Intestinal microbiota in infants at high risk for allergy: Effects of prebiotics and role in eczema development

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Tetrathiomolybdate induces doxorubicin sensitivity in resistant tumor cell lines

Objective

Doxorubicin is a potent anti-cancer agent with efficacy against a broad range of tumors, including endometrial cancer. Doxorubicin produces reactive oxygen species (ROS) resulting in cytotoxicity. Tetrathiomolybdate (TM), a copper-chelating agent, is known to target a cellular antioxidant enzyme copper/zinc-superoxide dismutase. This study tests the hypothesis that TM can modulate antioxidants in tumor cells and render doxorubicin resistant tumor cells sensitive to doxorubicin.

Methods

The anti-cancer activities of doxorubicin and TM, as single agents and in combination, were assessed. Flow cytometric and immunoblot analysis were conducted to investigate the induction of apoptosis and changes in apoptotic signaling pathways.

Results

Doxorubicin-induced growth inhibition was observed in each endometrial cancer cell line (ECC-1, AN3CA, and KLE) tested with cell specificity. ECC-1 and KLE cells were found to have increased resistance to doxorubicin than AN3CA cells. Moreover, doxorubicin mediated apoptosis was greater in the AN3CA cell line than ECC-1 and KLE. The combination of doxorubicin with a sub-cytotoxic level of TM was significantly more effective at inducing apoptosis in doxorubicin resistant cell lines.

Conclusion

Our results highlight the therapeutic potential of TM to sensitize tumor cells to doxorubicin for endometrial cancer treatment.     

An AscI boundary library for the studies of genetic and epigenetic alterations in CpG islands

Knudson's two-hit hypothesis postulates that genetic alterations in both alleles are required for the inactivation of tumor-suppressor genes. Genetic alterations include small or large deletions and mutations. Over the past years, it has become clear that epigenetic alterations such as DNA methylation are additional mechanisms for gene silencing. Restriction Landmark Genomic Scanning (RLGS) is a two-dimensional gel electrophoresis that assesses the methylation status of thousands of CpG islands. RLGS has been applied successfully to scan cancer genomes for aberrant DNA methylation patterns. So far, the majority of this work was done using NotI as the restriction landmark site. Here, we describe the development of RLGS using AscI as the restriction landmark site for genome-wide scans of cancer genomes. The availability of AscI as a restriction landmark for RLGS allows for scanning almost twice as many CpG islands in the human genome compared with using NotI only. We describe the development of an AscI-EcoRV boundary library that supports the cloning of novel methylated genes. Feasibility of this system is shown in three tumor types, medulloblastomas, lung cancers, and head and neck cancers. We report the cloning of 178 AscI RLGS fragments via two methods by use of this library.     

Percept-related changes in horizontal optokinetic nystagmus at different body orientations in space

Large-field motion of the visual environment is a powerful stimulus to induce the perception of contra-directional self-motion in a stationary observer. We investigated the interrelations between horizontal optokinetic nystagmus and subjective states of motion perception under variation of subjects' orientation with respect to gravity. Subjects were tested sitting upright and lying supine, and signalled transitions between object- and self-motion perception whilst viewing an optokinetic stimulus rotating about the subjects' longitudinal axis at a range of angular velocities. Optokinetic stimulation in the supine condition resulted in subjects perceiving a graviceptive conflict and the illusory perception of whole body tilt in a direction opposite to optokinetic stimulus rotation, whereas during upright viewing the axis of stimulus rotation was aligned with the direction of gravity and thus did not result in a conflict or perception of tilt. In both postures, self-motion perception coincided with an increased deviation of mean horizontal gaze position in the perceived direction of heading with a concurrent reduction in optokinetic nystagmus slow-phase gain. Slow-phase gain was also significantly reduced in the supine position as well as at increasing stimulus velocities. The results demonstrate that spontaneous transitions between the perception of object-motion and that of self-motion consistently coincide with spatial attentional and orientational strategies, shifting from passive monitoring to active oculomotor exploration and anticipation.     

Lipopolysaccharide-based enzyme-linked immunosorbent assay for experimental use in detection of antibodies to Lawsonia intracellularis in pigs

