A novel pathophysiologic phenomenon in cachexic patients with chronic obstructive pulmonary disease: the relationship between the circadian rhythm of circulating leptin and the very low-frequency component of heart rate variability.

Cachexic patients with chronic obstructive pulmonary disease (COPD) show abnormalities of the autonomic nervous system (ANS), neuroendocrine function, and energy expenditure. Leptin has been implicated in the regulation of ANS, neuroendocine function, and thermogenesis in humans. We assessed the physiologic significance of the circadian rhythm of circulating leptin using power spectrum analysis of heart rate variability (HRV) in nine cachexic male patients with COPD, eight noncachexic patients with COPD, and seven healthy control subjects. A diurnal pattern of 24-h leptin levels was present in both the control subjects (analysis of variance [ANOVA]; F = 7.80, p < 0.0001) and noncachexic COPD patients (F = 9.29, p < 0.0001), but was strikingly absent in the cachexic COPD patients (F = 2.09, p = NS). Analysis of HRV demonstrated that the diurnal rhythm of 24-h very low frequency (VLF; 0.003 to 0.04 Hz) showed significantly identical fluctuations with those of 24-h leptin levels, in all of the three groups (r = 0.388, p < 0.0001). Because VLF has been considered to reflect neuroendocrine and thermoregulatory influences, these data may suggest that the loss of circadian rhythm of circulating leptin has clinical importance in the pathophysiologic features in cachexic patients with COPD.

Percutaneous in vivo gene transfer to the peripheral lungs using plasmid-liposome complexes.

The purpose of this study was to investigate a new method of in vivo gene transfer to the lung parenchyma by the percutaneous approach. The plasmid that contains the gene for firefly luciferase driven by a cytomegalovirus (CMV) promoter (pCMVL) in combination with cationic lipids was percutaneously injected into the lung parenchyma. Luciferase activities were localized to the lobes of the lung where the plasmids with cationic lipids were injected. Percutaneous injection of the plasmid containing the human endothelin-1 (hET-1) gene driven by a CMV promoter (pRc/CMVhET-1) in combination with cationic lipids into the lungs caused pulmonary fibrosis localized to the injection site in the peripheral lungs. We concluded that percutaneous in vivo gene transfer to the lungs is a unique and important approach to introduce exogenous gene expression in the limited area of the lung parenchyma. This method of gene transfer will be applicable for human gene therapy for targeted areas of peripheral lung and will also be useful to assess the function of the proteins expressed by a gene in the local area of the lungs.

Levels of cytokeratin 19 fragments in bronchoalveolar lavage fluid correlate to the intensity of neutrophil and eosinophil-alveolitis in patients with idiopathic pulmonary fibrosis.

Cytokeratin 19 (CK19) is a specific cytoskeletal structure for simple epithelia, including bronchial and alveolar epithelial cells (BAEC). Since CK19 is abundant in alveolar epithelial cells, and could be released from injured alveolar epithelium in idiopathic pulmonary fibrosis (IPF), we investigated the levels of CK19 fragments in the bronchoalveolar lavage fluids (BALF) of 16 patients with idiopathic pulmonary fibrosis (IPF) and 12 patients with sarcoidosis using enzyme-linked immunoassay. There were also 19 control subjects (10 asymptomatic smokers and nine non-smokers). BALF from the non-smokers as well as the asymptomatic smokers contained few CK19 fragments (0.2+/-0.2, 1.3+/-0.5 pg ml(-1) respectively). There were significantly high levels of CK19 in the BALF from patients with IPF (7.3+/-1.4 pg/ml; P<0.01 vs. control non-smoker). Even if the levels of CK19 were expressed as relative to the albumin concentration, significantly increased levels of CK19 fragments were noted in BALF from patients with IPF. However, these levels were not found in BALF from patients with sarcoidosis. Importantly, levels of CK19 fragment in BALF were significantly correlated to the number of neutrophils (r = 0.791, P<0.001) and eosinophils (r = 0.771, P<0.001) but not to that of macrophages or lymphocytes in BALF from IPF patients. Our results suggest the usefulness of CK19 measurement in BALF for assessing the presence of bronchiolo-alveolar epithelial injuries in idiopathic pulmonary fibrosis.

Progressive bronchial obstruction associated with toxic epidermal necrolysis.

Toxic epidermal necrolysis (TEN) is an acute life-threatening condition, characterized by erosion of the mucous membranes, extensive detachment of the epidermis, and severe constitutional symptoms. Pulmonary complications of TEN are reported as rare, but are one of the most common causes of death. Our report focuses on an unusual case of toxic epidermal necrolysis which showed multiple bronchial obliteration during the chronic phase of the disease. Biopsied tissue of the obliterated bronchi demonstrated non-specific granulation. To improve the obliterated ventilatory function, we tried to reopen the bronchial obliteration using a balloon catheter under the guidance of fibreoptic bronchoscopy, however rapid restenosis of the bronchi ensued.

