Toll-like receptor 4 mediates LPS-induced release of nitric oxide and tumor necrosis factor-alpha by embryonal cardiomyocytes: biological significance and clinical implications in human pathology.

Lipopolysaccharide (LPS) the major structural component of the outer membrane of Gram-negative bacteria contributes to the cardiovascular collapse and death observed in septic patients, as well as in the immunocompromised host. LPS activates multiple cells to release proinflammatory cytokines, nitric oxide (NO) and other reactive molecules able to depress cardiac functions. It has been appreciated that the pattern recognition receptor, TLR4, is a signalling receptor for LPS, but its role in the embryonal cardiomyocytes is poorly understood. Here, we provide evidence for TLR4-dependent functional responses by LPS treated embryonal cardiomyocytes. It will be reported that LPS is able to induce TNF-alpha and NO release from cultured cardiomyocytes, while molecular and morphological evidence demonstrates the expression of TLR4 on surface membrane of embryonal cardiomyocytes. LPS-induced signalling was studied evaluating the expression of the extracellular signal-regulated kinase (ERK) and signal transducer and activators of transcription (STAT) protein families in response to LPS. The role of TLR4 was investigated with blocking assays using monoclonal antibody against this endotoxin receptor. Our results indicated that LPS-induced activation of signal transduction in embryonal cardiomyocytes occurs by a TLR4-dependent mechanism. In summary, chick embryonal cardiomyocytes may constitute a valid experimental model in order to study the LPS induced inflammatory responses by cardiomyocytes, useful not only to identify the signalling pathways evoked by endotoxin receptor, including TLR4, but also to suggest therapeutic targets for the control of myocardial dysfunction induced by infectious agents. In this respect, in elderly a continuous leakage of LPS from gut flora and/or external environment should be regarded as a possible cause of cardiac failure and, therefore, adequately prevented or treated.

Role of multidetector-row CT in assessing the source of arterial haemorrhage in patients with pelvic vascular trauma. Comparison with angiography.

PURPOSE: We investigated the role of multidetector-row computed tomography (MDCT) in identifying active bleeding and its source in polytrauma patients with pelvic vascular injuries with or without associated fractures of the pelvis. MATERIALS AND METHODS: From January 2003 to December 2007, 28 patients (19 men and nine women, age range 16-80 years) with acute symptoms from blunt pelvic trauma and a drop in haematocrit underwent MDCT and angiography. Conventional radiography of the pelvis was performed in all patients at the time of admission to the emergency department. MDCT was performed with a four-row unit in 15 patients and a 16-row unit in the remaining 13 patients. The study included whole-body CT to identify craniocerebral, vertebral, thoracic, abdominal and pelvic injuries. CT was performed before and after rapid infusion (4-5 ml/s) of intravenous contrast material (120 ml) using a power injector. A triphasic contrast-enhanced study was performed in all patients. MDCT images were transferred to a workstation to assess pelvic fracture, site of haematoma and active extravasation of contrast material, visibility of possible vascular injuries and associated traumatic lesions. At angiography, an abdominal and pelvic aortogram was obtained in all cases before selective catheterisation of the internal iliac arteries and superselective catheterisation of their branches for embolisation purposes. Results related to identifying the source of bleeding at MDCT were compared with sites of bleeding or vascular injury identified by selective pelvic angiography. The sensitivity and positive predictive value (PPV) of MDCT were determined. RESULTS: MDCT allowed us to identify pelvic bleeding in 21/28 patients (75%), with most cases being detected in the delayed contrast-enhanced phase (13/21 cases, 61.9%). Injured arteries were identified on MDCT in 12/21 cases (57%): the obturator artery (n=9), internal iliac artery (n=6), internal pudendal artery (n=6) and superior gluteal artery (n=5) were most frequently injured. In 8/21 patients (28.6%), more than one artery was injured. Among the 12 patients in whom MDCT showed the presence of pelvic haemorrhage, there was agreement between MDCT and angiography in ten cases. Angiography confirmed the site of bleeding detected on MDCT and identified a second arterial haemorrhage in one patient. There was no agreement between MDCT and angiography in the last patient. MDCT showed a sensitivity of 42.85% and a PPV of 100% in identifying the injured arteries. CONCLUSIONS: Arterial haemorrhage is one of the most serious problems associated with pelvic fracture, and it remains the leading cause of death attributable to such fractures. MDCT provides diagnostic information regarding the presence of small pelvic fractures and, thanks to the contrast-enhanced angiographic technique, it is capable of identifying pelvic bleeding, with the demonstration in some cases of it source. The presence of contrast material extravasation is an indicator of injury to a specific artery passing through the region of the pelvis where the extravasation is noted on MDCT. Urgent angiography and subsequent transcatheter embolisation are the most effective methods for controlling ongoing arterial bleeding in pelvic injuries.

