Real-time 3-D dark-field microscopy for the validation of the cross-linking process of alginate microcapsules

Alginate-based microencapsulation is a promising method for long-term maintenance of cellular and membrane function of the cells and tissue fragments required for in vitro and in vivo biosensors, for tissue engineering and particularly for immunoisolation of non-autologous transplants. Microcapsules of high mechanical strength and optimum permeability can be produced by injection of BaCl2 crystals into alginate droplets before they come into contact with external Ba2+. A key requirement is that the system parameters (number of crystals, speed of the crystal stream etc.) are properly adjusted according to the mannuronic and guluronic acid ratio and the average molecular mass of the alginate as well as to the diameter of the microcapsules. Robust, reliable, rapid and low-cost validation tools are, therefore, needed for assurance of the microcapsule quality. Here, we describe a novel three-dimensional (3-D) dark-field microscopy that allows the real-time measurement of the number and spatial distribution of the injected Ba2+ ions throughout the microcapsules after treatment with sulphate. This novel method requires only a conventional microscope equipped with three polarising filters and a double aperture stop. In contrast to confocal laser scanning microscopy images, peripherally attached BaSO4 precipitates can clearly be distinguished from internal ones. The data also demonstrate that several steps of the alginate gelling process must be improved before such immunoisolation can be used in patients.     

Analgesic doses of fentanyl impair oxidative metabolism of neonatal hepatocytes

BACKGROUND/PURPOSE: Studies in human surgical neonates have shown that intraoperative fentanyl analgesia results in greater fall in perioperative body core temperature compared with morphine analgesia. The aim of the study was to compare in a neonatal animal model the biochemical effect of fentanyl and morphine on hepatocyte oxidative metabolism. METHODS: Hepatocytes were isolated from suckling rats and the oxygen consumption from palmitate was measured polarographically. In experiment A, fentanyl and morphine within the respective analgesic serum ranges were added to hepatocytes to assess the effect on oxygen consumption. In experiment B, fentanyl was added to hepatocytes in the presence of inhibitors of mitochondrial respiration to investigate its site of action. In experiment C, hepatocytes were incubated with either fentanyl or morphine, centrifuged, and then examined ultrastructurally by electron microscopy. RESULTS: In experiment A, fentanyl inhibited oxygen consumption by up to 40% (P < .01). Morphine inhibited oxygen consumption to a maximum of 25% (P < .01). In experiment B, in the presence of oligomycin, fentanyl increased the inhibition of oxygen consumption; however, in the presence of myxothiazol, no further inhibition by fentanyl occurred. In experiment C, mild ultrastructural alterations to hepatocytes were observed after incubation with fentanyl but not with morphine. CONCLUSIONS: This study demonstrates that therapeutic doses of two commonly used analgesic drugs impair neonatal hepatic oxidative metabolism. Fentanyl exerts a greater effect than morphine by diminishing liver oxygen consumption by up to 40%. The inhibitory effect of fentanyl occurs directly on the mitochondrial respiratory chain, either on substrate oxidation or on the thermogenic proton leak. The findings of this study are relevant to the perioperative management of surgical neonates.     

Deletions in chromosome arms 3p and 11q are new prognostic markers in localized and 4s neuroblastoma.

PURPOSE: To find new nonrandom chromosomal changes in neuroblastoma (NB) with a potential to forecast the patient's outcome, alterations in chromosome arms 3p and 11q were investigated. EXPERIMENTAL DESIGN: Frequency and prognostic potential of 3p and 11q alterations in 144 NBs were analyzed using interphase fluorescence in situ hybridization with DNA probes for 3p26 and 11q23. Aberrations were defined as deletion (monosomy of a specific region) or imbalance (at least two intact and additional 3p26- or 11q23-deleted chromosomes). RESULTS: Forty-two of 144 cases (29%) displayed 11q alterations (21% deletions, 8% imbalances). Most aberrations were associated with stage 4 disease (28 of 59, 47%) but were also present in localized and 4s tumors (14 of 85, 16%; P = 0.007). Patients with 11q deletion/imbalance were significantly older at diagnosis (P < 0.001). Changes in 3p were detected in 26 of 144 (18%) samples (15% deletions, 3% imbalances). These alterations were also associated with stage 4 [20 of 59 (34%) versus 6 of 85 (7%) in stages 1-3 and 4s, P = 0.007], and the median age was increased (P < 0.001). Aberrations in both chromosomes were highly associated with each other (P < 0.001). MYCN amplification (MNA) was detected in 10% and 12% of tumors with 11q and 3p alterations, and changes in 1p36 occurred in 13% and 26% of the 3p- and 11q-aberrant tumors. MYCN amplification and 11q deletion/imbalance tended to show an inverse correlation (P = 0.07) as well as 1p and 3p deletion/imbalance (P = 0.07). Patients with 3p and 11q abnormalities in localized/4s tumors showed an inferior outcome compared with those without these alterations (P = 0.002 and P = 0.0027, respectively), in particular in MYCN single copy tumors (P < 0.0001 and P = 0.0006, respectively). CONCLUSION: Alterations in 3p and 11q are frequent nonrandom aberrations in NB and define a new high-risk subgroup in MYCN single copy stage 1-3 and 4s disease.     

