysaccharide or saline added to the perfusate., Results: Hypercapnia resulted in reduced pulmonary edema, lung stiffness, tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in the lavage and perfusate. The moderately high V(T) did not cause lung injury but increased lavage IL-6 and perfusate IL-6 as well as TNF-alpha. Pulmonary edema and respiratory mechanics improved, possibly as a result of a stretch-induced increase in surfactant turnover. Lipopolysaccharide did not induce significant lung injury., Conclusions: We conclude that hypercapnia exerts a protective effect by modulating inflammation, lung mechanics, and edema. The moderately high V(T) used in this study stimulated inflammation but paradoxically improved edema and lung mechanics with an associated increase in surfactant release.

Ventilatory-induced strain can exacerbate acute lung injury (ALI). Current ventilation strategies favour low tidal volumes and high end-expiratory volumes to 'rest' the lung, but can lead to an increase in CO2. Alveolar macrophages (AM) play a pivotal role in ALI through the release of inflammatory mediators. The effect of physical strain and CO2 on the release of pro-inflammatory mediators was examined in isolated rat AM. AM were cultured on IgG-coated silastic membranes with or without lipopolysaccharide (LPS) and 5% or 20% CO2 and subjected to a repetitive sinusoidal mechanical strain (30%, 60 cycles/min) for 4 h. Cell viability and metabolic activity were assessed. In both the presence and absence of LPS, physical strain increased metabolic activity by approximately 5%, while 20% CO2 decreased metabolic activity by approximately 40%. Twenty per cent CO2 decreased TNF-alpha secretion by approximately 45%, without affecting cell viability. Physical strain enhanced LPS-induced secretion of TNF-alpha by 1.5%, but not IL-6 or CINC-1. Hence, the effects of both CO2 and physical strain are mediated independently through changes in AM metabolic activity. Physical strain is not a major determinant of TNF-alpha, IL-6 or CINC-1 in AM. Our results confirm that high CO2 can lessen the TNF-alpha inflammatory response of AM.

1. As a result of its enormous surface area and necessary thinness for gas exchange, the alveolocapillary barrier is vulnerable to mechanical disruption from raised pulmonary microvascular pressure (Pmv). 2. Because surfactant protein-B (SP-B) leaks into the blood stream from the alveoli in response to alveolocapillary barrier damage and exercise leads to increased Pmv, we sought to determine whether exercise results in increased plasma SP-B. Moreover, in the setting of exercise-induced left ventricular dysfunction, the consequent increase in left heart filling pressure and, therefore, P(mv) would be expected to further increase plasma SP-B levels. 3. Twenty consecutive subjects referred for treadmill exercise stress echocardiography (ESE) had venous blood sampled immediately before and after ESE for batch atrial natriuretic peptide (ANP) and SP-B assay. Echocardiographic measures of pulmonary haemodynamics (pulmonary artery flow acceleration time (pafAT) and right ventricular outflow tract velocity time integral (rVTI)) were also taken pre- and post-exercise. 4. Although circulating ANP levels increased following exercise (P < 0.001), there was no change in circulating SP-B levels in the entire cohort. 5. Ten subjects had a positive ESE for ventricular dysfunction. Although circulating ANP was increased post-exercise in both the negative and positive ESE groups (P < 0.05 and P < 0.01, respectively), circulating SP-B only increased post-exercise in the positive ESE group (P < 0.05). Echocardiographic parameters supported an increment in P(mv) in the cohort with exercise-induced left ventricular dysfunction because this group had an increase in pafAT (P < 0.05; reflecting pulmonary artery pressure) and no change in rVTI. 6. Physical exertion associated with a Bruce protocol ESE is insufficient to increase circulating SP-B, despite evidence of increased left atrial and pulmonary vascular pressure. However, in the setting of exercise-induced myocardial dysfunction, there is a detectable increase in circulating SP-B. 7. The exaggerated increase in pulmonary vascular pressure in exercise-induced myocardial dysfunction may result in increased SP-B leakage from the alveoli into the circulation by altering the integrity of the alveolocapillary barrier to protein.

Alveolar macrophages (AM) may be exposed to a range of CO(2) and pH levels depending on their location in the alveoli and the health of the lung. Cytokines produced by AM contribute to inflammation in acute lung injury (ALI). Current ventilatory practices for the management of ALI favor low tidal volumes, which can give rise to increases in CO(2) and changes in pH of the alveolar microenvironment. Here we examined the effect of CO(2) on cytokine release from LPS-stimulated rat AM. AM were incubated for 1-4 h under different atmospheric gas mixtures ranging from 2.5-20% CO(2). To distinguish between effects of pH and CO(2), the culture media were also buffered to pH 7.2 with NaHCO(3). Cell metabolic activity, but not cell viability, decreased and increased significantly after 4 h at 20 and 2.5% CO(2), respectively. Increasing CO(2) decreased TNF-alpha secretion but had no effect on lysate TNF-alpha. Buffering the media abated the effects of CO(2) on TNF-alpha secretion. CO(2) increased cytokine-induced neutrophil chemoattractant factor-1 secretion only when the pH was buffered to 7.2. Effects of CO(2) on cytokine responses were reversible. In conclusion, the effects of CO(2) on cytokine lysate levels and/or secretion in AM are cytokine specific and, depending on both the cytokine and the immediate microenvironment, may be beneficial or detrimental to ALI.

