Monocytes and lymphocytes as active participants in the pathogenesis of experimental shock

We investigated the role played by monocytes and lymphocytes in the pathogenesis of experimental shock. Splanchnic artery occlusion (SAO) shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min followed by reperfusion. Sham operated animals were used as controls. SAO shocked rats had a decreased survival time (80±11 min, while sham shocked rats survived more than 4 h), increased serum (248±21 U/ml) and macrophage (145±15 U/ml) levels of TNF-, enhanced myeloperoxidase (MPO) activity in the ileum (3.38±0.2 U×10^–3/g tissue), decreased number of monocytes, lymphocytes and neutrophils and a profound hypotension. In addition we found an increased expression of vascular cell adhesion molecule-1 (VCAM-1) on aortic endothelium and a reduced percentage of VLA-4 positive monocytes and lymphocytes. Inhibition of TNF- synthesis, reversed the increased endothelial expression of VCAM-1, increased the percentage of integrin VLA-4 positive leukocytes and improved monocyte, lymphocyte and neutrophil count. Furthermore a passive immunization with specific antibodies raised against VCAM-1 (2 mg/kg, i.v. 3 h before SAO) increased survival, reduced MPO activity in the ileum (0.034±0.04 U×10^–3/g tissue) and improved mean arterial blood pressure. Our data suggest that monocytes and lymphocytes participate in the pathogenesis of splanchnic ischaemia-reperfusion injury and may amplify the adhesion of neutrophils to peripheral tissues.by I. Ahnfelt Rønne     

Limiting factors for cytopathological diagnosis of high-grade squamous intraepithelial lesions: a cytohistological correlation between findings in cervical smears and loop electrical excision procedure.

The present study sought possible factors leading to the cytological diagnosis of atypical squamous cells of uncertain significance (ASCUS) in cases of high-grade squamous intraepithelial lesions (HSIL). Based on retrospective histopathological analysis of loop electrical excision procedure (LEEP) products that diagnosed HSIL, two study groups were randomly selected. The first was consisted of cases with two consecutive Papanicolaou (Pap) smears with the diagnosis of ASCUS. The second (control) group was represented by cases diagnosed as HSIL by cytology. From the Pap smears diagnosed as ASCUS, the sampling limitations was different from control group (P < 0.05). The median size of the largest lesion in each case with ASCUS was 2.66 mm (+/- 1.71 mm). In the control group, the median size of the largest lesion was 5.15 mm (+/-2.58 mm) (P < 0.05). The size of the lesion and sample limitations led patients with cervical intraepithelial neoplasms to be diagnosed as ASCUS for two consecutive times, after a 6-mo period.     

Alloy telescopic crowns. Clinical indications

This work involves a partially edentate subject fitted with a denture on conical crowns. Of the methods at present used in patients with few teeth, this is considered one of the most satisfactory for dentist and patient. In cases in which the remaining teeth are moveable, a denture on magnets can be utilized. A correct oral hygiene is emphasized to favour the duration of the tooth-denture combination.     

Beneficial effects of Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic, in zymosan-induced shock

1 The therapeutic efficacy of Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), a novel superoxide dismutase mimetic which scavenges peroxynitrite, was investigated in rats subjected to shock induced by peritoneal injection of zymosan. 2 Our data show that MnTBAP (given at 1, 3 and 10 mg kg-1 intraperitoneally, 1 and 6 h after zymosan injection) significantly reduce in dose dependent manner the development of peritonitis (peritoneal exudation, high nitrate/nitrite and peroxynitrite plasma levels, leukocyte infiltration and histological examination). 3 Furthermore, our data suggest that there is a reduction in the lung, small intestine and liver myeloperoxidase (MPO) activity and lipid peroxidation activity from MnTBAP-treated rats. 4 MnTBAP also reduced the appearance of nitrotyrosine immunoreactivity in the inflamed tissues. 5 Furthermore, a significant reduction of suppression of mitochondrial respiration, DNA strand breakage and reduction of cellular levels of NAD+ was observed in ex vivo macrophages harvested from the peritoneal cavity of zymosan-treated rat. 6 In vivo treatment with MnTBAP significantly reduced in a dose-dependent manner peroxynitrite formation and prevented the appearance of DNA damage, the decrease in mitochondrial respiration and the loss of cellular levels of NAD+. 7 In conclusion our results showed that MnTBAP was effective in preventing the development of zymosan-induced shock.     

