High-grade dysplasia and superficial adenocarcinoma in Barrett's esophagus: histological mapping and expression of p53, p21 and Bcl-2 oncoproteins

In order to characterize the early morphological and molecular stages of the neoplastic progression of Barrett's mucosa, we performed the entire histological examination of ten specimens of resected Barrett's esophagus with high-grade dysplasia or superficial adenocarcinoma. The expression of p53, p21 and Bcl-2 proteins was assessed by immunohistochemistry. The surface of Barrett's mucosa ranged from 2.6 cm(2) to 31 cm(2). Dysplasia and adenocarcinoma always developed in specialized mucosa and often occupied small surfaces. High-grade dysplasia was multifocal in eight cases. There was no preferential site for neoplastic transformation into high-grade dysplasia or superficial adenocarcinoma in Barrett's mucosa. Three superficial adenocarcinomas and four high-grade dysplasias overexpressed p53 protein. p21 protein was focally expressed in nondysplastic mucosa and overexpressed in two superficial adenocarcinomas, one high-grade dysplasia and two low-grade dysplasias. In most cases, the expression of p21 and p53 proteins was unrelated. Bcl-2 protein was detected in only one area of low-grade dysplasia. In our study, high-grade dysplasia and superficial adenocarcinoma appeared as tiny lesions, often multifocal for high-grade dysplasia confirming the need for an extensive sampling of Barrett's mucosa in the endoscopic surveillance. p53 dysfunction plays a major role in the progression from dysplasia to carcinoma in Barrett's esophagus and appears unrelated to p21 and Bcl-2 expression.     

Tumor necrosis factor and granulocyte macrophage-colony stimulating factor stimulate human macrophages to restrict growth of virulent Mycobacterium avium and to kill avirulent M. avium: killing effector mechanism depends on the generation of reactive nitrogen intermediates

An avirulent and a virulent strain of Mycobacterium avium were selected on the basis of their growth patterns in human monocyte-derived macrophages. The virulent 7497 M. avium grew progressively in untreated macrophages, whereas the avirulent LR/149 M. avium was killed to a moderate extent by untreated human macrophages (50% of the original infectious inoculum killed 7 days after infection). We set out to investigate the possibility of modulating these growth patterns by cytokine treatment. Application of tumor necrosis factor (TNF) (100 U/ml) led to macrophages restricting significantly the growth of virulent M. avium 7497 (tenfold decrease at 7 days). TNF was also effective at modulating positively the interaction between avirulent LR/149 M. avium and macrophages inasmuch as TNF-treated cells killed 99% of infecting mycobacteria at 7 days. Granulocyte macrophage-colony stimulating factor (GM-CSF) (100-10,000 U/ml) treatment led to macrophages being as mycobacteriostatic for virulent 7497 M. avium as TNF-alpha-treated cells (i.e., tenfold reduction in growth). Treatment of macrophages with both GM-CSF and TNF-alpha was shown to have additive effects on bacteriostatic activity on M. avium. The mechanism of killing of avirulent M. avium by TNF-alpha was shown to be dependent on the generation of reactive nitrogen intermediates, as seen by inhibition of effector mechanisms by NG-monomethyl-arginine and arginase. Moreover, there was a correlation between NO2- generation and mycobactericidal activity of macrophages. Addition of superoxide dismutase reversed the killing of avirulent M. avium by untreated or TNF-treated macrophages. This abrogation was also apparent in chronic granulomatous disease (CGD) macrophages, which were inefficient at generating reactive oxygen intermediates. Moreover, macrophages from CGD patients killed avirulent M. avium as efficiently as cells from normal individuals. We conclude from these results that 1) GM-CSF and TNF-alpha, alone or in combination, increase effector functions of macrophages against virulent and avirulent strains of M. avium; 2) reactive nitrogen intermediates seem to be involved in this effector mechanism; and 3) superoxide dismutase protected M. avium against macrophage effector function, seemingly by protecting the bacteria against endogenous superoxide anion. The implications of these findings for host resistance to atypical mycobacteria are discussed.     

