Relationship between soluble CD14, lipopolysaccharide binding protein, and the alveolar inflammatory response in patients with acute respiratory distress syndrome

The effects of bacterial endotoxin (lipopolysaccharide, LPS) are amplified by lipopolysaccharide binding protein (LBP) and CD14, resulting in cellular activation at very low concentrations of LPS. To investigate the importance of this pathway in acute lung injury, we measured LPS, LBP, and soluble CD14 (sCD14) in the bronchoalveolar lavage fluid (BAL) of 82 patients with acute respiratory distress syndrome (ARDS). LBP and sCD14 increased markedly in BAL of patients with ARDS. sCD14 and LBP each were strongly related to BAL total protein and polymorphonuclear neutrophil (PMN) concentration, whereas LPS concentration was not. Multivariate analyses showed sCD14 to be strongly related to BAL total protein, even after controlling for LPS and LBP concentrations. sCD14 was strongly and independently related to PMN concentration, after controlling for BAL LPS, LBP, and interleukin-8 (IL-8). The BAL LPS concentration was not strongly related to either BAL total protein or BAL PMN. The BAL sCD14 and LBP values were similar in all subgroups of patients with ARDS, and were not related to survival. The serum LBP and sCD14 were elevated in ARDS, but were not related to BAL total protein, LBP, sCD14, PMN, or clinical outcome. Thus, LBP and sCD14 reach high concentrations in the lungs of patients with ARDS, and BAL sCD14 is strongly related to two major indices of lung inflammation: total protein and PMN concentration. CD14-dependent mechanisms may contribute to lung inflammation in ARDS.     

A new method for seed oil body purification and examination of oil body integrity following germination

Plant seeds store triacylglycerols as energy sources for germination and postgerminative growth of seedlings. The triacylglycerols are preserved in small, discrete, intracellular organelles called oil bodies. A new method was developed to purify seed oil bodies. The method included extraction, flotation by centrifugation, detergent washing, ionic elution, treatment with a chaotropic agent, and integrity testing by use of hexane. These processes subsequently removed non-specifically associated or trapped proteins within the oil bodies. Oil bodies purified by this method maintained their integrity and displayed electrostatic repulsion and steric hindrance on their surface. Compared with the previous procedure, this method allowed higher purification of oil bodies, as demonstrated by SDS-PAGE using five species of oilseeds. Oil bodies purified from sesame were further analyzed by two-dimensional gel electrophoresis and revealed two potential oleosin isoforms. The integrity of oil bodies in germinating sesame seedlings was examined by hexane extraction. Our results indicated that consumption of triacylglycerols reduced gradually the total amount of oil bodies in seedlings, whereas no alteration was observed in the integrity of remaining oil bodies. This observation implies that oil bodies in germinating seeds are not degraded simultaneously. It is suggested that glyoxisomes, with the assistance of mitochondria, fuse and digest oil bodies one at a time, while the remaining oil bodies are preserved intact during the whole period of germination.     

Scarring trachoma is associated with polymorphism in the tumor necrosis factor alpha (TNF-alpha) gene promoter and with elevated TNF-alpha levels in tear fluid

Tumor necrosis factor alpha (TNF-alpha) may play a central role in the disease pathogenesis which occurs as a consequence of chlamydial infection. To investigate the importance of TNF-alpha gene promoter polymorphisms and TNF-alpha levels in tear fluid in scarring trachoma, a large matched-pair case-control study was performed in The Gambia. The -308A allele was present in a higher proportion of patients (28.4%) than controls (18.4%), with an increasing association for homozygotes (chi2 for trend, P = 0.032; allele frequency, 0.163 in patients and 0.099 in controls; chi2, P = 0.025). For the -238A allele, the association was similar but not significant. The disease association was highly significant when the number of either -308A or -238A sites in an individual was considered (P = 0.003). TNF-alpha promoter alleles are tightly linked to some HLA class I and II alleles, but multivariate analysis confirmed that the disease associations were independent of HLA, although a class I allele, A*6802, is also associated with disease. TNF-alpha was more frequently detected in tear samples from patients (27.6%) than from controls (15.9%), increasingly so for higher levels of detectable TNF-alpha (P = 0.015). Among patients, detectable TNF-alpha in tears was highly associated with the presence of ocular chlamydial infection (P < 0.001). The results indicate that TNF-alpha plays a major role in the tissue damage and scarring which occurs as a consequence of Chlamydia trachomatis infection.     