Effects of zileuton,a new 5-lipoxygenase inhibitor,in experimentally induced colitis in rats

Intravenous injection of platelet-activating factor (PAF) (0.36 mol/kg b.w.) in mice induced severe hemoconcentration, leucopenia, thrombocytopenia and finally the death of 85% of the tested animals. Combined inhibition of histamine and serotonin by promethazine and chlorpromazine, 6.24 and 3.12 mg/kg b.w. subcutaneously, protected the mice from PAF in part, reducing the death rate to 43%. These drugs did not protect the mice against the PAF-induced hemoconcentration, leucopenia and thrombocytopenia. Sulfinpyrazone (100 mg/kg b.w.) intravenously was the most effective both in protecting mice from PAF-induced death, reducing the death rate to 17%, and from thrombocytopenia, although hemoconcentration persisted. These results indicated that an important component of the PAF-induced systemic effects is mediated by reactions which can be inhibited by sulfinpyrazone. Furthermore, PAF-induced thrombocytopenia is not a direct PAF effect since it can be inhibited by sulfinpyrazone.     

Interrater reliability of the Camberwell Assessment of Need Short Appraisal Schedule

OBJECTIVE: Australian mental health policy aims to introduce evidence-based practice within a community care approach. This aim requires reliable measures that can be used by a wide variety of professionals. The interrater reliability of the Camberwell Assessment of Need Short Appraisal Schedule (CANSAS) was assessed under routine conditions. METHOD: Three interviewer-observer dyads assessed the needs of 14 inpatients and 18 day patients of a psychiatric rehabilitation unit in New South Wales, Australia. RESULTS: Agreement on the identification of an area of need was high. However, agreement was higher on patient ratings than on staff ratings. Correlations on staff ratings of met needs were also moderate (r = 0.53), suggesting discrepancies in rating the level of need. CONCLUSION: Differences in staff ratings may be attributed to ambiguity in the definition of need and levels of need and/or the sources of information used by the rater making the assessment. An approach to establishing an operational definition of need is suggested, and an increase in the number of levels of need is recommended. Implications for Australian mental health policy are noted.     

Diagnosis of pulmonary embolism: Ventilation perfusion scintigraphy versus helical computed tomography pulmonary angiography

The present study compared the accuracy of ventilation perfusion scintigraphy (VQS) and CT pulmonary angiography (CTPA) for the diagnosis of pulmonary embolism. This was a prospective observational study of 112 patients with suspected pulmonary embolism (PE) who could be studied with both investigations within 24 h. Results were compared to final diagnosis at completion of 6-month follow up, using receiver operating characteristic (ROC) analysis. Pulmonary embolism was diagnosed in 27 referred patients (24%). The sensitivity and specificity of VQS and CTPA were similar to that reported from the literature. A normal VQ scan had the highest negative predictive value (100%), while a high-probability VQ scan had the highest positive predictive value (92%). There was no overall difference (area under the ROC curve (AUC)) between VQS (AUC (95% CI) = 0.82 (0.75,0.89)) and CTPA (AUC = 0.88 (0.81,0.94)) for the diagnosis of PE. Among patients with abnormal chest X-rays, CTPA (AUC 0.90 (0.83,0.97)) appeared somewhat better than VQS (AUC 0.78 (0.68,0.88)) but this difference did not reach statistical significance. In this instance, CTPA is at least as accurate as VQS and may provide an opportunity to make alternative diagnoses.     