Background: In chronic heart failure (CHF), elevated pulmonary microvascular pressure (P(mv)) results in pulmonary edema. Because elevated P(mv) may alter the integrity of the alveolocapillary barrier, allowing leakage of surfactant protein-B (SP-B) from the alveoli into the circulation, we aimed to determine plasma levels of SP-B in CHF and their relation to clinical status., Methods and Results: Fifty-three outpatients with CHF had plasma SP-B and N-terminal proBNP (NT-proBNP) assayed, in addition to a formalized clinical assessment at each clinic review over a period of 18 months. The control group comprised 19 normal volunteers. Plasma SP-B was elevated in CHF (P<0.001), and levels increased with New York Heart Association classification (P<0.001). SP-B correlated with objective clinical status parameters and NT-proBNP. During follow-up, major cardiovascular events occurred in patients with higher plasma SP-B (P<0.01) and NT-proBNP (P<0.05). Furthermore, on conditional logistic regression analysis, only SP-B was independently associated with CHF hospitalization (P=0.005). The 53 patients underwent a total of 210 outpatient visits. When the diuretic dosage was increased on clinical grounds, SP-B had increased 39% (P<0.001) and NT-proBNP had increased 32% (P<0.001). Conversely, at the next visit, SP-B fell 12% (P<0.001), whereas NT-proBNP fell 39% (P<0.001)., Conclusions: Plasma SP-B is increased in CHF, and levels are related to clinical severity. Furthermore, within individual patients, SP-B levels vary with dynamic clinical status and NT-proBNP levels. Because plasma SP-B is independently associated with CHF hospitalization, it may, by virtue of its differing release mechanism to NT-proBNP, be a clinically useful biomarker of the pulmonary consequences of raised P(mv).

Increased leakage of surfactant proteins A and B (SP-A and SP-B) and Clara cell secretory protein (CC16) from the air spaces into the circulation occurs in a range of respiratory conditions. However, circulating levels depend not only on the rate of entry into the circulation, but also on the rate of clearance. In order to clarify the role of the kidney in the clearance of these proteins, serum levels were related to markers of glomerular filtration in 54 non-smoking patients with varying degrees of renal dysfunction, none of whom had respiratory disease or were receiving dialysis at the time of sampling. Serum SP-A was related to SP-B (r = 0.53, p < 0.001) and to CC16 (r = 0.33, p < 0.02). Similarly, SP-B was related to CC16 (r = 0.39, p < 0.004). Stepwise multiple linear regression analysis suggested that serum SP-A and SP-B are influenced by age (approximately 20 and approximately 25% of variance, respectively), whereas CC16 is determined by renal function and, to a lesser extent, by body weight (approximately 63% of variance in total). We conclude that CC16 is cleared from blood by the renal route, whereas SP-A and SP-B are not. Serum SP-A and SP-B are influenced by age, which we speculate reflects increased damage to the alveolocapillary barrier.

Chronic heart failure (CHF) is associated with adaptive structural changes at the alveolocapillary barrier that may be associated with altered protein permeability. Bidirectional protein movement across the barrier was studied in anesthetized rats with infarct-induced CHF by following (125)I-labeled albumin ((125)I-albumin) flux into the alveoli and the leakage of surfactant protein (SP)-B from the alveoli into the circulation. Three groups were studied: controls [0% left ventricular (LV) infarction], moderate infarct (25-45% LV infarction), and large infarct (>46% LV infarction). Wet and dry lung weights increased in the large infarct group (both P < 0.001), consistent with increased lung water and solid lung tissue. (125)I-albumin flux increased across the endothelial (P < 0.001) and epithelial (P < 0.01) components of the alveolocapillary barrier in the large infarct group. Plasma SP-B increased 23% with moderate infarcts (P < 0.05) and 97% with large infarcts (P < 0.001), independent of alveolar levels. Lavage fluid immune cells (P < 0.01) and myeloperoxidase activity (P < 0.05) increased in the large infarct group, consistent with inflammation. Bidirectional protein movement across the alveolocapillary barrier is increased in CHF, and alveolar inflammation may contribute to this pathophysiological defect.

Objectives: To determine whether acute cardiogenic pulmonary edema is associated with damage to the alveolocapillary barrier, as evidenced by increased leakage of surfactant specific proteins into the circulation, to document the duration of alveolocapillary barrier damage in this setting, and to explore the role of pulmonary parenchymal inflammation by determining if circulating tumor necrosis factor-alpha is increased after acute cardiogenic pulmonary edema., Design: Prospective, observational study., Setting: Critical care, cardiac intensive care, and cardiology wards of a tertiary-care university teaching hospital., Patients: A total of 28 patients presenting with acute cardiogenic pulmonary edema and 13 age-matched normal volunteers., Interventions: Circulating surfactant protein-A and -B and tumor necrosis factor-alpha were measured on days 0 (presentation), 1, 3, 7, and 14. Clinical markers of pulmonary edema were documented at the same times., Measurements and Main Results: Surfactant protein-A and -B were elevated on day 0 compared with controls (367 +/- 17 ng/mL vs. 303 +/- 17 and 3821 +/- 266 ng/mL vs. 2747 +/- 157 [mean +/- sem], p <.05), and although clinical, hemodynamic and radiographic variables improved rapidly (p <.001), surfactant protein-A and -B rose further until day 3 (437 +/- 22, p <.001, 4642 +/- 353, p <.01). Tumor necrosis factor-alpha was elevated at presentation (p <.05), doubled by day 1 (6.98 +/- 1.36 pg/mL, p <.05), remained elevated on day 3 (5.72 +/- 0.96 pg/mL, p <.05), and peak levels were related to chest radiograph extravascular lung water score (r(p) = 0.64, p =.003)., Conclusions: Although the initial increase in plasma surfactant protein-A and -B may represent hydrostatic stress failure of the alveolocapillary barrier, the prolonged elevation, when hemodynamic abnormalities have resolved, and the delayed elevation of tumor necrosis factor-alpha are consistent with pulmonary parenchymal inflammation, which may further damage the alveolocapillary barrier. This prolonged physiologic defect at the alveolocapillary barrier after acute cardiogenic pulmonary edema may partly account for the vulnerability of these patients to recurrent pulmonary fluid accumulation.