An enzyme-linked immunosorbent assay (ELISA) for Lawsonia intracellularis was developed and compared with a whole-cell antigen-based immunofluorescence antibody test (IFAT). The antigen-containing lipopolysaccharide (LPS) was derived from Percoll gradient purified cultures of L. intracellularis by using a modification of the Westphal hot phenol procedure. The antigen was bound directly to polystyrene 96-well microtiter plates, and the assay was performed in an indirect ELISA format. Specificity and sensitivity values based on 80 known positive and 80 known negative serum samples from controlled experimental trials were 93.7% and 88.7%, respectively. Serological results from a controlled L. intracellularis challenge exposure study confirmed the high specificity and sensitivity of this assay (100% and 99.5%, respectively). Comparisons between the LPS ELISA and the IFAT in detecting anti-Lawsonia antibodies in this controlled study revealed significantly more LPS ELISA-positive pigs than IFAT-positive pigs on days 21, 28, 35, and 42 (P = 0.003, 0.030, 0.002, and 0.006, respectively). This indirect ELISA (LPS ELISA) test is an improved method of detecting antibodies in pigs soon after exposure to L. intracellularis, regardless of isolate type (vaccine or wild type) in experimental studies. The LPS ELISA may be used as a tool to support future research trials on vaccine efficacy and to further understand the immune response induced by L. intracellularis.     

Lipopolysaccharide-Based Enzyme-Linked Immunosorbent Assay for Experimental Use in Detection of Antibodies to Lawsonia intracellularis in Pigs

An enzyme-linked immunosorbent assay (ELISA) for Lawsonia intracellularis was developed and compared with a whole-cell antigen-based immunofluorescence antibody test (IFAT). The antigen-containing lipopolysaccharide (LPS) was derived from Percoll gradient purified cultures of L. intracellularis by using a modification of the Westphal hot phenol procedure. The antigen was bound directly to polystyrene 96-well microtiter plates, and the assay was performed in an indirect ELISA format. Specificity and sensitivity values based on 80 known positive and 80 known negative serum samples from controlled experimental trials were 93.7% and 88.7%, respectively. Serological results from a controlled L. intracellularis challenge exposure study confirmed the high specificity and sensitivity of this assay (100% and 99.5%, respectively). Comparisons between the LPS ELISA and the IFAT in detecting anti-Lawsonia antibodies in this controlled study revealed significantly more LPS ELISA-positive pigs than IFAT-positive pigs on days 21, 28, 35, and 42 (P = 0.003, 0.030, 0.002, and 0.006, respectively). This indirect ELISA (LPS ELISA) test is an improved method of detecting antibodies in pigs soon after exposure to L. intracellularis, regardless of isolate type (vaccine or wild type) in experimental studies. The LPS ELISA may be used as a tool to support future research trials on vaccine efficacy and to further understand the immune response induced by L. intracellularis.     

Geminin regulates neuronal differentiation by antagonizing Brg1 activity

Precise control of cell proliferation and differentiation is critical for organogenesis. Geminin (Gem) has been proposed to link cell cycle exit and differentiation as a prodifferentiation factor and plays a role in neural cell fate acquisition. Here, we identified the SWI/SNF chromatin-remodeling protein Brg1 as an interacting partner of Gem. Brg1 has been implicated in cell cycle withdrawal and cellular differentiation. Surprisingly, we discovered that Gem antagonizes Brg1 activity during neurogenesis to maintain the undifferentiated cell state. Down-regulation of Gem expression normally precedes neuronal differentiation, and gain- and loss-of-function experiments in Xenopus embryos and mouse P19 cells demonstrated that Gem was essential to prevent premature neurogenesis. Misexpression of Gem also suppressed ectopic neurogenesis driven by Ngn and NeuroD. Gem's activity to block differentiation depended upon its ability to bind Brg1 and could be mediated by Gem's inhibition of proneural basic helix-loop-helix (bHLH)-Brg1 interactions required for bHLH target gene activation. Our data demonstrate a novel mechanism of Gem activity, through regulation of SWI/SNF chromatin-remodeling proteins, and indicate that Gem is an essential regulator of neurogenesis that can control the timing of neural progenitor differentiation and maintain the undifferentiated cell state.     

Correlation between clinical scoring of allergic patch test reactions and optical coherence tomography

Noninvasive imaging techniques might be of particular diagnostic value for studying and monitoring cutaneous inflammatory conditions such as contact dermatitis. We evaluate acute allergic contact dermatitis (AACD) by means of optical coherence tomography (OCT) and correlate the clinical grading of patch test reactions with the findings obtained from OCT. Twenty positive patch test reactions (+, n = 6; ++, n = 7; +++, n = 7) are investigated using a conventional OCT scanner. In comparison to the control sites, OCT of AACD showed pronounced skin folds, thickened and/or disrupted entrance signals, and a significant increase in epidermal thickness. Moreover, clearly demarcated signal-free cavities within the epidermis and considerable reduction of dermal reflectivity are demonstrated by OCT. Notably, the latter findings strongly correlate with the clinical patch test grading. OCT may be a useful tool for visualization of micromorphological features of AACD. However, before OCT can be employed as an objective parameter in grading severity of patch test reactions, larger studies are required that correlate clinical patch test readings and OCT findings with histopathology.