Biosynthesis of iridoid sex pheromones in aphids

Iridoid monoterpenes, widely distributed in plants and insects, have many ecological functions. While the biosynthesis of iridoids has been extensively studied in plants, little is known about how insects synthesize these natural products. Here, we elucidated the biosynthesis of the iridoids cis-trans-nepetalactol and cis-trans-nepetalactone in the pea aphid Acyrthosiphon pisum (Harris), where they act as sex pheromones. The exclusive production of iridoids in hind legs of sexual female aphids allowed us to identify iridoid genes by searching for genes specifically expressed in this tissue. Biochemical characterization of candidate enzymes revealed that the iridoid pathway in aphids proceeds through the same sequence of intermediates as described for plants. The six identified aphid enzymes are unrelated to their counterparts in plants, conclusively demonstrating an independent evolution of the entire iridoid pathway in plants and insects. In contrast to the plant pathway, at least three of the aphid iridoid enzymes are likely membrane bound. We demonstrated that a lipid environment facilitates the cyclization of a reactive enol intermediate to the iridoid cyclopentanoid-pyran scaffold in vitro, suggesting that membranes are an essential component of the aphid iridoid pathway. Altogether, our discovery of this complex insect metabolic pathway establishes the genetic and biochemical basis for the formation of iridoid sex pheromones in aphids, and this discovery also serves as a foundation for understanding the convergent evolution of complex metabolic pathways between kingdoms.

Iridoids are a class of atypical bicyclic monoterpenoids that are widely distributed in flowering plants, but, notably, are also found in several insect orders, including Coleoptera, Hymenoptera, and Hemiptera (1). Iridoids therefore present an opportunity to compare and contrast the chemical logic of natural product biosynthesis between plants and insects.In plants, iridoids largely act as defensive metabolites or biosynthetic intermediates for other natural products (e.g., monoterpenoid indole alkaloids and isoquinoline alkaloids). The pathway leading to the cyclopentanoid-pyran (iridoid) scaffold was first elucidated in the plant Madagascar periwinkle (Catharanthus roseus) (2–6) and more recently in the two mint species Nepeta mussinii and Nepeta cataria (7–9). Iridoid biosynthesis in plants starts with the condensation of the universal terpene precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to form geranyl diphosphate (GPP), followed by hydrolysis to geraniol (Fig. 1A). Both reactions take place in the plastids and are catalyzed by trans-isoprenyl diphosphate synthase (IDS) and geraniol synthase (GES), respectively. Hydroxylation of geraniol by geraniol-8-hydroxylase (G8H) leads to 8-hydroxygeraniol, which is further oxidized in two consecutive reaction steps by 8-hydroxygeraniol oxidase (HGO) to 8-oxogeranial. This dialdehyde is then converted to the iridoid nepetalactol by a two-step reduction–cyclization sequence that involves the formation of a highly reactive 8-oxocitronellyl enol/enolate intermediate. Initially, reduction and cyclization of 8-oxogeranial were thought to be controlled by a single enzyme, iridoid synthase (ISY) (3), though later studies showed that ISY likely catalyzes only the NADPH-dependent reduction of 8-oxogeranial to the 8-oxocitronellyl enol/enolate intermediate (8). This intermediate can nonenzymatically cyclize, or, alternatively, the stereoselective cyclization of this intermediate to nepetalactol is enzymatically mediated by nepetalactol-related short-chain dehydrogenase (NEPS) or by major latex protein-like (MLPL) enzymes (8, 9). In C. roseus, nepetalactol is further metabolized to secologanin, which serves as a precursor for the formation of monoterpene indole alkaloids in this plant (10). In Nepeta, a NEPS protein oxidizes nepetalactol to nepetalactone (8), with both the alcohol and lactone released as volatiles.Open in a separate windowFig. 1.The formation of iridoids in plants and aphids. (A) Labeling studies suggest that the biosynthesis of iridoids in the pea aphid A. pisum mimics the biosynthetic pathway in iridoid-producing plants. IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; IDS, isoprenyl diphosphate synthase; GES, geranyl diphosphate synthase; G8H, geraniol 8-hydroxylase; HGO, 8-hydroxygeraniol oxidoreductase; ISY, iridoid synthase; NEPO, nepetalactol oxidase. (B) Relative expression of mevalonate and putative nepetalactone pathway genes in hind legs and front legs of different sexual stages of A. pisum. Relative expression data are based on RPKM values obtained by RNAseq. f-hl, hind legs of sexual females; f-fl, front legs of sexual females; af-hl, hind legs of asexual females; m-hl, hind legs of males.Insects utilize iridoids as both defense compounds and volatile pheromones, but in terms of biosynthesis, comparatively little is understood about insect-derived iridoids. Biosynthetic insights have been obtained from studies on larvae of chrysomelid leaf beetles, which accumulate the iridoid-related monocyclic dialdehydes chrysomelidial and plagiodial (11). Feeding experiments with isotopically labeled precursors and the discovery of some of the enzymes involved in chrysomelidial formation demonstrated that leaf beetles produce these compounds by a series of chemical reactions similar to those that occur in plants (12–15). Although the enzymatic basis for this pathway has not been completely established, the fact that the known enzymes are unrelated to their counterparts in plants suggests independent evolution of the pathway occurred (14).Cis-trans-nepetalactol and cis-trans-nepetalactone are the major iridoids produced by catnip (N. mussinii) and catmint (N. cataria) (16). These molecules are responsible for the euphoric effect these plants have on cats, but their ecological function is unclear, though they may play roles in mediating interactions with insects (17). Interestingly, cis-trans-nepetalactol and cis-trans-nepetalactone occur also in aphids, which produce these compounds as volatile sex pheromones (18, 19). The pea aphid Acyrthosiphon pisum, for example, has been reported to biosynthesize (1R,4aS,7S,7aR)-cis-trans-nepetalactol and (4aS,7S,7aR)-cis-trans-nepetalactone in glandular structures on the hind legs of sexual female aphids, from where they are released to attract male conspecifics (18, 20). Recent studies with isotopically labeled iridoid precursors suggest that the iridoid pathway in aphids follows the reaction sequence described for plants (21). However, the underlying enzymatic machinery of this pathway is completely unknown.Here, we report the elucidation of the entire iridoid pathway in the pea aphid A. pisum. By searching for genes expressed exclusively in hind legs of sexual female aphids, the site of iridoid production, we could rapidly identify all six biosynthetic genes/enzymes responsible for the conversion of IPP and DMAPP to cis-trans-nepetalactone. The discovery of the insect nepetalactone pathway in its entirety now allows a comparison of the chemical solutions that have evolved for nepetalactone biosynthesis in plants and animals. Although the chemical steps from GPP to nepetalactone are the same in both Nepeta and pea aphids, the enzymes of these pathways have clearly evolved independently.     