Background: Conventional measures of the severity of alveolar proteinosis (AP) include alveolar-arterial oxygen gradient ([A - a]DO(2)), vital capacity (VC), and carbon monoxide transfer factor (TLCO), but alternative serological measures have been sought. Granulocyte-macrophage colony stimulating factor (GM-CSF) neutralising autoantibody is found in patients with idiopathic acquired AP. We have investigated the interrelationships between the levels of this antibody and those of surfactant protein (SP)-A and -B, lactate dehydrogenase (LDH), and conventional measures of disease severity, and the capacity of these parameters to predict the response to rhGM-CSF treatment., Methods: Blood levels of anti-GM-CSF antibodies, SP-A, SP-B, LDH, and [A - a]DO(2), VC, and TLCO were measured before rhGM-CSF treatment and every 2 weeks thereafter in 14 patients with AP., Results: At baseline, high levels of anti-GM-CSF antibodies and increased SP-A and SP-B levels were seen in all patients, and LDH was raised in 83%. SP-A was highly correlated with [A - a]DO(2), VC, and TLCO (p

Since the 16-kDa bronchiolar Clara cell protein (CC16) and the alveolar surfactant-associated proteins (SP)-A and -B leak into the circulation when parenchymal health is disturbed, the aim of this study was to determine whether their serum levels could serve as early peripheral markers of tobacco smoke-induced epithelial injury. Sixty-nine (51 yrs (32-54) median (25-75th percentile)) nonsmokers and 54 (42 yrs (31-53)) asymptomatic smokers were enrolled in the study. Serum levels of SP-A did not differ between subjects (270 (208-389) versus 259 (168-392) microg x L(-1)), however, CC16 levels decreased (10.6 (8.7-14.6) versus 7.6 (6.0-11.2) microg x L(-1)) and SP-B levels increased (2,529 (2,091-2,943) versus 3,053 (2,613-4,188) microg x L(-1)) in the smokers. When tobacco smoke exposure, serum creatinine (renal index), age and sex were used as independent variables, CC16 was negatively influenced by cumulative smoking and positively influenced by age. SP-A and -B were negatively influenced by creatinine and positively influenced by cumulative smoking. Serum SP-B was inversely correlated with forced expiratory volume in one second/vital capacity, suggesting an association between obstructive disease and parenchymal lung health. The authors suggest that serum surfactant-associated proteins-A and -B reflect increased alveolocapillary leakage whereas Clara cell secretory protein 16 reflects tobacco smoke-induced Clara cell toxicity. Their evaluation may allow the effects of tobacco smoke on different levels of the respiratory tract, cellular toxicity and epithelial leakage to be distinguished.

Although endotoxin-induced acute lung injury is associated with inflammation, alveolocapillary injury, surfactant dysfunction, and altered lung mechanics, the precise sequence of these changes is polemic. We have studied the early pathogenesis of acute lung injury in spontaneously breathing anesthetized rats after intravenous infusion of Salmonella abortus equi endotoxin. The animals became hypoxic, and airway resistance, tissue resistance, lung elastance, and static compliance all deteriorated well before any change in alveolar neutrophils, macrophages, lung fluid (99mTc-labeled diethylenetriamine pentaacetic acid), or 125I-albumin flux, which were only appreciably increased at 8.5 hours. Lung elastance deteriorated before airway resistance, indicating that the compliance change was specific rather than caused by reduced lung volume. The subcellular and alveolar content of surfactant proteins A and B, cholesterol, disaturated phospholipids, and phospholipid classes remained normal in the face of a dramatic increase in the synthesis and turnover of 3H-disaturated phosphatidylcholine. Our findings indicate that the increase in surfactant disaturated phospholipid turnover reflects, at least in part, an approximately five-fold increase in "sigh frequency." We suggest that endotoxin has direct effects on tissue resistance and lung elastance independent of surfactant composition and that the initial respiratory failure results primarily from endotoxin-induced ventilation/perfusion mismatch independent of edema or alveolocapillary injury per se.

