Development of a bispecific F(ab′)2 conjugate against the complement receptor CR3 of macrophages and a variant CD44 antigen of rat pancreatic adenocarcinoma for redirecting macrophage-mediated tumor cytotoxicity

 A bispecific F(ab′)₂ antibody conjugate (BAC) was constructed against the complement receptor CR3 of macrophages and a variant CD44 (CD44v6) antigen of rat pancreatic adenocarcinoma cells to redirect macrophage-mediated tumor cytotoxicity. The Fab′ fragments of monoclonal antibodies (mAb) 1.1ASML and OX42, recognizing the CD44v6 and the CR3 antigens respectively, were chemically coupled at the hinge region using 5,5′-dithiobis(2-nitrobenzoate). The BAC was characterized in vitro for its specific, dual binding capacity to CD44v6 and CR3 antigens. Although the monovalence of the BAC resulted in lower avidities to both the antigens as expected, it was still able to form stable cross-linkages between tumor cells and macrophages in culture leading to the formation of “clump-like” cell aggregates. The in vitro and in vivo tumor-targeting capacity of the BAC was compared with that of the parental antitumor mAb 1.1ASML, which mediates tumor killing by antibody-dependent cell cytotoxicity. These results showed that, even though the bivalent mAb 1.1ASML did not mediate stable cross-linking of target and effector cells, its Fc-receptor-mediated killing of tumor cells was more effective when compared to the BAC. Thus, this study strongly supports the hypothesis that firm persistent binding between effector and target cells per se is not as important as the choice of trigger molecule used for macrophage activation to redirect their tumor cytotoxic potential effectively. Received: 2 May 1996 / Accepted: 21 May 1996     

Predicting patterns of invasion by black cherry (Prunus serotina Ehrh.) in Flanders (Belgium) and its impact on the forest understorey community

The design of cost-efficient control strategies for invasive species that are too widespread and abundant for complete eradication, at least in the short term, will benefit from a rigorous analysis of invasion patterns and associated effects on native biodiversity. In this paper, the case of the invasive North American tree Prunus serotina in Flanders (Belgium) is presented. Our main objectives were to determine the susceptibility of forest stands to invasion by P. serotina and the subsequent effects of invasion on the understorey community. We used the large database of the first Flemish Forest Inventory. Multiple logistic regressions indicated that P. serotina occurred more frequently in privately owned, younger forest on coarse-textured, dry soils (podzols), and the combination of these factors allowed us to correctly predict presence/absence of P. serotina in 70% of the validation plots. However, locational variables proved to be important as well, indicating that the invasion process is still ongoing. Prediction of P. serotina densities by means of multiple linear regressions was less successful. Effects on the understorey richness were analysed by comparing the number of species and the mean Ellenberg values between pairs of plots, only differing by the presence of P. serotina in the shrub layer. A reduction of the understorey richness following invasion was only pronounced on the more moist soils, while compositional changes mainly occurred on drier soils. It is concluded that priority for control should be given to landscapes with a low fraction of invaded stands and to forest stands located on more moist soils. However, using its potential to threaten native biodiversity as an argument for control should be done with care as further research is needed whether the observed negative effects are due to a species (i.e. native vs. non-native) or a density effect (high vs. low).     

Smoking cessation and bronchial epithelial remodelling in COPD: a cross-sectional study

Background

Chronic Obstructive Pulmonary Disease (COPD) is associated with bronchial epithelial changes, including squamous cell metaplasia and goblet cell hyperplasia. These features are partially attributed to activation of the epidermal growth factor receptor (EGFR). Whereas smoking cessation reduces respiratory symptoms and lung function decline in COPD, inflammation persists. We determined epithelial proliferation and composition in bronchial biopsies from current and ex-smokers with COPD, and its relation to duration of smoking cessation.

Methods

114 COPD patients were studied cross-sectionally: 99 males/15 females, age 62 ± 8 years, median 42 pack-years, no corticosteroids, current (n = 72) or ex-smokers (n = 42, median cessation duration 3.5 years), postbronchodilator FEV₁ 63 ± 9% predicted. Squamous cell metaplasia (%), goblet cell (PAS/Alcian Blue⁺) area (%), proliferating (Ki-67⁺) cell numbers (/mm basement membrane), and EGFR expression (%) were measured in intact epithelium of bronchial biopsies.