Polymorphisms in the glutathione S-transferase class mu and theta genes interact and increase susceptibility to lung cancer in minority populations (Texas, United States)

The genes coding for separate isoforms of both the human glutathioneS-transferase class mu and class theta enzymes (GSTM1and GSTT1) arepolymorphic with a variable ethnic distribution. These enzymes detoxifyreactive epoxides, including carcinogens produced by tobacco smoke. Becauseof this, the null polymorphism in the GSTM1 gene (coding for the glutathioneS-transferase class mu enzyme) has been studied widely as a possible sourceof inherited susceptibility to smoking-related lung cancer. The more recentlydescribed null polymorphism in the GSTT1 gene also could contribute to anincreased risk of smoking-related lung cancer. As the incidence of lungcancer is known to differ by ethnicity, we have conducted a case-controlstudy in the United States of 108 African-Americans (Blacks) and 60Mexican-Americans (Hispanics) with lung cancer and 132 African-American(Black) and 146 Mexican-American (Hispanic) controls to investigate theassociation of the GSTT1 and GSTM1 polymorphi sms with lung cancer inminority populations. In the unadjusted data, there was a borderlinesignificant association of the GSTM1 null polymorphism with lung cancer inMexican-Americans (odds ratio [OR] = 1.8, 95 percent confidence interval [CI]= 1.0-3.3 ) that was not observed in African-Americans. The GSTT1 nullpolymorphism also had a higher prevalence in cases than controls in bothracial/ethnic groups, but this increase was not statistically significant.When the data were analyzed using logistic regression controlling for age,gender, race, and smoking, no significant association of either trait withlung cancer was observed, with ORs for both traits of approximately 1.3.However, when the prevalence of individuals who were null for bothpolymorphisms was compared by case status, a significant interaction wasobserved. Logistic regression models showed the OR for the association oflung cancer and the presence of both null polymorphisms compared with one(either GSTT1 or GSTM1) or no null genotype to be 2.9 (P < 0.04). Theseresults suggest that there may be carcinogenic intermediates in cigarettesmoke that are substrates for both the GSTT1 and GSTM1 enzymes, and that lungcancer risk is increased more than additively for individuals who have bothGSTT1 and GSTM1 null polymorphisms.     

Median nerve compression in Proteus syndrome

Proteus syndrome is a multi–organ disorder, a prime feature of which is localized gigantism, usually clinically obvious. Symptoms secondary to hypertrophy of nerves has not been previously recognized as a part of the syndrome. Accepted: 16 May 1997     

Nitric oxide inhibits neonatal hepatocyte oxidative metabolism

BACKGROUND/PURPOSE: Liver function is frequently impaired in neonates with sepsis. Nitric oxide (NO) is thought to be a mediator of organ dysfunction and liver oxidative metabolism during sepsis. The authors developed an in vitro model to investigate the effect of NO and the combined effect of NO plus H2O2 on neonatal hepatocyte oxidative metabolism. METHODS: Hepatocytes were isolated from neonatal rats. Oxygen consumption was measured polarographically. In Study A, cells were exposed to S-Nitroso-N-acetylpenicillamine (SNAP), an NO donor, at various concentrations. In study B, myxothiazol and oligomycin, inhibitors of mitochondrial respiration, were added to investigate the site of action of NO. In study C, hepatocytes were incubated in the presence of both SNAP (300 micromol/L) and H2O2 (1.5 mmol/L). In study D, morphological alterations induced by NO and NO plus H2O2 were investigated by hepatocyte electron microscopy. RESULTS: In study A, SNAP caused a dose-dependent decrease in oxygen consumption. A significant inhibition was reached at 300 micromol/L SNAP. In study B, the lack of further inhibition when SNAP was given together with myxothiazol indicates that NO acts intramitochondrially. Similarly, no further inhibition occurred when the NO donor was given together with oligomycin, suggesting that the effect of NO is mainly at the level of ATP synthase. In study C, concomitant addition of 300 micromol/L SNAP and 1.5 mmol/L H2O2 to hepatocytes caused further inhibition of oxygen consumption compared with either SNAP or H2O2 alone. In study D, mild alterations in hepatocyte morphology were noted in the presence of SNAP or SNAP plus H2O2. CONCLUSIONS: In neonatal hepatocytes, NO significantly inhibits mitochondrial oxygen consumption, possibly at the level of ATP synthase. The effect of NO is additive to that of H2O2. Morphological findings were consistent with these biochemical effects and suggest that NO and H2O2 are important mediators of liver damage during sepsis.     