Hydrogen peroxide enhances shedding of type I soluble tumor necrosis factor receptor from pulmonary epithelial cells.

Reactive oxygen intermediates (ROIs) are among the important mediators in the pathogenesis of lung diseases in which tumor necrosis factor (TNF) plays a pivotal role. However, the effects of ROIs on the TNF- TNF receptor system remain unclear. Effects of hydrogen peroxide on the shedding of soluble tumor necrosis factor receptor (sTNF-R) were investigated in a pulmonary epithelial cell line (A549) using enzyme-linked immunoassay. A549 cells spontaneously released type I sTNF-R (sTNF-RI) into the culture medium. Hydrogen peroxide accelerated the release of sTNF-RI from the A549 cells time- and dose- dependently. Stimulated release of sTNF-RI by hydrogen peroxide or phorbol myristate acetate (PMA) was inhibited by pretreatment with the intracellular hydroxyl radical scavengers dimethyl sulfoxide and dimethyl thiourea. A synthetic metalloproteinase inhibitor (KB-R8301) inhibited not only spontaneous release of sTNF-RI but also shedding enhanced by hydrogen peroxide and PMA. Preincubation with a protein kinase C inhibitor, calphostin C, downregulated the hydrogen peroxide- or PMA-induced shedding of sTNF-RI. Neither genistein, a tyrosine kinase inhibitor, nor H-89, a protein kinase A inhibitor, inhibited shedding of sTNF-RI by hydrogen peroxide and PMA. Although the surface expression of TNF-R assessed by 125I-TNF specific binding was decreased in the presence of hydrogen peroxide or PMA, TNF-RI mRNA transcript levels remained unchanged. These results show that hydrogen peroxide is involved in the activation of metalloproteinase and protein kinase C responsible for the shedding of sTNF-RI. Accordingly, ROIs may alter TNF action by enhanced shedding of sTNF-RI and reducing its surface receptor expression.

Hypersensitivity pneumonitis induced by spores of Pholiota nameko.

A 42-year-old man experienced recurrent episodes of nonproductive cough, fever, and dyspnea on exertion. He had worked as a mushroom farmer for 10 years. The diagnosis of hypersensitivity pneumonitis was confirmed immunologically by detecting a precipitin to spores of Pholiota nameko but not to other antigens. After separation from the antigen along with an addition of corticosteroid therapy, the symptoms, inflammatory findings and a reduced level of PaO2 quickly subsided.

[Thirteen-week repeated dose toxicity by oral administration of trandolapril (RU44570) with 4-week recovery test in beagles].

Trandolapril (RU44570) was orally administered to dogs at a daily dose of 2.5, 25 or 250 mg/kg for 13 weeks. After the administration period, 25 and 250 mg/kg groups were observed for recovery for 4 weeks. The results obtained are as follows: 1. One male in the 250 mg/kg group showed decrease of food consumption and body weight, stomatitis, hematemesis, decumbence, hypothermia, and finally loss of reactivity to stimuli. This animal was killed because of these severe changes on the 39th day of administration. Among the surviving animals, a temporary loss of body weight was observed in a few animals of the 25 and 250 mg/kg groups, and a decreased food consumption was sporadically seen in a few animals of the 250 mg/kg group during the administration period. No abnormal changes were found in the clinical observation and water intake in the surviving animals. 2. The changes attributable to the pharmacological effect of RU44570 were a decreased activity of the angiotensin-converting enzyme, increases in plasma renin activity and urine volume, and decreases in specific gravity and concentrations of Na, K and Cl in the urine of every administration group. A decrease in blood pressure and an increase in the PAS and Bowie positive granules in the juxtaglomerular cells were also found in the 25 and 250 mg/kg groups. In addition, thickening of the afferent arteriolar wall of the glomeruli, a basophilic change of the renal tubular epithelial cells, and localized atrophy and hypertrophy of the renal tubules were observed in the 25 and 250 mg/kg groups, and increases in BUN, ALP and creatinine, and a slight dilation of the renal tubules were seen in the 250 mg/kg group. These observations indicated that RU44570 affected renal structure at a dose of 25 mg/kg or more renal function at a dose of 250 mg/kg. The animal killed in a moribund state showed nephrosis which consisted mainly of a moderate dilation of the renal tubules and vacuolation of the renal tubular epithelial cells, stomatitis, severe hemorrhage and necrosis with neutrophil infiltration in the fundus of the glandular stomach, atrophy of the hemopoietic system, and ectopic calcification in the heart, kidneys, stomach, trachea and alveolar wall. Changes in the kidneys similar to those observed in other animals were also detected. These changes suggested that this animal lapsed into a moribund state due to renal dysfunction and the resultant uremia.(ABSTRACT TRUNCATED AT 400 WORDS)

Recent developments in the organic synthesis of lipid A in relation to biologic activities.