Assessment of Hageman factor activation in human plasma: quantification of activated Hageman factor-C1 inactivator complexes by an enzyme- linked differential antibody immunosorbent assay

An enzyme-linked immunosorbent assay has been developed for the quantitation of activated Hageman factor-C1 inactivator (HF-C1 INH) complexes. Addition of increasing quantities of either of the major forms of activated Hageman factor (HFa or HFf) to normal plasma or to Hageman factor-deficient plasma leads to a dose-dependent increase in activated HF-C1 INH complexes. As little as 0.5 micrograms/mL of activated HF added to plasma can be detected, corresponding to activation of approximately 2% of plasma HF. The sensitivity of the assay is increased at least tenfold when complexes are formed in HF- deficient plasma, indicating competition between unactivated HF and activated HF-C1 INH complexes for binding to the antibody. Specificity is demonstrated in that addition of activated HF to hereditary angioedema plasma yields less than 1% of the activated HF-C1 INH complex formation obtained with normal plasma. Kaolin activation of HF- deficient plasma yields no detectable complex formation. Kaolin activation of prekallikrein-deficient plasma demonstrates a time- dependent increase in formation of activated HF-C1 INH complex consistent with the ability of HF in this plasma to autoactivate as the time of incubation with the surface is increased. Kaolin treatment of high-molecular weight (HMW) kininogen-deficient plasma yields an even more profound abnormality in the rate of formation of activated HF-C1 INH complexes reflecting the complex role of HMW kininogen in the initiation of contact activation. Although addition of corn inhibitor to plasma prevents activated HF-C1 INH complex formation, it does not inhibit activated HF sufficiently fast to prevent prekallikrein activation.