Surfactant protein-B is a lung specific protein secreted into the air spaces by pulmonary epithelial type II cells that leaks into the bloodstream in increased amounts in patients with ARDS. To test whether elevated plasma levels of surfactant protein-B would predict the development of ARDS in patients with acute hypoxemic respiratory failure, plasma and lung injury scores were collected at study entry and daily thereafter for 3 d from 54 patients admitted to our intensive care unit. ARDS was defined as a new bilateral infiltrate on chest radiograph and a lung injury score > or = 2.5. Twenty patients developed ARDS, of whom seven died. Although the initial lung injury score was not predictive of ARDS, the initial plasma surfactant protein-B was predictive (area under the curve = 0.77 [0.63 to 0.90], nonparametric receiver-operating characteristic analysis). In this cohort, plasma surfactant protein-B was particularly predictive of ARDS when applied to patients suffering a direct lung insult (area under the curve = 0.87 [0.72 to 1.02]), with a sensitivity of 85% (95% CI: 55 to 98%) and specificity of 78% (40 to 97%) at a cutoff of 4,994 ng/ml.

The external appearance of urinary calcium oxalate (CaOx) crystals suggests that they are solid, homogeneous structures, despite their known association with proteins. Our aim was to determine whether proteins comprising the organic matrix of CaOx crystals are superficial or intracrystalline in order to clarify the role of urinary proteins in the formation of kidney stones. CaOx crystals were precipitated from centrifuged and filtered, or ultrafiltered, healthy human urine. They were then treated with dilute NaOH to remove bound proteins, partially demineralized with EDTA, or fractured and subjected to limited proteolysis before examination by low-resolution scanning electron microscopy or field emission scanning electron microscopy. Crystals precipitated from centrifuged and filtered urine had a complex interior network of protein distributed throughout the mineral phase, which appeared to comprise closely packed subcrystalline particles stacked in an orderly array among an amorphous organic matrix. This ultrastructure was not evident in crystals deposited in the absence of macromolecules, which were completely solid. This is the first direct evidence that crystals generated from cell-free systems contain significant amounts of protein distributed throughout a complex internal cribriform ultrastructure. Combined with mineral erosion in the acidic lysosomal environment, proteins inside CaOx crystals would render them susceptible to attack by urinary and intracellular renal proteases and facilitate their further dissolution or disruption into small particles and ions for removal by exocytosis. The findings also have broader ramifications for industry and the materials sciences, as well as the development and resorption of crystals in biomineralization systems throughout nature., (Copyright 2001 Academic Press.)

Alveolar proteinosis (AP) is characterized by excessive surfactant accumulation, and most cases are of unknown etiology. Standard therapy for AP is whole-lung lavage, which may not correct the underlying defect. Because the hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is required for normal surfactant homeostasis, we evaluated the therapeutic activity of GM-CSF in patients with idiopathic AP. Fourteen patients received 5 microg/kg/d GM-CSF for 6 to 12 wk with serial monitoring of the alveolar-arterial oxygen gradient ([A-a]DO2), diffusing capacity of carbon monoxide, computed tomographic scans, and exercise testing. Patients not responding to 5 microg/kg/d GM-CSF underwent stepwise dose escalation, and responding patients were retreated at disease recurrence. Stored pretreatment sera were assayed for GM-CSF-neutralizing autoantibodies. According to prospective criteria, five of 14 patients responded to 5 microg/kg/d GM- CSF, and one of four patients responded after dose escalation (20 microg/kg/d). The overall response rate was 43% (mean improvement in [A-a]DO2 = 23.2 mm Hg). Responses lasted a median of 39 wk, and were reproducible with retreatment. GM-CSF was well-tolerated, with no late toxicity seen. The only treatment-related factor predictive of response was GM-CSF-induced eosinophilia (p = 0.01). Each of 12 patients tested had GM-CSF-neutralizing autoantibodies present in pretreatment serum. We conclude that GM- CSF has therapeutic activity in idiopathic AP, providing a potential alternative to whole-lung lavage.

Although acute lung injury (ALI) is associated with inflammation and surfactant dysfunction, the precise sequence of these changes remains poorly described. We used oleic acid to study the pathogenesis of ALI in spontaneously breathing anesthetized rats. We found that lung pathology can occur far more rapidly than previously appreciated. Lung neutrophils were increased approximately threefold within 5 min, and surfactant composition was dramatically altered within 15 min. Alveolar cholesterol increased by approximately 200%, and even though disaturated phospholipids increased by approximately 30% over 4 h, the disaturated phospholipid-to-total phospholipid ratio fell. Although the alveolocapillary barrier was profoundly disrupted after just 15 min, with marked elevations in lung fluid ((99m)Tc-labeled diethylenetriamine pentaacetic acid) and (125)I-labeled albumin flux, the lung rapidly began to regain its sieving properties. Despite the restoration in lung permeability, the animals remained hypoxic even though minute ventilation was increased approximately twofold and static compliance progressively deteriorated. This study highlights that ALI can set in motion a sequence of events continuing the respiratory failure irrespective of the alveolar surfactant pool size and the status of the alveolocapillary barrier.

Objective: We test the hypothesis that the changes we observed previously in the relative amounts of disaturated phospholipids (DSP), cholesterol (CHOL), and surfactant protein-A (SP-A) in human alveolar surfactant in response to acute exercise, and which were related to fitness, can be induced by training., Methodology: We examine the effect of 7 weeks' training on these major surfactant components, together with surfactant protein-B (SP-B), in bronchoalveolar lavage fluid harvested from 17 males, both at rest and after acute exercise. Fitness was assessed as workload/heart rate achieved during cycling for 30 min at 90% of theoretical maximal heart rate, and was increased in all subjects following training (mean increase 22.2+/-3.91%; P = 0.001)., Results: Training significantly increased the SP-A/DSP, SP-B/DSP, SP-A/CHOL and SP-A/SP-B ratios in whole surfactant harvested from subjects both at rest and immediately following exercise. Training also increased the SP-B/CHOL ratio at rest. Changes were particularly marked at rest in the SP-A/DSP, SP-A/CHOL, and SP-B/CHOL ratios in the tubular myelin-rich fraction, and after exercise in the SP-A/DSP, SP-A/CHOL, and SP-A/SP-B ratios in the tubular myelin-poor fraction., Conclusion: We conclude that training markedly alters the composition of alveolar surfactant both at rest and with exercise; the physiological significance of these changes remains to be determined.

