Complement factor C5a exerts an anti-inflammatory effect in acute pancreatitis and associated lung injury

Complement factor C5a acting via C5a receptors (C5aR) is recognized as an anaphylotoxin and chemoattractant that exerts proinflammatory effects in many pathological states. The effects of C5a and C5aR in acute pancreatitis and in pancreatitis-associated lung injury were evaluated using genetically altered mice that either lack C5aR or do not express C5. Pancreatitis was induced by administration of 12 hourly injections of cerulein (50 microg/kg ip). The severity of pancreatitis was determined by measuring serum amylase, neutrophil sequestration in the pancreas, and acinar cell necrosis. The severity of lung injury was evaluated by measuring neutrophil sequestration in the lung and pulmonary microvascular permeability. In both strains of genetically altered mice, the severity of pancreatitis and pancreatitis-associated lung injury was greater than that noted in the comparison wild-type strains of C5aR- and C5-sufficient animals. This exacerbation of injury in the absence of C5a function indicates that, in pancreatitis, C5a exerts an anti-inflammatory effect. Potentially, C5a and its receptor are capable of both promoting and reducing the extent of acute inflammation.     

CpG penta- and hexadeoxyribonucleotides as potent immunomodulatory agents

We demonstrate a new design for immunomodulatory CpG DNA containing two sequences each with as few as five or six-nucleotides joined together via 3(')-3(') linkers. These do not require the -PuPu(Py)CGPyPy- hexameric motif generally found essential for CpG DNA immune stimulation. These novel, short-immunomers show potent immunostimulatory activity manifested by IL-12 and IL-6 secretion in murine spleen cell and PBMC cultures and splenomegaly in vivo. Short-immunomers show strong activation of NF-kappaB and stress-activated signaling pathways and induce cytokines in J774 cell cultures. The same sequences also induce cytokines in healthy human PBMC cultures whereas conventional CpG DNA requires different optimal sequences for murine and human immune cells. Additionally, short-immunomers inhibit IL-5 secretion and induce IFN-gamma secretion in conalbumin-sensitized mouse spleen cell cultures, suggesting reversal of established Th2 responses to Th1 type responses. Short-immunomer also inhibits growth of MCF-7 human tumor xenograft in nude mice. This is the first report of activity with such short DNA sequences and also of sequences lacking hexameric motifs proposed in earlier studies.     

Inhibition of cyclooxygenase-2 ameliorates the severity of pancreatitis and associated lung injury

Cyclooxygenase-2 (COX-2), a widely distributed enzyme, plays an important role in inflammation. We have studied the role of COX-2 in acute pancreatitis and pancreatitis-associated lung injury using both the pharmacological inhibition of COX-2 and genetic deletion of COX-2. Pancreatitis was induced in mice by 12 hourly injections of cerulein. The severity of pancreatitis was assessed by measuring serum amylase, pancreatic trypsin activity, intrapancreatic sequestration of neutrophils, and acinar cell necrosis. The severity of lung injury was evaluated by measuring lactate dehydrogenase levels in the bronchoalveolar lavage fluid and by quantitating neutrophil sequestration in the lung. In both the pharmacologically inhibited and genetically altered mice, the severity of pancreatitis and pancreatitis-associated lung injury was reduced compared with the noninhibited strains of COX-2-sufficient mice. This reduction in injury indicates that COX-2 plays an important proinflammatory role in pancreatitis and its associated lung injury. Our findings support the concept that COX-2 inhibitors may play a beneficial role in the prevention of acute pancreatitis or in the reduction of its severity.     

Propagation of stress waves in inflated sheep lungs

If the lung is an elastic continuum, both longitudinal and transverse stress waves should be propagated in the medium with distinct velocities. In five isolated sheep lungs, we investigated the propagation of stress waves. The lungs were degassed and then inflated to a constant transpulmonary pressure (Ptp). We measured signals transmitted at locations approximately 1.5, 6, and 11 cm from an impulse surface distortion with the use of small microphones embedded in the pleural surface. Two transit times were computed from the first two significant peaks of the cross-correlation of microphone signal pairs. The "fast" wave velocities averaged 301 +/- 92, 445 +/- 80, and 577 +/- 211 (SD) cm/s for Ptp values of 5, 10, and 15 cmH2O, respectively. Corresponding "slow" wave velocities averaged 139 +/- 22, 217 +/- 36, and 255 +/- 89 cm/s. The fast waves were consistent with longitudinal waves of velocity [(K + 4G/3)/p]1/2, where bulk modulus K = 4 Ptp and shear modulus G = 0.7 Ptp. The slow waves were consistent with transverse (and/or Rayleigh) waves of velocity (G/p)1/2, with a G value of 0.9 Ptp. Measured values of K were 5 Ptp and values of G measured by indentation tests were 0.7 Ptp. Thus, stress wave velocities measured on pleural surface of isolated lungs correlated well with elastic moduli of lung parenchyma.     

Comparative Utilization of Paraffins by a Trichosporon Species

Utilization of normal and isoparaffins, separately and in mixtures, by a Trichosporon sp. was investigated. From a mixture of normal paraffins and isoparaffins, the organism consumed straight-chain paraffins, leaving the branched paraffins relatively unchanged. When offered separately, the highest utilization of n-alkanes by the organism was obtained in the range of undecane to octadecane; n-pentadecane was poorly utilized. From a mixture of n-alkanes, the rate of consumption of shorter-chain alkanes, n-decane to n-dodecane, was found to be relatively faster and more uniform than that of longer-chain alkanes.     

Effect of vascular volume and edema on wave propagation in canine lungs

The velocities of longitudinal and transverse stress waves transmitted through inflated lung parenchyma depend on the lung stiffness, as defined by the bulk and shear moduli, and the lung density. We examined the relationship between stress wave velocities and lung density. A saline-filled reservoir was connected to the vessels of caudal dog lobes held inflated at 5 cmH2O transpulmonary pressure, and vascular volume and extravascular lung water were increased in steps by increasing vascular pressure. At each step, we measured the transmitted signals at locations 2 and 7 cm from an impulse surface distortion by means of microphones embedded in the lung surface. Longitudinal and transverse wave velocities were computed by using cross-correlation analysis of microphone signal pairs. Both wave velocities decreased as lung density increased: as a first approximation, wave velocities were inversely proportional to the square root of lung density. This behavior is consistent with the propagation of small-amplitude stress waves through an elastic continuum. Estimated bulk and shear moduli were 26 and 3.5 cmH2O, respectively, and were consistent with results from quasi-static deformation tests.     

Optimization and validation of analytical conditions for bovine serum albumin using capillary electrophoresis.

A quick and reproducible capillary electrophoresis method was optimized and validated for the assay of bovine serum albumin (BSA). The effects of various parameters such as pH of buffer, concentration of buffer, capillary dimensions, use of coated capillaries, and additives such as surfactants and protein solubilizers were evaluated. The capillary coatings or additives did not give any advantage in reducing the surface adsorption of BSA on the capillary walls. The optimized conditions include use of borate buffer, pH 8.5 having a concentration of 150 mM in a 27 cm capillary with an aperture window of 100 x 200 microns for detection. The optimized method for the detection of BSA was validated. The interday and intraday coefficient of variation was not greater than 7.59% at BSA concentrations of 25-1000 micrograms/ml. The method developed was reproducible and accurate.     

Ultrastructure of the sodium pump: Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+, K+)-ATPase

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 μm. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per μm(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per μm(2) and 390 +/- 170 per μm(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.