Etiologic and other factors predicting nevus-associated cutaneous malignant melanoma.

Cutaneous malignant melanomas with histologic evidence of an associated nevus (N+) may have a different risk factor profile from that of melanomas without it (N-). To address this question, a case-only analysis of 932 people with cutaneous malignant melanoma was done to identify etiologic and other factors associated with N+ melanoma. Evidence of an associated nevus was found in 36% of melanomas. N+ melanomas were thinner (Ptrend=0.0009) and more likely to be of the superficial spreading type than other types of melanoma. Subjects with N+ melanomas were younger (Ptrend<0.0001) and reported a higher nevus density on their skin than subjects with N- melanomas [odds ratio (OR), 3.1; 95% confidence interval (CI), 1.6-6.0, for high nevus density versus no nevi]. Indicators of high accumulated sun exposure were less prevalent among subjects with N+ melanomas (OR, 0.3; 95% CI, 0.2-0.4, for melanoma location on the head and neck versus location on trunk; OR, 0.2; 95% CI, 0.1-0.4, for severe solar elastosis adjacent to the melanoma versus no elastosis; OR, 0.2; 95% CI, 0.1-0.4, for lentigo maligna melanoma subtype versus superficial spreading subtype). With the exception of solar elastosis and age, all of the aforementioned variables remained significantly associated with N+ melanomas in multivariate analyses. No associations with self-reported measures of sun exposure, sunburn, or pigmentation phenotype were apparent. Our findings provide some support for the hypothesis of etiologically separate pathways for melanoma, with N+ melanomas appearing less likely to develop in the presence of characteristics suggesting high accumulated sun exposure than N- melanomas. However, it is possible that high UV exposure causes involution of nevi, thus reducing the density of nevi in exposed skin and thereby the probability of N+ melanoma.     

Heparin octasaccharides inhibit angiogenesis in vivo.

BACKGROUND: In previous experiments, we showed that heparin oligosaccharides inhibit the angiogenic cytokine fibroblast growth factor-2. Here, we present the first in vivo study of size-fractionated heparin oligosaccharides in four models of angiogenesis that are progressively less dependent on fibroblast growth factor-2. EXPERIMENTAL DESIGN: Heparin oligosaccharides were prepared using size-exclusion gel filtration chromatography and characterized through depolymerization and strong anion exchange high-performance liquid chromatography. Size-defined oligosaccharides (20 mg/kg/d) were given to mice bearing s.c. sponges that were injected with fibroblast growth factor-2 (100 ng/d). After 14 days, octasaccharides and decasaccharides reduced the microvessel density to levels below control. In a second experiment, HEC-FGF2 human endometrial cancer cells that overexpress fibroblast growth factor-2 were implanted in a hollow fiber placed s.c. in vivo. Oligosaccharides were given at 20 mg/kg/d for 2 weeks and the data again showed that octasaccharides significantly reduced microvessel density around the fiber (P = 0.03). In a more complex model, where angiogenesis was induced by a broad spectrum of growth factors, including vascular endothelial growth factor, we implanted H460 lung carcinoma cells in hollow fibers and treated the animals with oligosaccharides at 20 mg/kg/d over 3 weeks. Octasaccharides reduced the microvessel density to that of control. Preliminary investigation of 6-O-desulfated heparins showed that these also had antiangiogenic activity. RESULTS: Finally, we examined the inhibitory potential of hexasaccharides and octasaccharides given at 20 mg/kg/d and these inhibited the growth of H460 lung carcinoma in vivo. At clinically attainable concentrations, significant anticoagulation (activated partial thromboplastin time, anti-factor Xa, and anti-factor IIa) was not observed in vitro unless species containing > or =16 saccharide residues were investigated. CONCLUSIONS: Thus, our preclinical data show that heparin octasaccharides represent novel antiangiogenic compounds that can be given without the anticoagulant effects of low molecular weight heparin.     

Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells

PURPOSE: Phosphorylation of the regulatory light chain of myosin II (referred to as myosin light chain or MLC) leads to a loss of barrier integrity in cellular monolayers by an increase in the contractility of the cortical actin cytoskeleton. This effect has been examined in corneal endothelial (CE) cells. METHODS: Experiments were performed using cultured bovine CE cells (BCEC). MLC phosphorylation was induced by a thrombin-mediated activation of the proteinase-activated receptor-1 (PAR-1). Expression of MLC kinase (MLCK), a Ca2+/calmodulin-dependent protein kinase that phosphorylates MLC at its Ser-19 and Thr-18 residues, was determined by RT-PCR and Western blotting. Expression of PAR-1, RhoA, and Rho kinase-1 (effector of RhoA) was ascertained by RT-PCR. MLC phosphorylation was assessed by urea-glycerol gel electrophoresis followed by immunoblotting. The effects of Rho kinase-1 and PKC were characterized by using their selective inhibitors, Y-27632 and chelerythrine, respectively. Reorganization of the cytoskeleton was evaluated by the phalloidin staining of actin. [Ca2+]i was measured using Fura-2. The barrier integrity was assayed as permeability of BCEC monolayers to horseradish peroxidase (HRP; 44 kDa). RESULTS: RT-PCR showed expression of MLCK, PAR-1, Rho kinase-1, and RhoA. Western blotting indicated expression of the non-muscle and smooth muscle isoforms of MLCK. Exposure to thrombin induced an increase in [Ca2+]i with the peak unaffected by an absence of extracellular Ca2+. Pre-exposure to thrombin (2 U ml(-1); 2 min) led to mono- and di-phosphorylation of MLC. Under both basal conditions and in the presence of thrombin, MLC phosphorylation was prevented by chelerythrine (10 microm) and Y-27632 (<25 microm). Thrombin led to inter-endothelial gaps secondary to the disruption of the cortical actin cytoskeleton, which under resting conditions was organized as a perijunctional actomyosin ring (PAMR). These responses were blocked by pre-treatment with Y-27632. Thrombin also increased permeability to HRP, which was abolished by pre-treatment with Y-27632. CONCLUSIONS: Thrombin induces MLC phosphorylation in BCEC. The consequent increase in the contractility of the actin cytoskeleton produces a centripetal force resulting in inter-endothelial gaps and a breakdown of barrier integrity. These responses are PKC- and Rho kinase-dependent. [Ca2+]i increase, as well as sensitivity of the thrombin response to PKC and Rho kinase inhibitors, are consistent with the expression of PAR-1 receptors in BCEC. Thrombin-induced hyperpermeability is a model to investigate barrier dysfunction induced by MLC phosphorylation.     

Size-fractionated heparins have differential effects on human neutrophil function in vitro

BACKGROUND AND PURPOSE: Heparin is known to possess a range of activities, other than effects on blood coagulation, many of which are anti-inflammatory. Effects with potential anti-inflammatory applications include the inhibition of elastase release from neutrophils, as well as the adhesion of these cells to vascular endothelium. In the present study we aimed to investigate whether fractionation of heparin may yield molecules with enhanced or specific effects on human neutrophil function. EXPERIMENTAL APPROACH: Fractions of defined molecular size were obtained from heparin by different methods and assessed for their effects on elastase release induced by formyl Met-Leu-Phe (fMLP), from neutrophils, in some cases following the priming of these cells with tumour necrosis factor-alpha (TNF-alpha). Effects of the fractions on neutrophil adhesion to interleukin-1beta (IL-beta)-stimulated human umbilical vein endothelial cells (HUVECs) were also examined. KEY RESULTS: Elastase release was inhibited by very low molecular weight fractions of heparin, with an apparent minimum chain length of 10 saccharides required for full effect. In contrast, neutrophil-endothelial adhesion was unaffected by these fractionated heparins, suggesting that certain non-anticoagulant actions of heparin may be lost by such an approach. CONCLUSIONS AND IMPLICATIONS: These data suggest that an optimum chain length of heparin possibly exists for certain non-anticoagulant actions of heparin, which may prove to be useful in the design of novel drugs with specific anti-inflammatory actions.     

Antibiotic-resistant organisms in the neonatal intensive care unit

Neonates, particularly those born prematurely, are at an increased risk of bacterial infection. Empiric treatment with antimicrobials occurs frequently in the neonatal intensive care unit (NICU). Repeated and/or prolonged courses of antibiotic exposure have resulted in an increase in the prevalence of hospital-acquired, antibiotic-resistant organisms such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and multidrug-resistant Gram-negative rods. As bacterial strains become increasingly resistant to standard antimicrobial therapy, measures to control and prevent this problem are essential. Current efforts have focused on monitoring and restricting the use of antimicrobials, proper hand hygiene, evaluation of potential reservoirs of bacterial acquisition and transmission, cohorting and isolation of colonized infants, decolonization strategies, and fostering of effective inter- and intrahospital communication.