Pulmonary alveolar proteinosis is a rare condition characterized by the abnormal accumulation of surfactant-like material within the alveolar spaces and distal bronchioles. Two cases with contrasting modes of presentation, course, and response to therapeutic whole lung lavage are described. Both cases were in hypoxaemic respiratory failure at the time the definitive diagnosis was made, and in both cases the diagnosis was made by segmental bronchoalveolar lavage following negative open lung biopsy. In neither was an underlying causative organism or agent identified. In one case the alveolar proteinosis developed in late pregnancy, a presentation that is previously unreported. Clinical improvement in this case required repeated whole lung lavages and was accompanied by a trend towards normalization of the ratios of surfactant protein-A and surfactant protein-B to disaturated phospholipid, ratios which may be useful as prognostic indicators. The response to therapeutic lavage was markedly different in the two cases, and it is postulated that this may relate to the fact that alveolar proteinosis is a heterogeneous disease and that the course and response to treatment may relate in part to the specific composition of the abnormal proteinaceous fluid.

Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis. However the pathophysiology of bronchiolitis is unclear. Leukocytes, especially neutrophils, may play an important role in the pathogenesis of bronchiolitis. Whereas we have previously shown that neutrophils augment epithelial leakage and detachment in RSV infection in vitro, it is unknown whether epithelial damage occurs in vivo in infants with RSV bronchiolitis. We hypothesized that respiratory epithelial damage occurs in infants with RSV bronchiolitis and that surfactant proteins leak into the circulation. The plasma concentrations of surfactant protein-A and surfactant protein-B in infants with RSV bronchiolitis were measured by ELISA. Plasma immunoreactive surfactant protein-B in infants with RSV bronchiolitis was markedly higher than that in matching controls. Our study suggests that alveolocapillary permeability is increased in infants with RSV bronchiolitis in vivo and that surfactant protein-B may be a sensitive marker for lung injury in such infants.

The Australian lungfish Neoceratodus forsteri is the most primitive member of the lungfish family, with a surfactant lipid composition similar to the actinopterygiian fishes, which evolved 400 million years ago. We have analysed the proteins associated with surfactant isolated from lung lavage of this species, and used electron microscopy and immunohistochemistry to examine the surfactant structures and the subcellular localisation of these proteins. The epithelial lining of the gas-exchange region of the lungfish lung consists of one basic cell type, which has characteristics of both mammalian alveolar type I and type II cells and may be the common ancestor of both. It has long cytoplasmic plates containing microvilli, large osmiophilic bodies resembling mammalian lamellar bodies and a cytoplasm rich in metabolic organelles. Extracellular structures reminiscent of mammalian surfactant forms, but not including tubular myelin, were observed in the airspaces. Immunochemical analysis of the lungfish surfactant and lung tissue, using antibodies to human SP-A and SP-B, showed a similar staining pattern to human surfactant, indicating that SP-A- and SP-B-like proteins are present. Immunohistochemistry revealed that both SP-A and SP-B reactivity was present in the secretory cell osmiophilic bodies. In conclusion, our results suggest that, despite the great diversity in present day lung structures, a common cellular mechanism may have evolved to overcome fundamental problems associated with air-breathing.

The application of impedance pneumography for monitoring respiration in small animals has been limited by problems with calibration. With improved instrumentation, we describe the calibration of tidal volume in anesthetized rats. The detection of changes in voltage, reflecting the electrical impedance variations associated with respiration, was optimized by using disposable adhesive silver-silver chloride electrodes, advanced circuitry, and analog-to-digital recording instrumentation. We found a linear relationship between change in impedance and tidal volume in individual rats (R2 >/= 98%), which was strongly influenced by rat weight. Consequently, a calibration equation incorporating change in impedance and rat weight was derived to predict tidal volume. Comparison of the predicted and true tidal volumes revealed a mean R2 >/= 98%, slopes of approximately 1, intercepts of approximately 0, and bias of approximately 0.07 ml. The predicted volumes were not significantly affected by either frequency of respiration or pulmonary edema. We conclude that impedance pneumography provides a valuable tool for the noninvasive measurement of tidal volume in anesthetized rats.