Stimulation of neutrophil oxidative metabolism by indomethacin

Human neutrophils exposed to indomethacin demonstrate an enhanced capacity for superoxide ion (O ₂ ^– ) generation when stimulated with opsonized zymosan. Enhancement is not seen with indomethacin-treated cells exposed to solube oxidative stimuli. To further investigate this phenomenon, O ₂ ^– generation, chemiluminescence, and phagocytosis were assessed in human neutrophils preincubated with indomethacin. Zymosan-stimulated O ₂ ^– release was increased from 150 to 300% of controls in neutrophils exposed to 400 g/ml. indomethacin. Enhancement was not reversed by removal of indomethacin from the medium prior to addition of the stimulus and was dose-dependent at drug concentrations of 5 to 400 /ml. Neutrophils exposed to methacin alone also generated more O ₂ ^– than control cells, although this increment was not sufficient to account for the degree of enhancement seen when indomethacintreated cells were exposed to zymosan. Neutrophil cehmiluminescence induced by zymosan was also increased by exposure to indomethacin, and at a drug concentration of 400 g/ml (1.1 mM), enhancement randed from 253 to 333% of controls. As was observed with O ₂ ^– generation, chemiluminescence of neutrophils was increased in the presence of indomethacin alone, although, to a degree far less than was seen when drug-treated cells were stimulated with zymosan. Phagocytosis of radiolabeledS. aureus by neutrophils incubated with indomethacin was increased 13±5% over controls (P<0.01,n=5), but was unaltered by incubation of cells with the buffer used to solubilize the drug. The modest degree of enhancement of phagocytosis suggests that increased particle uptake is not the sole mechanism of oxidative enhancement. The data are in keeping with the hypothesis that indomethacin has a direct effect on the neutrophil plasma membrane and/or the O ₂ ^– -forming oxidase.     

Detection and identification of group B streptococci by use of pigment production

Pigment production by group B streptococci (GBS) is a useful test for identification of the organisms. The test is positive in 99.5% of beta-haemolytic strains. No false-positives are noted. Non-haemolytic strains do not produce pigment. Islam's media less agar can be used as a one-step broth detector of GBS in mixed cultures. This may have application for the detection of GBS in women in labour. When used as an identification system for GBS, serum-starch broth can be further modified by reduction of serum and starch concentrations by at least 80%.     

Alternative methods to analyse the impact of HIV mutations on virological response to antiviral therapy

Background  

Principal component analysis (PCA) and partial least square (PLS) regression may be useful to summarize the HIV genotypic information. Without pre-selection each mutation presented in at least one patient is considered with a different weight. We compared these two strategies with the construction of a usual genotypic score.     

High selectivity of the i f channel to Na+ and K+ in rabbit isolated sinoatrial node cells

The ionic selectivity of the hyperpolarizationactivated inward current (i _f) channel to monovalent cations was investigated in single isolated sinoatrial node cells of the rabbit using the whole-cell patch-clamp technique. With a 140 mM K⁺ pipette, replacement of 90% external Na⁺ by Li⁺ caused a –24.5 mV shift of the fully activated current/voltage I/V curve without a significant decrease of the slope conductance. With a 140 mM Cs⁺ pipette, the i _f current decreased almost proportionally to the decrease in external [Na⁺]_o as Li⁺ was substituted. These responses are practically the same as those observed with N-methyl glucamine (NMG⁺) substitution, suggesting that the relative permeability of Li⁺ compared with Na⁺ for the i _f channel is as low as that of NMG⁺. When Cs⁺ or Rb⁺ was substituted for internal K⁺, the fully activated I/V relationship for i _f showed strong inward rectification with a positive reversal potential, indicating low permeability of the i _f channel for Cs⁺ and Rb⁺. These results show that the i _f channel is highly selective for Na⁺ and K⁺ and will not pass the similar ions Li⁺ and Rb⁺. Such a high degree of selectivity is unique and may imply that the structure of the i _f channel differs greatly from that of other Na⁺ and K⁺ conducting channels.     