Results

Ex-smokers with COPD had significantly less epithelial squamous cell metaplasia, proliferating cell numbers, and a trend towards reduced goblet cell area than current smokers with COPD (p = 0.025, p = 0.001, p = 0.081, respectively), but no significant difference in EGFR expression. Epithelial features were not different between short-term quitters (<3.5 years) and current smokers. Long-term quitters (≥3.5 years) had less goblet cell area than both current smokers and short-term quitters (medians: 7.9% vs. 14.4%, p = 0.005; 7.9% vs. 13.5%, p = 0.008; respectively), and less proliferating cell numbers than current smokers (2.8% vs. 18.6%, p < 0.001).

Conclusion

Ex-smokers with COPD had less bronchial epithelial remodelling than current smokers, which was only observed after long-term smoking cessation (>3.5 years).

Trial registration

NCT00158847     

The methylation patterns of the IGF2 and IGF2R genes in bovine spermatozoa are not affected by flow-cytometric sex sorting

The objectives of this study were to investigate the effect of sexing by flow cytometry on the methylation patterns of the IGF2 and IGF2R genes. Frozen‐thawed, unsorted, and sex‐sorted sperm samples from four Nellore bulls were used. Each ejaculate was separated into three fractions: non‐sexed (NS), sexed for X‐sperm (SX), and sexed for Y‐sperm (SY). Sperm were isolated from the extender, cryoprotectant, and other cell types by centrifugation on a 40:70% Percoll gradient, and sperm pellets were used for genomic DNA isolation. DNA was used for analyses of the methylation patterns by bisulfite sequencing. Methylation status of the IGF2 and IGF2R genes were evaluated by sequencing 195 and 147 individual clones, respectively. No global differences in DNA methylation were found between NS, SX, and SY groups for the IGF2 (P = 0.09) or IGF2R genes (P = 0.38). Very specific methylation patterns were observed in the 25th and 26th CpG sites in the IGF2R gene. representing higher methylation in NS than in the SX and SY groups compared with the other CpG sites. Further, individual variation in methylation patterns was found among bulls. In conclusion, the sex‐sorting procedure by flow cytometry did not affect the overall DNA methylation patterns of the IGF2 and IGF2R genes, although individual variation in their methylation patterns among bulls was observed. Mol. Reprod. Dev. 79:77–84, 2012. © 2011 Wiley Periodicals, Inc.     

Regulation of the stimulant actions of neurokinin a and human hemokinin-1 on the human uterus: a comparison with histamine

Regulation of the contractile effects of tachykinins and histamine on the human uterus was investigated with biopsy sections of the outer myometrial layer. The effects of neurokinin A (NKA) and human hemokinin-1 (hHK-1) in tissues from pregnant but not from nonpregnant women were enhanced by the inhibition of neprilysin. The effects of NKA and eledoisin were blocked by the NK2 receptor antagonist SR 48968 but not by the NK1 receptor antagonist SR 140333 in tissues from both groups of women. Human HK-1 acted as a partial agonist blocked by SR 48968 and, to a lesser extent, by SR 140333; endokinin D was inactive. In tissues from pregnant women, responses to high potassium-containing Krebs solution were 2-3-fold higher than those from nonpregnant women. Mepyramine-sensitive maximal responses to histamine were similarly enhanced. The absolute maximum responses to NKA and its stable NK2 receptor-selective analogue, [Lys5MeLeu9Nle10]NKA(4-10), were increased in pregnancy, but their efficacies relative to potassium responses were decreased. Tachykinin potencies were lower in tissues from pregnant women than in those from nonpregnant women. These data 1) show for the first time that hHK-1 is a uterine stimulant in the human, 2) confirm that the NK2 receptor is predominant in mediating tachykinin actions on the human myometrium, and 3) indicate that mammalian tachykinin effects are tightly regulated during pregnancy in a manner that would negate an inappropriate uterotonic effect. The potencies of these peptides in tissues from nonpregnant women undergoing hysterectomy are consistent with their possible role in menstrual and menopausal disorders.     