Surfactant proteins A and B (SP-A and SP-B) enter the circulation in a manner that acutely reflects changes in pulmonary function in patients with acute respiratory failure (ARF). There is a small but significant gradient in SP-A and SP-B from arterial to mixed venous (A-V) blood, and since we have detected both proteins in urine, the kidney may be a major site of their systemic clearance. Clara cell secretory protein 16 (CC16), which leaks from the respiratory tract, is known to be freely eliminated by the kidney. Lung plasma protein levels will depend on the rates of both protein entry into and clearance from plasma. In order to study the limiting variable determining these levels, we compared plasma CC16, SP-A, and SP-B in matching A-V blood samples from 37 ARF patients with indices of lung dysfunction and glomerular filtration rate (GFR) (of plasma cystatin C and creatinine). Cystatin C, CC16, SP-A, and SP-B were reduced in mixed venous plasma (all p < 0.001) and their A-V gradients were directly related to their arterial levels (all p < 0.03). Whereas CC16, SP-A, and SP-B reflected blood oxygenation (all p < 0.05), only SP-A and SP-B were related to lung injury score (LIS) (both p < 0.05). In contrast, whereas the clearances of both CC16 and cystatin C were related to that of creatinine (p < 0.02 for both), the clearances of SP-A and SP-B were not. Our study confirms that all three lung proteins are acutely cleared from the circulation of patients with ARF (half-lives < 18 min), and we conclude that whereas the plasma concentration of CC16 depends on GFR, plasma concentrations of SP-A and SP-B reflect lung function independently of this variable.

Alveolar proteinosis (AP) is an idiopathic condition characterized by excess alveolar surfactant. Although the surfactant proteins (SP) are known to be aberrant, little is known of their variation between patients or their abundance relative to the lipids. We have examined surfactant composition in lavage fluid from 16 normal subjects and 13 patients with AP, one of whom was lavaged on 11 occasions over approximately 13 mo. In this patient we have examined composition on each occasion and in each sequential lavage aliquot. Composition was constant between right and left lung, but it differed markedly between patients. The cholesterol/disaturated phospholipid ratios (CHOL/DSP) were invariably elevated, on average by approximately 7-fold, whereas the SP-A/DSP and SP-B/DSP ratios were generally elevated, in some cases by as much as approximately 40- and approximately 100-fold, respectively. Although AP lavage generally contained more non-thiol-dependent SP-A aggregates and low Mr isoforms, the two-dimensional immunochemical staining patterns varied between patients and right and left lung. In the patient lavaged on multiple occasions, the SP-A/DSP and SP-B/DSP ratios progressively decreased as the patient's condition resolved. Because the SP-B/SP-A ratio was normal in all cases, we suggest that structural changes to the proteins occurred secondarily and that caution must be used in comparing functional data derived using SP-A obtained from patients with AP.

Surfactant protein-A (SP-A) leaks into the circulation of patients with acute respiratory distress syndrome (ARDS) or acute cardiogenic pulmonary edema (APE) in a manner inversely related to lung function. Since surfactant protein-B (SP-B) is synthesized as a precursor considerably smaller than alveolar SP-A, we investigated whether it enters the circulation more readily. Reactivities consistent with SP-B proprotein (approximately 42 to approximately 45 kD) and the approximately 25 kD processing intermediate were detected in plasma. Plasma immunoreactive SP-B levels were significantly higher in ARDS (8,007+/-1,654 ng/ml [mean+/-SEM], n = 22) and APE (3,646+/-635 ng/ml, n = 10) patients compared with normal subjects (1,685+/-58 ng/ml, n = 33) and ventilated patients with no cardiorespiratory disease (1,829+/-184 ng/ml, n = 7). All groups had plasma SP-B/SP-A ratios approximately 6- to approximately 8-fold higher than in normal lavage or ARDS tracheal aspirate fluid, consistent with protein sieving. During admission, both plasma SP-B and the SP-B/SP-A ratio were inversely related to blood oxygenation (PaO2/FIO2) (p < 0.0001 and p < 0.025, n = 260 from 39 patients; Spearman) and static respiratory system compliance (deltaV/deltaP) (p < 0.0001 and p < 0.01, n = 168 from 25 patients). We describe in detail three patients and conclude that immunoreactive SP-B enters more readily than SP-A, is cleared acutely, and provides a better indicator of lung trauma.

Although one would predict that surfactant replacement therapy would be effective in acute respiratory distress syndrome (ARDS), a recent large trial proved unsuccessful, possibly reflecting the nature of the surfactant used. Given the importance of the unique proteins in the action of surfactant, these would seem vital components of any exogenous surfactant. The ability to identify patients at risk of ARDS and to characterise their surfactant might allow prophylactic treatment with a nebulised, complementary, tailor-made preparation of surfactant. Advanced cases might undergo bronchoscopic focal lavage to remove plasma proteins and inflammatory mediators prior to focal instillation of surfactant to areas of greatest need. Ventilation regimens might be adjusted both to minimise trauma and to conserve endogenous surfactant.

Alveolar disaturated phospholipids (DSPA) increase in vivo in rats with hyperpnea and in isolated perfused lungs (IPL) in response to either salbutamol or increasing tidal volume (VT). Because surfactant protein-A (SP-A) may play a role in surfactant homeostasis, we have examined the relationship between SP-A and DSP in the alveolus lamellar bodies (LB-A), and in a vesicular (LB-B) lung subfraction. Whereas 2 h swimming increased total DSPA (approximately 48%), it had no effect on alveolar SP-A (SP-AA). In the IPL, salbutamol increased total DSPA (approximately 30%) and SP-AA (approximately 41%); increasing VT (2.5-fold) only increased DSPA (approximately 22%). SP-A and DSP also varied differentially in the tubular myelin-rich and -poor subfractions. In both the IPL and in vivo, we found inverse relationships between DSPA and SP-AA/DSPA, indicating that although SP-AA and DSPA are related, they vary independently. Whereas total SP-AA/DSPA varied between 0.046 and 0.074, it remained constant in LB-A (approximately 0.015) and LB-B (approximately 0.010), suggesting that DSP and SP-A are secreted differentially and that only a small portion of SP-AA is derived from lamellar bodies.

