Improved Diagnostic Sensitivity of Bowel Disease of Prematurity on Contrast-Enhanced Ultrasound

Bowel diseases of prematurity, including necrotizing enterocolitis, are dreaded ailments of neonates. Early diagnosis is difficult, with clinical and radiographic findings often inconclusive. We present a novel use of contrast-enhanced ultrasound in detection of pediatric bowel disease. Early identification of compromised blood flow or an at-risk bowel can be quantitatively detected and monitored. This ability has implications for guidance of emerging therapies, allowing targeting of inflammation. These findings represent an advancement in detection of bowel disease in neonates.     

Pheochromocytoma and pregnancy: a case report and review of anesthetic management

PURPOSE: To describe a patient diagnosed with pheochromocytoma in the third trimester of pregnancy and discuss the perioperative and anesthetic management. CLINICAL FEATURES: A 32-yr-old previously healthy woman (gravida 4, para 2) presented to our tertiary care obstetrical hospital at 34 weeks five days gestation with a history of labile blood pressure and severe hypertension. A week prior to admission she began having episodes of severe headache, dizziness, sweating and nausea. On a routine obstetric visit she was noted to be severely hypertensive with a blood pressure of 200/120 mmHg. Biochemical investigations confirmed the diagnosis of pheochromocytoma and magnetic resonance imaging demonstrated a 3 cm x 3 cm right adrenal mass. The patient was invasively monitored in the intensive care unit and treated with alpha- followed by beta-blockade with phenoxybenzamine and metoprolol. A multidisciplinary conference was organized involving endocrinology, anesthesiology, general surgery and obstetrics to determine the most appropriate management of the patient. An uncomplicated laparoscopic adrenalectomy was performed following a period of recovery after an uneventful elective Cesarean delivery. CONCLUSIONS: The primary goals in the management of pheochromocytoma in pregnancy are early diagnosis, avoidance of a hypertensive crisis during delivery and definitive surgical treatment. Timing of surgical resection will depend on the gestational age at which diagnosis is made. Cesarean section is the preferred mode of delivery when the tumour is still present. This case illustrates that with antenatal diagnosis, advanced methods of tumour localization, adequate preoperative adrenergic blockade and team planning, pheochromocytoma in pregnancy can be treated successfully.     

Roles of specific amino acids in the N terminus of Pseudomonas aeruginosa flagellin and of flagellin glycosylation in the innate immune response

The Toll-like receptor 5 (TLR5) binding site has been predicted to be in the N terminus of the flagellin molecule. In order to better define the interaction between the N-terminal amino acids of Pseudomonas aeruginosa flagellin and TLR5, site-specific mutations were generated between residues 88 and 97 of P. aeruginosa PAK flagellin as well as outside of this region. The mutant flagellins were expressed in Escherichia coli BL21(plysS), purified by affinity chromatography, and passed through a polymyxin B column to remove contaminating lipopolysaccharide (LPS). Their ability to stimulate interleukin-8 (IL-8) release from A549 cells was examined. The cloned mutated genes were used to complement a PAK fliC mutant in order to test for effects on motility and on IL-8 release by purified flagellar preparations. All the mutations, single or double, in the predicted TLR5 binding region reduced IL-8 signaling to less than 95% of the wild-type flagellin levels, but the single mutation outside the binding region had no effect. Changes made at two amino acid sites resulted in loss/reduction of motility; however, changes made at single sites, i.e., Q83A, L88A, R90A, M91A, L94A, and Q97A, had no effect on motility. The mutated genes encoding two of the motile but poorly signaling flagellins had no compensatory mutations to allow motility. Thus, while it is speculated that pathogen-associated molecular patterns (PAMPs) have evolved in locations that are essential to maintain function, it appears that there is tolerance for at least single amino acid changes in the PAMP of P. aeruginosa flagellin. The purpose of flagellin glycosylation in P. aeruginosa is unknown. In order to examine its role, if any, in signaling an inflammatory response, we used whole flagella from the motile chromosomal mutant strains PAKrfbC and PAO1rfbC, which are defective in flagellin glycosylation. IL-8 release from A549 cells stimulated with nonglycosylated flagellar preparations (having less then 1 picogram of LPS/mug) was significantly reduced compared to their respective wild-type flagellar preparations, indicating a role of flagellar glycosylation in the proinflammatory action of Pseudomonas flagellin. The basis of the latter activity is unknown, since the glycosylation sites are found in the D3 domain of flagellins and the TLR5 binding site is located in the D1 domain. Thus, P. aeruginosa flagellin has evolved additional flagellar signaling mechanisms over that described for Salmonella flagellin.     