For elucidation of a relationship between chemical structure and biologic activity of lipid A, syntheses of 13 derivatives of glucosamine beta (1-6) disaccharide, which were acylated at 3, 4, and 6'-hydroxyl groups and/or phosphorylated at positions 1 and 4', were carried out. The results showed the importance of the presence of the phosphate group at either position 1 or 4' and of the beta-hydroxyl group in acyl function, particularly in N-linked fatty acids, for the exhibition of biologic activities characteristic of lipid A. New evidence on the positions of fatty acids in natural lipid A of Escherichia coli species obtained by means of 2D-H nuclear magnetic resonance was also demonstrated; the findings indicate that the 3'-hydroxyl group is acylated and the 6'-hydroxyl group in the molecule must be free.

Further study of biological activities of chemically synthesized analogues of lipid A in artificial membrane vesicles.

In the previous paper [Eur. J. Biochem. 124, 405 (1982)], we demonstrated that chemically synthesized lipid-A analogues such as the 1-monophosphate or 1,4'-diphosphate of 6-O-(2-deoxy-2-tetradecanoylamino-6-O-tetradecanoyl-D-glucopyra nos yl)-2-deoxy-2-tetradecanolyamino-3,4-di-O-tetradecanoyl-D-gluco pyr anose enhanced immunogenicity of liposomal model membranes sensitized with amphipathic antigen when they were incorporated in the same liposomes. Here we extend the observation by testing the recently synthesized analogues including diglucosamine analogues carrying hydroxy and acyloxy fatty acids. Among the analogues tested, those which showed higher adjuvant and mitogenic activities in the liposomal system were N-acylated and O-acylated beta-1,6-linked D-glucosamine disaccharides carrying either amide-bound 3-hydroxytetradecanoic acids in addition to phosphate in position 1 of the reducing sugar or amide-bound 3-tetradecanoyloxytetradecanoic acids. The analogue carrying both amide-bound 3-hydroxytetradecanoic acids and phosphate in position 4 of the non-reducing sugar showed weak adjuvant activity and marginal mitogenic activity.

Mitogenic and polyclonal B cell activation activities of synthetic lipid A analogues.

The activation of murine B cells and macrophages by synthetic lipid A analogues, solubilized with triethylamine and complexed with bovine serum albumin, were investigated in vitro. The analogues used are nonphosphorylated, C-1 or C-4' monophosphorylated and C-1,4' diphosphorylated derivatives of beta-1,6-linked D-glucosamine disaccharide possessing both ester- and amide-bound fatty acid substituents. They were divided into 4 groups, A, B, C and D in terms of the fatty acid substitution. Ester- and amine-bound fatty acids of the analogues are both tetradecanoic acids (C14) in group A, C14 and (R)-3-hydroxytetradecanoic acids (C14-OH) in group B, both C14-OH in group C and C14-OH and (R)-3-tetradecanoyloxytetradecanoic acids in group D. Mitogenic activity was exhibited in spleen cells from C3H/HeN mice by the C-1 monophosphorylated analogues in groups A and B, and by the C-4' monophosphorylated analogues in groups B, C and D, but not in cells from C3H/HeJ mice. Nonphosphorylated analogues in groups A, B and C, and C-4' monophosphorylated analogue in group A showed negative mitogenic activity. Only the nonphosphorylated analogue in group D exhibited mitogenic activity in spleen cells from C3H/HeJ mice as well as those from C3H/HeN mice. None of the other analogues exhibited the activity in C3H/HeJ spleen cells. Polyclonal B cell activation activity was exhibited by the C-1 monophosphorylated analogues in groups A and B, and by the C-4' monophosphorylated analogues in groups C and D. Nonphosphorylated analogues in all groups and C-4' monophosphorylated analogues in groups A and B showed negative PBA activity. None of the analogues tested could induce any cytostatic macrophages from thioglycollate-elicited peritoneal macrophages.

Interferon-inducing, pyrogenic and proclotting enzyme of horseshoe crab activation activities of chemically synthesized lipid A analogues.

