Custom design of a GeXP multiplexed assay used to assess expression profiles of inflammatory gene targets in normal colon, polyp, and tumor tissue

Colon cancers are characterized by aberrant gene expression signatures associated with disease initiation and progression. Identification of aberrant gene expression associated with colon carcinogenesis has increased significantly with application of gene array technologies. Downstream processing of these data has been hindered by the lack of robust multiplexed gene quantitative technologies facilitating study of the identified multiple gene targets. The GenomeLab Genetic Analysis System presents a novel technology platform for quantitative multiplexed gene expression analysis. This report describes the custom design of a GeXP multiplexed assay used to assess expression profiles of 14 inflammatory gene targets in normal, polyp, and tumor tissue. Characteristic normal, polyp, and tumor tissue gene expression profiles were obtained. Statistical analysis confirmed comparable relative quantitation of gene expression using the GeXP, macroarray, and single-plex real-time polymerase chain reaction assays. GeXP assays may be usefully applied in clinical and regulatory studies of multiple gene targets. This system permits custom-design options for relative quantification of multiple gene target expression, simultaneously in a single reaction, using nanogram quantities of total RNA template. The system provides an approach to advance the study of multiple targets identified from gene array analysis with potential for characterizing gene expression signatures in clinical diagnostics.     

Genetic variations of the melatonin pathway in patients with attention-deficit and hyperactivity disorders

Melatonin is a powerful antioxidant and a synchronizer of many physiological processes. Alteration in melatonin signaling has been reported in a broad range of diseases, but little is known about the genetic variability of this pathway in humans. Here, we sequenced all the genes of the melatonin pathway -AA-NAT, ASMT, MTNR1A, MTNR1B and GPR50 - in 321 individuals from Sweden including 101 patients with attention-deficit/hyperactivity disorder (ADHD) and 220 controls from the general population. We could find several damaging mutations in patients with ADHD, but no significant enrichment compared with the general population. Among these variations, we found a splice site mutation in ASMT (IVS5+2T>C) and one stop mutation in MTNR1A (Y170X) - detected exclusively in patients with ADHD - for which biochemical analyses indicated that they abolish the activity of ASMT and MTNR1A. These genetic and functional results represent the first comprehensive ascertainment of melatonin signaling deficiency in ADHD.     

Erythropoietin, GDF15, IL6, hepcidin and testosterone levels in a large cohort of elderly individuals with anaemia of known and unknown cause

Epidemiologic studies have documented an increasing frequency of anaemia in individuals 65 yrs and older. Elderly individuals with anaemia have been categorised into the following: those with chronic disease, those with iron, B12 or folate deficiency and those with anaemia of unknown aetiology (AUE). There is considerable interest and debate as to whether AUE has an inflammatory component, is caused by cytokine dysregulation affecting production or response to erythropoietin (EPO) or iron availability or represents a novel pathologic process. Here, we compare a large cohort of AUE cases with a matched, non-anaemic control group and with individuals who have anaemia of defined cause. IL-6, hepcidin, GDF15, EPO and testosterone levels were compared. IL6 and hepcidin levels did not differ significantly between AUE and control groups, indicating that inflammation or iron restriction is not central feature of anaemia in this group. GDF15 levels were significantly elevated when comparing AUE with controls and were markedly elevated in patients with renal disease. Testosterone levels were lower in men from the AUE group compared with non-anaemic controls. EPO levels in the AUE group were increased relative to controls but were inappropriately low for the degree of anaemia. Our data indicate that an impaired EPO response, in the absence of evidence for iron restriction or inflammation, is characteristic of AUE.     

Serum Fetuin-A Levels Are Associated with Vascular Calcifications and Predict Cardiovascular Events in Renal Transplant Recipients

Summary

Background and objectives

Vascular calcifications predict cardiovascular disease, the major cause of death in renal transplant recipients (RTRs). We studied the determinants of fetuin-A, a potent circulating calcification inhibitor encoded by the AHSG gene, and tested its association with vascular calcifications and long-term survival and cardiovascular events (CVEs) in RTRs.

Design, setting, participants, & measurements

Two hundred seventy-seven prevalent RTRs from a single center were included. CVEs and deaths were prospectively recorded during a 5-year follow-up.