Retrospective analysis of 19 cases of isolated torsion of the splenic pedicle in dogs

Nineteen cases of isolated splenic torsion in the dog were reviewed retrospectively. Perioperative data were analysed in order to determine prognostic indicators of morbidity and mortality for animals with this disease. This Great Dane and German shepherd dog were found to be at increased risk for this condition. The historical features of splenic torsion were non-specific and included intermittent signs of gastrointestinal disease and abdominal discomfort. Mean duration of malaise was eight days, but 17 dogs were presented because of a rapid deterioration in their condition. Abdominal ultrasound findings assisted in the decision to perform surgery in all the dogs on which it was performed. Eighteen animals had splenic torsion confirmed at surgery and were treated by total splenectomy. All 18 surgically managed dogs survived. Analysis of pre- and postoperative data failed to reveal preoperative parameters that consistently predicted postoperative complications.     

8-epi PGF2 alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo

BACKGROUND: Myocardial reperfusion is believed to be associated with free radical injury. However, indexes of oxidative stress in vivo have been limited by their poor specificity and sensitivity. Isoprostanes are stable products of arachidonic acid formed in a nonenzymatic, free radical-catalyzed manner. We have developed a sensitive and specific assay for one of these compounds, 8-epi prostaglandin (PG) F2 alpha. METHODS AND RESULTS: To address its utility as an index of oxidative stress during coronary reperfusion, we measured urinary levels by gas chromatography/mass spectrometry in a canine model of coronary thrombolysis, in patients with acute myocardial infarction treated with thrombolytic therapy, and in patients after elective coronary artery bypass surgery. Urinary 8-epi PGF2 alpha was unchanged after circumflex artery occlusion in a canine model of coronary thrombolysis (n = 13; 437.2 +/- 56.4 versus 432.7 +/- 55.2 pmol/mmol creatinine) but increased significantly (P < .05) immediately after reperfusion (553.8 +/- 64.7 pmol/mmol). Urinary levels were increased (P < .001) in patients (n = 12) with acute myocardial infarction given lytic therapy (265.8 +/- 40.8 pmol/mmol) compared with age-matched control subjects (n = 20; 91.5 +/- 11.8 pmol/mmol) and patients with stable coronary disease (n = 20; 95.7 +/- 6.3 pmol/mmol). Preoperative levels rose from 113.2 +/- 11.8 to 248.2 +/- 86.3 pmol/mmol at 30 minutes into revascularization to 332.2 +/- 82.6 pmol/mmol by 15 minutes after global myocardial reperfusion (P < .05) and dropped to 181.2 +/- 50.4 pmol/mmol at 30 minutes and 120.2 +/- 9.9 pmol/mmol at 24 hours after bypass surgery (n = 5). Corresponding changes in spin adduct formation, found with electron paramagnetic resonance, were noted in 2 patients. CONCLUSIONS: These data support the hypothesis that free radical generation occurs during myocardial reperfusion. Measurement of isoprostane production may serve as a noninvasive index of oxidative stress.     

Long-term observation of subcutaneous tissue reaction to synthetic auditory ossicle (Apaceram) in rats

The present study evaluates histological characteristics of the soft tissue response to long-term implantation of Apaceram discs composed of dense hydroxyapatite in rats. Discs were implanted into the subcutaneous tissue of 76 rats for six to 20 months. Decalcified histological sections stained with haematoxylin and eosin (H & E) and Mallory's azan were examined. Different cell types surrounding implants were counted. The greatest proportion of macrophages was found at six months (13.5 per cent). This proportion gradually decreased to four per cent at 20 months. Small numbers of lymphocytes and foreign body giant cells were observed in every group, but neither neutrophils nor osteogenesis were observed in any specimens. Results of the present study and previous related studies indicate that despite reappearance of a small number of macrophages six months after implantation, Apaceram is useful for reconstructive surgery.     

Two mechanisms for the recapture of extracellular GM2 activator protein: evidence for a major secretory form of the protein

The GM2 activator protein is a small monomeric protein containing a single site for Asn-linked glycosylation. Its only proven in vivo function is to act as a substrate specific cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A. However, we and others have shown it can act as a general glycolipid transporter at neutral pH in vitro. Any other possible in vivo functions would require that some of the newly synthesized activator molecules not be targeted to the lysosome. The lysosomal targeting mechanism for the activator has not been conclusively identified. While earlier reports suggested that it is likely through the mannose-6-phosphate receptor, another more recent report demonstrated that deficient human cells could recapture nonglycosylated, bacterially produced activator, suggesting its use of an alternate targeting pathway. Here, we demonstrate that the mannose-6-phosphate pathway is likely the major intracellular, biosynthetic route to the lysosome, as well as a high affinity recapture pathway for the endocytosis of activator protein from extracellular fluids. Additionally, we show that there exists a second lower affinity recapture pathway that requires its native protein structure, is carbohydrate independent, and likely does not involve its ability to bind glycosphingolipids in the plasma membrane. Finally, we document that the pool of newly synthesized precursor activator protein contains a majority of molecules with a complex-type oligosaccharide, which cannot contain a functional mannose-6-phosphate targeting signal. These molecules makeup the secreted forms of the protein in normal human fibroblasts.     