Methylated polyphenols are poor "chemical" antioxidants but can still effectively protect cells from hydrogen peroxide-induced cytotoxicity

Several polyphenolic compounds, including flavonoids and phenolic acids, were compared with their per-methylated forms in both chemical and cell-based assays for antioxidant capacity. Methylation largely eliminated "chemical" antioxidant capacity, according to ferric reducing antioxidant power and oxygen radical absorbance capacity assays. Methylation, however, only moderately reduced protection of human Jurkat cells in culture, from hydrogen peroxide-mediated cytotoxicity, at physiologically relevant concentrations. Neither methylated nor un-methylated compounds were detectably metabolized by the cells. It appears that the protective mechanism of polyphenolic antioxidants against high concentrations of hydrogen peroxide in human cells may be largely unrelated to chemical antioxidant capacity.     

A two-entropies analysis to identify functional positions in the transmembrane region of class A G protein-coupled receptors

Residues in the transmembrane region of G protein-coupled receptors (GPCRs) are important for ligand binding and activation, but the function of individual positions is poorly understood. Using a sequence alignment of class A GPCRs (grouped in subfamilies), we propose a so-called "two-entropies analysis" to determine the potential role of individual positions in the transmembrane region of class A GPCRs. In our approach, such positions appear scattered, while largely clustered according to their biological function. Our method appears superior when compared to other bioinformatics approaches, such as the evolutionary trace method, entropy-variability plot, and correlated mutation analysis, both qualitatively and quantitatively.     

Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge <Emphasis Type="Italic">Microciona prolifera</Emphasis>

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca²⁺-independent cell-binding domain and (ii) a Ca²⁺-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide β-d-GlcpNAc3S-(1→3)-α-l-Fucp and a pyruvated trisaccharide β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly demonstrated self-recognition on the disaccharide level in the presence of Ca²⁺-ions. To determine binding forces of the carbohydrate–carbohydrate interactions for both synthetic MAF oligosaccharides, atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca²⁺-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- was found to be quantized in integer multiples of 30 ± 6 pN. No binding was observed between the two monolayers in the absence of Ca²⁺-ions. Cd²⁺-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- did not show a binding in the presence of Ca²⁺-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation in the presence of Ca²⁺-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between the pyruvated trisaccharide.     

Nitrosamine exposure exacerbates high fat diet-mediated type 2 diabetes mellitus, non-alcoholic steatohepatitis, and neurodegeneration with cognitive impairment

Background

The current epidemics of type 2 diabetes mellitus (T2DM), non-alcoholic steatohepatitis (NASH), and Alzheimer's disease (AD) all represent insulin-resistance diseases. Previous studies linked insulin resistance diseases to high fat diets or exposure to streptozotocin, a nitrosamine-related compound that causes T2DM, NASH, and AD-type neurodegeneration. We hypothesize that low-level exposure to nitrosamines that are widely present in processed foods, amplifies the deleterious effects of high fat intake in promoting T2DM, NASH, and neurodegeneration.

Methods

Long Evans rat pups were treated with N-nitrosodiethylamine (NDEA) by i.p. Injection, and upon weaning, they were fed with high fat (60%; HFD) or low fat (5%; LFD) chow for 6 weeks. Rats were evaluated for cognitive impairment, insulin resistance, and neurodegeneration using behavioral, biochemical, molecular, and histological methods.

Results

NDEA and HFD ± NDEA caused T2DM, NASH, deficits in spatial learning, and neurodegeneration with hepatic and brain insulin and/or IGF resistance, and reductions in tau and choline acetyltransferase levels in the temporal lobe. In addition, pro-ceramide genes, which promote insulin resistance, were increased in livers and brains of rats exposed to NDEA, HFD, or both. In nearly all assays, the adverse effects of HFD+NDEA were worse than either treatment alone.

Conclusions

Environmental and food contaminant exposures to low, sub-mutagenic levels of nitrosamines, together with chronic HFD feeding, function synergistically to promote major insulin resistance diseases including T2DM, NASH, and AD-type neurodegeneration. Steps to minimize human exposure to nitrosamines and consumption of high-fat content foods are needed to quell these costly and devastating epidemics.