We have induced prolonged hyperpnea in rats and examined the distribution of surfactant-associated proteins (SP-A and SP-B) and lysozyme in lamellar bodies (lb) and two alveolar fractions, one tubular myelin rich (alv-1) and the other tubular myelin poor (alv-2). We have also examined the expression of SP-A, SP-B, SP-C, and lysozyme mRNA in lung tissue and alveolar type II cells. Hyperpnea resulted in significant increases in lb SP-A, lysozyme, and phospholipid (PL) but no change in the protein-to-PH ratios, suggesting that lb stoichiometry is constant. The SP-A and SP-B-to-PL ratios were 33 and 18 times greater, respectively, in control alv-1 than in lb, suggesting that alv-1 is enriched with these proteins. In contrast, the lysozyme-to-PL ratio was similar in control alv-1 and lb. Hyperpnea did not alter the alv-1 SP-A or SP-B-to-PL ratios, suggesting some constant stoichiometry to their lipid association; however, the lysozyme-to-PL ratio was reduced. Whereas hyperpnea significantly elevated the PL, SP-A, and lysozyme levels in alv-2, the SP-B level was unchanged. We suggest that surfactant-associated lysozyme is secreted with lb, the majority of SP-A is linked to lipid secretion but not necessarily with lb, and the majority of SP-B secretion is independent of PL secretion. Hyperpnea did not alter the mRNA expression of SP-A, SP-B, SP-C, or lysozyme in alveolar type II cells, but expression of SP-A and SP-B mRNA was significantly increased in lung tissue.

Detection of alveolo-capillary damage has important implications for treatment modalities in ventilated patients. Although surfactant protein-A (SP-A) is normally only found in appreciable amounts in the lung, we describe significantly elevated concentrations in the sera of patients with acute cardiogenic pulmonary edema (median, 250 ng/ml; range, 180 to 364; n = 10) and in those with the adult respiratory distress syndrome (ARDS) (median, 378 ng/ml; range, 215 to 1,378; n = 15) relative to healthy control subjects (median, 175 ng/ml; range, 123 to 248; n = 15) and ventilated patients with no cardiorespiratory disease (median, 169 ng/ml; range, 126 to 253; n = 6) (p < 0.01, in all cases). Serum SP-A was inversely related to blood oxygenation and to static respiratory system compliance both at the time of the patient's entry into the study (p < 0.005, rs = -0.51, n = 31; p < 0.001, rs = 0.82, n = 17; respectively) and during the course of admission (p < 0.001, rs = -0.34, n = 168; p < 0.001, rs = -0.50, n = 111; respectively). In addition, we describe in detail three cases of ARDS where lung function either improved, remained static, or deteriorated. We conclude that serum SP-A is an acute indicator of lung function and alveolo-capillary membrane injury.

Demineralization of calcium oxalate (CaOx) crystals precipitated from human urine in vitro yields an organic crystal matrix extract (CME) consisting predominantly of a single protein which we originally named crystal matrix protein but have subsequently shown to be a urinary form of prothrombin activation peptide fragment 1 (F1). The aim of this study was to determine whether CME is a promoter or inhibitor of CaOx crystallization. The effect of CME on CaOx crystal growth and aggregation was tested using a standard seeded crystallization system, and its effect quantified by use of particle size analysis and a computer model. In addition, the effect of CME on the crystallization of CaOx was tested in undiluted, ultrafiltered human urine using Coulter Counter analysis and scanning electron microscopy. It was shown that CME is a potent inhibitor of CaOx crystal growth and aggregation in a seeded metastable solution. However, of greater significance is that at a concentration of 10 mg/l it completely reversed the formation of large crystalline aggregates that form upon the removal of urinary macromolecules from undiluted urine. It was concluded that CME is the most potent macromolecular urinary inhibitor yet to be tested in urine in vitro. By preventing the aggregation of newly formed crystals, the components of CME may significantly reduce the probability of particle retention in vivo and therefore the occurrence of urolithiasis.

Although surfactant protein A (SP-A) is an integral component of alveolar surfactant, its relative abundance in lamellar bodies, regarded as the intracellular storage organelles for surfactant, remains contentious. We have previously shown that lamellar bodies, isolated from rat lung by upward flotation on a sucrose gradient, can be subfractionated into classic-appearing lamellar bodies (Lb-A) and a vesicular fraction (Lb-B), which we have speculated may be a second release form of surfactant. In the present study, we have used two-dimensional protein electrophoresis and immunochemical analysis to clarify the origin and the composition of these two subcellular fractions. In addition, we have examined the hypothesis that the secretion of SP-A and surfactant phospholipids occurs by independent pathways by examining the distribution of SP-A, total protein, and disaturated phospholipids (DSP) in the tubular myelin-rich (Alv-1) and tubular myelin-poor (Alv-2) fractions separated from lavaged material and in Lb-A and Lb-B isolated from both lung homogenate and purified alveolar type II cells. Our findings indicate that Lb-B is derived from type II cells, although they do not indicate whether it is a secretory form of surfactant, a reuptake vesicle, or a mixture of both. We found that the lung has a large tissue pool of immunoreactive SP-A. The %SP-A/DSP of total lamellar bodies isolated from type II cells was 0.96 +/- 0.1 (mean +/- SE), intermediate between that in Lb-A (1.67 +/- 0.13) and in Lb-B (0.65 +/- 0.04). In contrast, the %SP-A/DSP was 11.16 +/- 0.84 in whole lung homogenate and 13.14 +/- 1.71 in whole type II cells. In the alveolar compartment, the %SP-A/DSP was 17.38 +/- 3.40 in Alv-1, 6.34 +/- 0.31 in Alv-2, and 10.49 +/- 1.43 in macrophages, values an order of magnitude greater than found with the lamellar bodies. Our results indicate that only a relatively small portion of alveolar SP-A is derived from lamellar bodies, and we suggest that secretion of SP-A and DSP occurs via independent pathways.