Interferon-inducing, pyrogenic and proclotting enzyme of horseshoe crab activation activities of chemically synthesized lipid A analogues were investigated and compared with the same activities of a natural lipid A. These analogues are nonphosphorylated, C-1 or C-4' monophosphorylated and C-1,4' bisphosphorylated derivatives of beta-1,6-linked D-glucosamine disaccharide possessing both ester-bound and amide-bound fatty acid substituents. Fatty acid substituents of the analogues are tetradecanoyl (C14), (R)-3-hydroxytetradecanoyl (C14-OH) or (R)-3-tetradecanoyloxytetradecanoyl [C14-O-(C14)] groups. The biological activities of the samples were assayed after solubilization with triethylamine and complexing with bovine serum albumin. Interferon-inducing activity was exhibited by both the C-1 monophosphorylated compounds examined. Ester-bound and amide-bound fatty acid substituents of these compounds are both C14 or C14 and C14-OH, respectively. Nonphosphorylated, C-4' monophosphorylated and C-1,4' bisphosphorylated compounds possessing the same fatty acid substituents as those of the C-1 monophosphorylated compounds showed no detectable interferon-inducing activity. C-4' monophosphorylated compounds possessing C14-OH as ester-bound and C14-OH or C14-O-(C14) as amide-bound fatty acid substituents exhibited interferon-inducing activity, but nonphosphorylated compounds possessing the same fatty acid substituents did not. None of the analogues exhibited significant pyrogenicity nor proclotting enzyme of horseshoe crab activation activity under the conditions employed in this study.

Immunobiological activities of synthetic lipid A analogs and related compounds as compared with those of bacterial lipopolysaccharide, re-glycolipid, lipid A, and muramyl dipeptide.

Thirteen acylated and phosphorylated derivatives of beta-1,6-linked glucosamine disaccharide (lipid A analogs), which were synthesized after the structural model of Salmonella-type lipid A, and seven similar derivatives of glucosamine monosaccharide (lipid A-related compounds) were studied for their immunobiological activities. These included mitogenicity and polyclonal B cell activation enhancement of migration of monocytes and polymorphonuclear leukocytes derived from human peripheral blood, stimulation of guinea pig peritoneal macrophages, activation of human complement, and stimulation of serum antibody production and induction of delayed-type hypersensitivity against ovalbumin in guinea pigs. Comparisons were made with lipid A, RE-glycolipid, lipopolysaccharide of natural sources, and a well-known synthetic adjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine. Some of the lipid A analogs were found to manifest the mitogenic, polyclonal B cell-activating macrophage-stimulating, complement-activating, and immunostimulating activities, although the observed activities were generally far less than those of natural products in intensity and efficiency. Other immunobiological effects exhibited by most of the synthetic lipid A analogs were the enhancement of migration of monocytes and polymorphonuclear leukocytes. It is premature to draw definite conclusions on structure-activity relationships, since a few compounds which were active in some assay systems were scarcely active in other assays. However, an indisputable fact was that beta-1,6-glucosamine disaccharide 1 alpha,4'-diphosphate, which carries two amide-bound (R)-3-hydroxytetradecanoyl and three ester-bound tetradecanoyl residues, and thus has the structure most closely resembling natural lipid A among test compounds in this study, was definitely active in all of the present assay systems. However, its potency was generally much less than natural products. Some of glucosamine monosaccharide derivatives, especially N-(R)-3-[(R)-3-hydroxytetradecanoyloxy]tetradecanoyl glucosamine, also exerted all of the in vitro activities described above. This fact suggests that a glucosamine disaccharide structure may not necessarily be a prerequisite as far as the in vitro immunobiological activities tested are concerned.

Biological activity of chemically synthesized analogues of lipid A. Demonstration of adjuvant effect in hapten-sensitized liposomal system.

The adjuvant effect of synthetic analogues of lipid A was investigated in mice in vivo. Two of the five synthetic lipid A analogues showed adjuvant activity when they were incorporated into dipalmitoylglycerophosphocholine-cholesterol-dipalmitoylglycerophosphate liposomes sensitized with trinitrophenylaminocaproylglycerophosphoethanolamine. The minimum structure for an adjuvant effect in the liposomal immunogen system was 1-monophosphate of 6-0-(2-deoxy-2-tetradecanoylamino-6-0-tetradecanoyl-D-glucopyranosyl)-2-deoxy-2 -tetradecanoyl-amino-3,4-di-O-tetradecanoyl-D-glucopyranose.

Stimulation of nonspecific resistance to infection induced by 6-O-acyl muramyl dipeptide analogs in mice.