[Diaphragmatic rupture with intrathoracic hepatic dislocation. Two case report]. / Rottura diaframmatrica con erniazione intratoracica del fegato. Presentazione di due casi clinici.

The authors show two cases of diaphragmatic rupture by blunt trauma with intra thoracic liver dislocation due to an accident on the street. The preoperative diagnosis has been based on the CT scan. The laparotomy, central for a patient and sub costal bilateral for the other one, gave the chance to repair the diaphragmatic defect directly and to deal with the associated lesions existing in both the casualties. No prosthetic material has been used and the reconstruction of the diaphragmatic defect has been conducted through a interrupted suture with non absorbable material. According to our experience the reparation of the diaphragmatic defect has always been conducted through a laparotomic approach and without using any prosthetic material. We repute that in these cases the laparotomic approach is the best one, which permits not only the reparation of the diaphragmatic defect but also the correct management of the related lesions which could be present. Usually we use a interrupted suture with non absorbable material for minor lesions and a continuous one, double layered if possible, for the major ones.

Structural relatedness between the 18S rRNA genes and the formyl peptide receptor genes: new insights into the phylogenesis of immune receptors.

In this study the authors examined the sequences of the ribosomal 18S rRNA of Drosophila and man and 16 mRNA sequences coding for different members of the family of the mammalian formyl peptide receptors (FPRs). The positions in the sequences of all >or=7-base oligonucleotide identities occurring in at least one of the 18S rRNAs and one of the FPR mRNAs were recorded. On the basis of the positional data, the Drosophila 18S-FPR and human 18S-FPR distances (in nucleotides) were determined for each identity. Then the actual frequency distribution of the distances (grouped into 200-unit classes) was derived. The theoretical frequency distribution of distances was also calculated under the assumption of non-relatedness between the 18S and FPR sequences. Comparison between the theoretical and the actual distributions showed that at class -500 (range from - 400 to - 600) of the 18S-FPR values the actual frequency was significantly (p < 0.01) higher than the theoretical frequency, in both Drosophila and man, suggesting that the second section of the FPR genes (approximately from nucleotide 400 to the end of sequence) may be structurally related to the first section of the ribosomal 18S genes (approximately nucelotides 1-650). The authors advance the hypothesis that the two families of genes may have used common ancestral raw genetic materials in the building of the extant sequences.

Multidetector-row CT evaluation of nontraumatic acute thoracic aortic syndromes.

Acute thoracic aortic syndromes encompass a spectrum of emergencies presenting with acute chest pain and marked by a high risk of aortic rupture and sudden death. These include nontraumatic disease entities of the thoracic aorta, namely, dissection, intramural haematoma, penetrating atherosclerotic ulcer and aneurysm rupture. In clinical practice, the most frequent imaging procedure used in the diagnostic assessment of these diseases is computed tomography (CT), which, thanks to recent technological developments [multidetector-row computed tomography (MDCT)], affords important diagnostic possibilities and very interesting future perspectives. This paper on the use of MDCT in the evaluation of acute thoracic nontraumatic aortic syndromes illustrates the examination technique and the key imaging findings related to each disease. Moreover, the role of MDCT for planning specific treatment is also highlighted.

Assessment of the enzymuria resulting from gentamicin alone and combinations of gentamicin with various beta-lactam antibiotics.

OBJECTIVE: To determine the propensity of beta-lactam antimicrobials to ameliorate or potentiate aminoglycoside-induced renal enzymuria. DESIGN: Two open, randomized, double-blind, parallel-group studies were conducted in young, healthy, male volunteer subjects. Using a common protocol, 24-hour urine collections were analyzed for the renal tubular enzymes alanine aminopeptidase (AAP) and N-acetyl-beta-D-glucosaminidase (NAG), as well as for creatinine. Antimicrobial combinations studied included gentamicin plus placebo and gentamicin plus ticarcillin/clavulanate (protocol 1); and gentamicin plus placebo, gentamicin plus piperacillin, and gentamicin plus ceftazidime (protocol 2). The antimicrobial regimens were administered for 7 days. Eight subjects completed each treatment group. RESULTS: There were no significant differences between treatment groups with regard to urine creatinine excretion or serum gentamicin concentrations in either protocol. Enzymuria (AAP [p = 0.039] and NAG [p = 0.337]) was decreased in the gentamicin plus ticarcillin/clavulanate treatment compared with that in the gentamicin plus placebo treatment. Increased enzymuria, as indicated by increased urine concentrations of AAP and NAG, was observed in the gentamicin plus ceftazidime treatment (p < 0.05) compared with the other two treatments. CONCLUSIONS: Based on relative enzymuria, ticarcillin/clavulanate may be renal protective. Piperacillin neither potentiated nor ameliorated aminoglycoside-induced enzymuria. Since acute elevations in AAP and NAG reflect insults to the kidney, these studies suggest that ceftazidime may enhance aminoglycoside-induced renal injury. Piperacillin had no effect on enzymuria and would appear not to enhance or protect against aminoglycoside-induced renal injury.