Polymorphisms in XPD exons 10 and 23 and bladder cancer risk.

INTRODUCTION: The nucleotide excision repair gene, xeroderma pigmentosum complementation group D (XPD), has been hypothesized to have a role in cancer risk, but results from prior molecular epidemiologic studies and genotype-phenotype analyses are conflicting. MATERIALS AND METHODS: We examined the frequency of the XPD Asp312Asn polymorphism in exon 10 and the XPD Lys751Gln polymorphism in exon 23 in 505 incident bladder cancer cases and 486 healthy controls. RESULTS: Overall, the XPD exon 10 and 23 genotypes were not associated with bladder cancer risk, after adjusting for age, sex, ethnicity, and smoking status. A gender-specific role was evident that showed an increased risk for women, but not for men, associated with the variant genotypes for both exons. For example, when the exon 23 variant allele genotypes were combined (Lys/Gln + Gln/Gln), there was an increased bladder cancer risk in women [odds ratio (OR), 1.69; 95% confidence interval (95% CI), 1.12-2.58] but not in men (OR, 0.99; 95% CI, 0.79-1.24; P(interaction) = 0.041; OR, 1.62; 95% CI, 1.02-2.58). There was also a gene-smoking interaction that showed the variant alleles for either exon or the combination of both increase the risk of bladder cancer for light and heavy smokers. For exon 23 (P(interaction) = 0.057; OR, 1.21; 95% CI, 0.99-1.47), heavy smokers (> or = 20 pack-years) who carried the exon 23 variant allele genotypes had an OR of 4.13 (95% CI, 2.53-6.73), whereas heavy smokers with the wild-type genotypes were at lower risk (OR, 3.55; 95% CI, 2.19-5.75). Moderate smokers (1-19 pack-years) with the variant allele genotypes had an OR of 1.54 (95% CI, 0.94-2.53), whereas moderate smokers with the wild-type genotypes had an OR of 1.12 (95% CI, 0.63-1.98). CONCLUSIONS: Although we did not observe main effects associated with the XPD genotypes, these results do suggest the variant allele genotypes were associated with increased bladder cancer risk in women and smokers with statistically significant interactions in the exon 23 polymorphism. Although there is biological plausibility, these novel findings for gender and smoking should be interpreted with caution upon confirmation in larger studies.     

Association of Pre-S/S and Polymerase Mutations with Acute and Chronic Hepatitis B Virus Infections in Patients from Rio de Janeiro,Brazil

Several hepatitis B virus (HBV)-related factors, including the viral load, genotype, and genomic mutations, have been linked to the development of liver diseases. Therefore, in this study we aimed to investigate the influence of HBV genetic variability during acute and chronic infection phases. A real-time nested PCR was used to detect HBV DNA in all samples (acute, n = 22; chronic, n = 49). All samples were sequenced for phylogenetic and mutation analyses. Genotype A, sub-genotype A1, was the most common genotype in the study population. A total of 190 mutations were found in the pre-S/S gene area and the acute profile revealed a greater number of nucleotide mutations (p < 0.05). However, both profiles contained nucleotide mutations linked to immune escape and an increased risk of hepatocellular carcinomas (acute, A7T; chronic, A7Q). Furthermore, 17 amino acid substitutions were identified in the viral polymerase region, including the drug resistance mutations lamivudine and entecavir (rtL180M), with statistically significant differences between the mutant and wild type strains. Owing to the natural occurrence of these mutations, it is important to screen for resistance mutations before beginning therapy.