Prenatal diagnosis of an extensive fetal lymphangioma using ultrasonography, magnetic resonance imaging and cytology

An unusual case of fetal lymphangioma diagnosed before delivery is reported in a second trimester pregnant woman. The lymphangioma was suspected at 28 weeks on the basis of the ultrasound appearances and progression of the lesions with advancing gestation. MRI was used to evaluate the extent and the tissue characteristics of the lesions. Cytology of the fluid aspirated from the cystic lesions showed abundant lymphocytes and macrophages, confirming the diagnosis of a lymphangioma. The parents opted for a pregnancy termination because of the rapid growth of the lesions and the poor prognosis. It is suggested that the combination of these tests could enable the early diagnosis of these tumours at a stage when the lesion is relatively limited and accessible to therapy in utero.     

Rehabilitation of dogs with surgically treated cranial cruciate ligament-deficient stifles by use of electrical stimulation of muscles

OBJECTIVE: To determine effect of electrical muscle stimulation (EMS) on rate and degree of return to function of the limb and development of degenerative joint disease (DJD) after surgical creation and subsequent stabilization of the cranial cruciate ligament (CrCL)-deficient stifle. ANIMALS: 12 clinically normal adult large (19.5 to 31.5 kg) dogs. PROCEDURE: Dogs were anesthetized, and the right CrCL was severed via arthrotomy, destabilizing the stifle. After 3 weeks, the stifle was surgically stabilized. Three weeks later, 6 dogs were subjected to an EMS treatment protocol for the thigh muscles. At 5, 9, 13, and 19 weeks after stifle destabilization, treated (n = 6) and control (n = 6) dogs were evaluated for return of stifle function. Gross and histologic evaluations of the stifles were performed at 19 weeks after stifle destabilization. RESULTS: Treated dogs had significantly (P = 0.001) better lameness score than did control dogs. There was less palpable crepitation of the stifle in treated dogs (P = 0.06); treated dogs also had significantly (P = 0.01) fewer radiographic signs of bone changes. Thigh circumference was significantly (P = 0.02) larger in treated dogs. There was less gross cartilage damage (P = 0.07) in the EMS-treated dogs, but more medial meniscal damage (P = 0.058, cranial pole; P = 0.051, caudal pole). CONCLUSIONS: Improved lameness scores, larger thigh circumference, and decreased radiographically apparent bony changes observed for the treated group of dogs support the hypothesis that dogs treated by EMS after surgical stabilization of the CrCL-deficient stifle had improved limb function, with less DJD, than did dogs treated with the currently accepted clinical protocol of cage rest and slow return to normal activity. However, results of force plate evaluation did not support the hypothesis. Increased meniscal damage in dogs treated by EMS may be cause for concern.     

Incorporation of nitric oxide-releasing crosslinked polyethyleneimine microspheres into vascular grafts

Over the years, many attempts have been made to increase the patency of small- to medium-sized prosthetic vascular grafts. However, none of them has greatly affected long-term rates. Recently, nitric oxide (NO) has been shown to inhibit thrombus formation in such grafts, suggesting that local delivery of NO may help to increase graft patency. This study describes the site-specific delivery of NO by entrapping NO-releasing microspheres in the pores of a vascular graft. NO-releasing polyethyleneimine microspheres (PEIX) were developed using a novel water-in-oil emulsion technique involving chemical crosslinking with a bis-epoxide. The PEIX microspheres were then derivatized with NO forming the [N(O)NO]- moiety of the diazeniumdiolates formerly known as NONOates. These polymeric NO-releasing particles were found to spontaneously release 194 nmol NO/mg with a half-life of over 66 h under physiologic conditions. Fluorescein isothiocyanate-labeled microspheres were then embedded into the pores of a 60-micron nonreinforced Gore-tex vascular graft using a simple evacuation technique and evaluated for microsphere placement and NO release. Scanning electron microscopic analysis showed the microspheres entrapped in the pores of the vascular graft releasing 10 nmol NO/mg with a half-life of 51 h. The microspheres remained entrapped in the graft even after immersion and NO release, as confirmed by fluorescence of the medium. These results suggest that NO-releasing particles can be incorporated into the pores of a vascular graft to deliver therapeutic amounts of NO for the prevention of thrombosis in small-diameter prosthetic grafts.