The experimental system utilized in investigating the correlation between the chemical structures of muramyl peptides and their protective activities in the sepsis type of systemic infections caused by Escherichia coli was applied in evaluating the enhancement of resistance to infection induced by 32 synthetic glycopeptide analogs, including 6-O-acyl derivatives and 1-alpha-O-benzyl derivatives of muramyl dipeptide (N-acetyl muramyl-L-alanyl-D-isoglutamine). In assessing the 6-O-acyl derivatives of muramyl dipeptide, we found that the degree of protective activity was attributable to the kinds of fatty acids introduced. Acylation of the 6-hydroxy group on the muramic acid moiety in muramyl dipeptide with natural mycolic acid or a synthetic fatty acid possessing either an alpha-branched or an alpha-branched, beta-hydroxylated group resulted in a decrease in or a disappearance of the protective activity of muramyl dipeptide. Acylation with a normal fatty acid or an iso fatty acid resulted in a retention or enhancement of muramyl dipeptide activity. The activity of acylated derivatives containing linear fatty acids was stimulated by increasing the chain length up to 18 carbon atoms. The highest degree of protective activity occurred with the derivatives acylated with straight-chain fatty acids, particularly with the derivatives acylated with palmitic acid and arachidic acid. Benzylation of the 1-hydroxy group of muramyl dipeptide resulted in a decrease in or a loss of protective activity.

Mitogenic effects of bacterial cell walls, their fragments, and related synthetic compounds on thymocytes and splenocytes of guinea pigs.

Stimulation of [(3)H]thymidine incorporation of thymocytes and splenocytes from guinea pigs by various bacterial cell walls and their peptidoglycans, by enzymatic digests, and by synthetic muramyl dipeptides was studied as an indication of mitogenic activity. Cell wall and peptidoglycan preparations, isolated from 19 strains belonging to 18 different species, definitely increased [(3)H]thymidine incorporation of thymocytes as well as splenocytes, regardless of mycolic acid contents as a non-peptidoglycan component. Both the cell walls from Nocardia corynebacteriodes (containing mycolic acids) and those from Streptomyces gardneri (lacking mycolic acids) showed far stronger mitogenic activities on splenocytes than other cell walls (stimulation index, 25 to 30). Furthermore, water-soluble enzymatic digests, notably the endopeptidase digests, which generally were greater in degree of polymerization of peptidoglycan subunits than the glycosidase digests obtained from representative cell walls, were found to have as distinct a stimulating activity on splenocytes as the original cell walls. In contrast, solubilization of the cell walls by enzymes, irrespective of endopeptidases or glycosidases, was accompanied by disappearance of the mitogenic activity on thymocytes. On the other hand, studies with synthetic 6-O-acyl-MurNAc-l-Ala-d-isoGln preparations (6-O-acyl-MDPs) revealed that 6-O-stearoyl-MDP and 6-O-(2-tetradecylhexadecanoyl)-MDP, unlike MDP, had distinct mitogenic activity on thymocytes, whereas their activity on splenocytes was rather weaker than MDP itself. The findings presented here suggest that MDP is the minimal structure for the mitogenic activities of bacterial cell walls on guinea pig splenocytes, but that MDP, though distinctively active by itself, requires a polymerized form to exert effectively its inherent stimulating activities on splenocytes. On the other hand, on thymocytes, MDP, unless it takes a particular form or has appropriate additive groups, cannot exert its mitogenic activities.

Macrophage activation by bacterial cell walls and related synthetic compounds.

Activation of peritoneal macrophages from guinea pigs by various bacterial cell walls, M-1 endo-N-acetylmuramidase enzymatically digested bacterial cell walls and synthetic muramyl dipeptides was studied in terms of stimulation of [14C] glucosamine incorporation. All test bacterial cell wall preparations significantly increased a [14C]glucosamine uptake by the macrophages. Some of the water-soluble M-1 enzyme digests also exerted stimulating effects on macrophages, although the activity of the digests was found to be weaker than those of original cell walls. Furthermore, an adjuvant-active synthetic MurNAc-L-Ala-D-isoGln (MDP) showed a weak but significant activity, whereas an adjuvant-inactive analog, MurNAc-L-Ala-L-iso-Gln, did not show a significant activity, at least with the dose of 100 microgram. Additional studies with 6-O-acyl derivatives of MDP revealed that 6-O-(2-tetradecylhexadecanoyl)-MDP and 6-O-(3-hydroxy-2-tetradecyl-octadecanoyl)-MDP exhibit stronger macrophage-stimulating effects than MDP. It can be concluded from the above findings that MDP is the essential structure responsible for stimulating the activity of cell walls on guinea pig peritoneal macrophages, but it requires a particle state, which results from an additive character of lipophilicity, to exert the activity fully and effectively.