Long-term in vivo biostability of poly(dimethylsiloxane)/poly(hexamethylene oxide) mixed macrodiol-based polyurethane elastomers

The long-term biostability of a novel thermoplastic polyurethane elastomer (Elast-Eon 2 80A) synthesized using poly(hexamethylene oxide) (PHMO) and poly(dimethylsiloxane) (PDMS) macrodiols has been studied using an in vivo ovine model. The material's biostability was compared with that of three commercially available control materials, Pellethane 2363-80A, Pellethane 2363-55D and Bionate 55D, after subcutaneous implantation of strained compression moulded flat sheet dumbbells in sheep for periods ranging from 3 to 24 months. Scanning electron microscopy, attenuated total reflectance-Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy were used to assess changes in the surface chemical structure and morphology of the materials. Gel permeation chromatography, differential scanning calorimetry and tensile testing were used to examine changes in bulk characteristics of the materials. The results showed that the biostability of the soft flexible PDMS-based test polyurethane was significantly better than the control material of similar softness, Pellethane 80A, and as good as or better than both of the harder commercially available negative control polyurethanes, Pellethane 55D and Bionate 55D. Changes observed in the surface of the Pellethane materials were consistent with oxidation of the aliphatic polyether soft segment and hydrolysis of the urethane bonds joining hard to soft segment with degradation in Pellethane 80A significantly more severe than that observed in Pellethane 55D. Very minor changes were seen on the surfaces of the Elast-Eon 2 80A and Bionate 55D materials. There was a general trend of molecular weight decreasing with time across all polymers and the molecular weights of all materials decreased at a similar relative rate. The polydispersity ratio, Mw/Mn, increased with time for all materials. Tensile tests indicated that UTS increased in Elast-Eon 2 80A and Bionate 55D following implantation under strained conditions. However, ultimate strain decreased and elastic modulus increased in the explanted specimens of all three materials when compared with their unimplanted unstrained counterparts. The results indicate that a soft, flexible PDMS-based polyurethane synthesized using 20% PHMO and 80% PDMS macrodiols has excellent long-term biostability compared with commercially available polyurethanes.     

Lack of protection in mice and necrotizing bronchointerstitial pneumonia with bronchiolitis in guinea pigs immunized with vaccines directed against the hsp60 molecule of Mycobacterium tuberculosis

In this study, the hsp60 and hsp70 heat shock protein antigens of Mycobacterium tuberculosis were tested as potential vaccine candidates, using purified recombinant protein antigens or antigens encoded in the form of a DNA plasmid vaccine. Guinea pigs vaccinated with a mixture of the two proteins showed no evidence of resistance to low-dose aerosol challenge infection and quickly developed severe lung damage characterized by necrotizing bronchointerstitial pneumonia and bronchiolitis. As a result, we turned instead to a DNA vaccination approach using a plasmid encoding the hsp60 antigen of M. tuberculosis. Although immunogenic in mice, vaccination with plasmid DNA encoding hsp60 was not protective in that model or in the guinea pig model and again gave rise to similar severe lung damage. This study seriously questions the safety of vaccines against tuberculosis that target highly conserved heat shock proteins.     

An engineered biopolymer prevents mucositis induced by 5-fluorouracil in hamsters

Oral mucositis is a common, treatment-limiting, and costly side effect of cancer treatments whose biological underpinnings remain poorly understood. In this study, mucositis induced in hamsters by 5-fluorouracil (5-FU) was observed after cheek-pouch scarifications, with and without administration of RGTA (RG1503), a polymer engineered to mimic the protective effects of heparan sulfate. RG1503 had no effects on 5-FU-induced decreases in body weight, blood cell counts, or cheek-pouch and jejunum epithelium proliferation rates, suggesting absence of interference with the cytotoxic effects of 5-FU. Extensive mucositis occurred in all of the untreated animals, and consisted of severe damage to cheek pouch tissues (epithelium, underlying connective tissue, and muscle bundles). Only half of the RG1503-treated animals had mucositis, over a mean area 70% smaller than in the untreated animals. Basement membranes were almost completely destroyed in the untreated group but was preserved in the RG1503 group. RG1503 blunted or abolished the following 5-FU-induced effects: increases in matrix metalloproteinase (MMP)-2, MMP-9, and plasmin, and decreases in tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. These data indicate that mucositis lesions are related to massive release of proteolytic enzymes and are improved by RG1503 treatment, this effect being ascribable in part to restoration of the MMP-TIMP balance. RG1503 given with cancer treatment might protect patients from mucositis.