M1 protein triggers a phosphoinositide cascade for group A Streptococcus invasion of epithelial cells

Invasion of nonphagocytic cells by bacteria provides a favorable niche for persistence and evasion of host defenses and antibiotics. M protein is a major virulence factor because it promotes high-frequency invasion of epithelial cells by group A Streptococcus (GAS) and also renders the bacterium resistant to phagocytosis. In this study, we investigated the role of M1 protein from serotype M1 strain 90-226 in regulating mammalian signal transduction and cytoskeletal rearrangement for bacterial entry. LY294002 and wortmannin, which are inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked invasion of epithelial cells by GAS by 75 and 80%, respectively, but failed to inhibit invasion by Salmonella enterica serovar Typhimurium. Also, epithelial cells transiently transfected with dominant negative p85 and p110 genes, the regulatory and catalytic subunits of PI 3-K, respectively, were less able to be invaded by GAS. To separate the influence of other streptococcal virulence factors from M protein, Lactococcus lactis was engineered to express M1 protein on its surface. L. lactis(pLM1) invaded epithelial cells efficiently in vitro, and PI 3-K inhibitors blocked 90% of this invasion. Purified soluble M1 protein stimulated the formation of stress fibers and actin tuffs on epithelial cells. LY294002 and wortmannin inhibited these cellular changes. A phosphoinositide analogue also inhibited the invasion of epithelial cells by GAS. Therefore, M1 protein, either directly or via bound fibronectin, initiates signals that depend on the lipid kinase PI 3-K pathway, which paves the way for cytoskeletal rearrangement that internalize the bacterium.     

Molecular and genetic characterizations of five pathogenic and two non-pathogenic monoclonal antiphospholipid antibodies

Antiphospholipid syndrome (APS) is an autoimmune disease that is characterized by thrombosis, recurrent fetal loss and thrombocytopenia. Antiphospholipid antibodies, detected by enzyme-linked immunoabsorbent assays (aCL) and/or in vitro blood clotting assays (LAC) are strongly associated with APS. Both the molecular structures used by pathogenic antiphospholipid antibodies and the genetic mechanisms leading to their production are unknown. We describe here the variable region genes of seven IgG antiphospholipid antibodies derived from two APS patients. Of these, five are pathogenic as defined in a mouse model of thrombosis and two are not. Analyses of the expressed variable region genes show no preferential V gene usage. However, similar to anti-DNA antibodies, pathogenic antiphospholipid antibodies contain an increased number of arginine residues in the third complimentarity-determining region (CDR3) of their H chains. The increased accumulation of arginine residues in the V(H) CDR3 may act to enhance antigen binding, promote disease and point to the importance of the H chain in the pathogenic potential of certain antiphospholipid antibodies.     

Polarimetric imaging of subsurface tissue-numerical results

Tissue images obtained at deeper depths lack significant contrast. To enhance the contrast of these images and to increase the visibility of subsurface tissues, a method is proposed. This technique is based on the principle that photons at longer wavelengths penetrate deeper than photons at shorter wavelengths. In this technique, images in the original and orthogonal polarized states are recorded with the source illumination in linear polarized state at two different wavelengths. Image subtraction of a fraction of the copolarized image from the perpendicular polarized state is done on these images. The images obtained after the first processing are subjected to the next image subtraction where the shorter wavelength image is subtracted from the longer wavelength image. Monte Carlo simulations show that the resultant image has marked contrast up to 2.5 cm.     

Vascularization and tissue infiltration of a biodegradable polyurethane matrix

Urethanes are frequently used in biomedical applications because of their excellent biocompatibility. However, their use has been limited to bioresistant polyurethanes. The aim of this study was to develop a nontoxic biodegradable polyurethane and to test its potential for tissue compatibility. A matrix was synthesized with pentane diisocyanate (PDI) as a hard segment and sucrose as a hydroxyl group donor to obtain a microtextured spongy urethane matrix. The matrix was biodegradable in an aqueous solution at 37 degrees C in vitro as well as in vivo. The polymer was mechanically stable at body temperatures and exhibited a glass transition temperature (Tg) of 67 degrees C. The porosity of the polymer network was between 10 and 2000 microm, with the majority of pores between 100 and 300 microm in diameter. This porosity was found to be adequate to support the adherence and proliferation of bone-marrow stromal cells (BMSC) and chondrocytes in vitro. The degradation products of the polymer were nontoxic to cells in vitro. Subdermal implants of the PDI-sucrose matrix did not exhibit toxicity in vivo and did not induce an acute inflammatory response in the host. However, some foreign-body giant cells did accumulate around the polymer and in its pores, suggesting its degradation is facilitated by hydrolysis as well as by giant cells. More important, subdermal implants of the polymer allowed marked infiltration of vascular and connective tissue, suggesting the free flow of fluids and nutrients in the implants. Because of the flexibility of the mechanical strength that can be obtained in urethanes and because of the ease with which a porous microtexture can be achieved, this matrix may be useful in many tissue-engineering applications.