A rapid sensitive monoclonal assay for lipid A in solution.

Lipid A is the toxic component of bacterial endotoxins (LPS) and LPS has been thought to be clinically important in Gram-negative bacterial sepsis, as well as in non-bacteremic states where endotoxemia of enteric origin may be deleterious. The presently accepted method of detecting both LPS and its common lipid A moiety is the Limulus lysate amebocyte assay (LAL), but this test cross-reacts with non-bacterial antigens and serum contains natural inhibitors to the reaction. As an extension of previous work using a polyclonal antibody, we have developed a rapid and sensitive monoclonal antibody assay for lipid A. This 3-step inhibition ELISA is reproducible in aqueous solutions and capable of detecting less than 10 pg/ml of this shared toxic endotoxin component. The assay does not detect intact LPS but acid hydrolysis releases the active lipid A for reaction. While already valuable in detecting lipid A in biological solutions, the presence of naturally occurring anti-lipid A immunoglobulins in serum interfere with the reaction and cause false positives in this inhibition assay. Clinical usefulness of the assay will depend on removal of these antibodies from serum prior to testing.

IgA antibody to lipid A in alcoholic liver disease.

To find out whether the increased immunoglobulin A (IgA) levels commonly reported in patients with alcoholic liver disease (ALD) are part of the immune response to gut-derived endotoxin antigen, an enzyme-linked immunoabsorbent assay was used to measure serum levels of antibodies to lipid A (the shared core of various lipopolysaccharides and the biologically active component of endotoxins) in patients with various diseases and in controls. Of 41 patients with ALD, 35 had significantly raised titres of IgA anti-lipid A. Rises of antilipid A were found in the secretory fraction of IgA as well, and titres of IgG antibody were also consistently increased. IgM titres were high in only 7 of 35 ALD patients tested. Patients with other diseases did not show significantly high titres of IgA antibodies to lipid A even though many had rises in total concentration of their serum IgA.

Antigenic heterogeneity of the non-serogroup antigen structure of Neisseria gonorrhoeae lipopolysaccharides.

Studies of the antigenic structure of the polysaccharide component of gonococcal lipopolysaccaride (LPS) indicated that the non-serogroup antigen structure is antigenically heterogeneous. Immunodiffusion studies of Gc4 strain 8551 indicated that in addition to the Gc serogroup determinent, this polysaccharide contains two other sets of determinants, one which is shared with the other five Gc serogroups and a second which is shared by the Gc1 and Gc3 serogroups. Enzyme-linked immunosorbent assay (ELISA) studies confirmed the immunodiffusion, documented qualitative differences, and suggested that quantitative differences may exist in the common determinant from serogroup to serogroup. As measured by ELISA inhibition, native LPS showed the same antigenic arrangement as the purified LPS polysaccharides. Studies with LPS-derived polysaccharides from nonprototype strains in immunodiffusion gave results identical to the prototype strains. This antigen variation, although distinct from serogroup specificity, was related to the serogroup determinant.

A rapid, semi-automated procedure for the evaluation of leukocyte locomotion in the micropore filter assay.

A commerically available bacterial colony counter has been modified to allow rapid, accurate, semi-automated evaluation of cell numbers in the micropore filter assay for chemotaxis. The method is valuable for objective, rapid evaluation of cell counts at various levels through the filter, as well as counts on the distal surface of the filter. Coupled with a programmable calculator, this instrument had made feasible a new method of assessing random migration by the regression line analysis, which discriminates between migration rate and mass migration of cells. This combination of equipment may thus serve as a considerable time saving accessory to laboratories engaged in cell locomotion research, but also will allow more rigorous assessment of differences among specific populations of cells.

Degradation of the polysaccharide component of gonococcal lipopolysaccharide by gonococcal and meningococcal sonic extracts.

An extract made from the supernatant of Neisseria gonorrhoeae Gc2 strain 1291 degraded the Gc2 polysaccharide antigen. Chemical analysis of this polysaccharide indicated it contains glucose, galactose, glucosamine, galactosamine, glucosamine-6-phosphate, heptose, 2-keto-3-deoxyotonate, and ethanolamine and is the polysaccharide component of gonococcal lipopolysaccharide. Degradation of the polysaccharide by sonic extracts resulted either in complete loss of antigenicity and immunogenicity or in partial degradation to subunits that could inhibit the Gc2-specific hemagglutination inhibition. The factors responsible for degradation were destroyed by heating at 100 degrees C for 5 min or by Pronase digestion, but were unaffected by ribonuclease, deoxyribonuclease, Mg2+, Ca2+, or ethylenediaminetetraacetic acid. The process was pH dependent, with optimal activity occurring at pH 7. Sonic extract supernatants from group B and C meningococcal strains contained degrading properties, whereas similar extracts produced from Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Streptococcus pneumoniae type II failed to degrade the Gc2 polysaccharide.