We have tested the hypothesis that the composition of alveolar surfactant varies with pattern of breathing and level of fitness. We examined three major components of surfactant, surfactant protein A (SP-A), disaturated phospholipids (DSP), and cholesterol (CHOL) in bronchoalveolar lavage (BAL) fluid from 12 healthy men before and after exercise. Fitness was assessed as work load/heart rate ([kpm.min-1]/[HR.HRmax-1]) achieved during cycling for 30 min at 90% theoretical maximal heart rate. Using a bronchoscope, four 20-ml vols of 0.15 M NaCl at 37 degrees C were instilled and then recovered from first a right upper and then a right lower lobe segmental bronchus. As we found no differences in the BAL from upper and lower lobes, the fluid was combined. We found a direct relationship between CHOL and DSP (rs = 0.84, p < 0.001), SP-A and CHOL (rs = 0.40, p < 0.025), and between SP-A and DSP (rs = 0.44, p < 0.025). The change in the ratios CHOL/DSP, SP-A/CHOL, and SP-A/DSP immediately after exercise was correlated with fitness (rs = -0.56, p < 0.025; rs = 0.75, p < 0.005; rs = 0.62, p < 0.025, respectively). We conclude that the composition of surfactant can change rapidly with exercise in a manner related to fitness, and we suggest that this is consistent with the existence of at least two pools of tissue surfactant of different composition supplying the alveolar compartment.

Urinary glycosaminoglycans are selectively included into calcium oxalate (CaOx) crystals precipitated from whole human urine: The presence of glycosaminoglycans (GAGs) in the organic matrix of urinary stones, and their known effects on CaOx crystallization have prompted speculation regarding their role in CaOx urolithiasis. The aim of this study was to examine the involvement of GAGs in the early stages of CaOx crystallization in human urine. Urine samples were collected from healthy men and CaOx crystallization was induced by the addition of a sodium oxalate load. The crystals were harvested and demineralized, and the GAG content of the resulting extract analysed by cellulose acetate electrophoresis. Only one GAG, heparan sulphate (HS) was detected in the organic matrix of the crystals; chondroitin sulphate (ChS), the most abundant urinary GAG, was conspicuously absent. Further experiments, in which varying amounts of HS and ChS were added to ultrafiltered (10,000 Da) urine prior to induction of calcium oxalate crystallization, showed that ChS was included into the crystals only when HS was absent from the urine. It was concluded that the selective inclusion of GAGs into crystals and stones is a function related more to relative binding affinity than to ambient GAG concentration and that HS and ChS compete for specific binding sites on the crystal surface.

The aim of this study was to determine the immunohistochemical distribution and quantification of crystal matrix protein (CMP). CMP, a 31 kDa glycoprotein, is the principal macromolecule found in calcium oxalate crystals generated in human urine, and is a potent inhibitor of crystal aggregation. A polyclonal rabbit anti-human CMP antibody was used to examine renal tissue by immunohistochemical techniques and light microscopy (N = 45). Twenty-five other human organs were similarly assessed. Quantification was performed using a visual analogue scale. CMP was visible as cytoplasmic staining in the epithelial cells of the TALH and the distal convoluted tubule including the macula densa in a subgroup of nephrons. CMP was not identified elsewhere in the urinary tract or in the extrarenal organs examined. Despite a trend indicating that the kidneys of normal men had more CMP than those of normal women, the difference failed to reach significance (P = 0.11). There was, however, more CMP in the stone formers group compared with either normal men (P < 0.01) or normal women (P < 0.01). This protein may be an important determinant of calcium oxalate kidney stone disease.

The abundance of protein in the matrix of calcium oxalate uroliths has fueled speculation regarding its role in stone genesis. In this study, we wanted to characterize the composition of the proteins associated with early stages of calcium oxalate crystallization in urine. Calcium oxalate crystallization was induced in urine from healthy men and women by the addition of an oxalate load. The crystals were harvested and demineralized, and the proteins remaining were separated and characterized by polyacrylamide gel electrophoresis and Western blotting. Most urinary proteins were not detected in the crystals or were present in only small quantities. The most abundant urinary macromolecule, Tamm-Horsfall glycoprotein, was notably absent from the crystal extracts. The predominant protein associated with the crystals, a previously unknown urinary constituent that we call crystal matrix protein (CMP; molecular mass, 30,000 Da), was more prevalent in the crystals derived from female urine. We conclude that most urinary proteins play no direct role in calcium oxalate crystal formation. However, the protein CMP exhibits a remarkable affinity for calcium oxalate crystals and may be important in stone pathogenesis.