Results

Independent determinants of lower serum fetuin-A levels were lower plasma cholesterol, the AHSG rs4918 G allele, and history of smoking. Low serum fetuin-A level was a determinant of aortic calcifications (assessed using spiral CT). Low fetuin-A levels (≤0.47 g/L, first quintile) were independently associated with CVEs and deaths (hazard ratio = 1.83; 95% confidence interval, 1.07 to 3.04). The association was confirmed for all-cause mortality, and the major adverse cardiovascular endpoints were analyzed separately. Patients with low fetuin-A and high high-sensitivity C-reactive protein (>4.36 mg/L, fourth quintile) levels had a 3.5-fold increased risk of all-cause mortality and CVEs. In the presence of inflammation, CVE-free survival was influenced by common variants in the AHSG gene.

Conclusions

These data show that low fetuin-A levels are independently associated with aortic calcifications and a higher risk of CVEs and mortality. They support fetuin-A as a circulating biomarker able to identify RTRs at risk for vascular calcifications and CVEs.     

Serotonin syndrome associated with polypharmacy in the elderly

The increasing use of serotonergic agents, alone and in combination, across multiple disciplines, makes it likely that the prevalence of serotonin syndrome will rise. Caution should be used, especially in the elderly, to avoid unnecessary and potentially harmful polypharmacy. We describe a case of serotonin syndrome in a 79-year-old man taking mirtazapine, venlafaxine and quetiapine. As this case illustrates, serotonin syndrome can be caused by combinations of direct serotonin agonists (e.g., serotonergic antidepressants) and indirect serotonin agonists (e.g., atypical antipsychotics).     

Binding characteristics of thrombin-activatable fibrinolysis inhibitor to streptococcal surface collagen-like proteins A and B

Streptococcus pyogenes is the causative agent in a wide range of diseases in humans. Thrombin-activatable fibrinolysis inhibitor (TAFI) binds to collagen-like proteins SclA and SclB at the surface of S. pyogenes. Activation of TAFI at this surface redirects inflammation from a transient to chronic state by modulation of the kallikrein/kinin system. We investigated TAFI binding characteristics to SclA/SclB. Thirty-four overlapping TAFI peptides of ~20 amino acids were generated. Two of these peptides (P18: residues G205-S221, and P19: R214-D232) specifically bound to SclA/SclB with high affinity, and competed in a dose-dependent manner with TAFI binding to SclA/SclB. In another series of experiments, the binding properties of activated TAFI (TAFIa) to SclA/SclB were studied with a quadruple TAFI mutant (TAFI-IIYQ) that after activation is a 70-fold more stable enzyme than wild-type TAFIa. TAFI and TAFI-IIYQ bound to the bacterial proteins with similar affinities. The rate of dissociation was different between the proenzyme (both TAFI and TAFI-IIYQ) and the stable enzyme TAFIa-IIYQ. TAFIa-IIYQ bound to SclA/SclB, but dissociated faster than TAFI-IIYQ. In conclusion, the bacterial proteins SclA and SclB bind to a TAFI fragment encompassing residues G205-D232. Binding of TAFI to the bacteria may allow activation of TAFI, whereafter the enzyme easily dissociates.     

Focal central white matter lesions in Alexander disease

Alexander disease is a neurodegenerative disorder of the central white matter caused by dominant mutations in GFAP, the gene encoding glial fibrillary acidic protein. Magnetic resonance imaging pattern recognition studies have established characteristic radiologic phenotypes for this disorder. In some cases, however, genetically confirmed cases do not express these features, and several reports have identified "atypical" radiologic findings in Alexander disease patients. Here, the authors report 3 genetically confirmed Alexander disease cases with focal central white matter lesions that, upon longitudinal clinical and radiologic evaluation, appear to reflect an atypical Alexander disease magnetic resonance imaging phenotype and not another pathophysiologic process such as encephalitis, infarction, or neoplasm.     

Differential patterns of initial and sustained responses in amygdala and cortical regions to emotional stimuli in schizophrenia patients and healthy participants

Background

We sought to investigate the altered brain responses to emotional stimuli in patients with schizophrenia.

Methods

We analyzed data from 14 patients with schizophrenia and 14 healthy controls who performed an emotional face matching task. We evaluated brain activity and connectivity in the amygdala and cortical regions during the initial (first 21 seconds of each stimulation block) and sustained (last 21 seconds) stages of an emotional processing task, and we determined changes in amygdala activity across the emotional processing task.

Results

The patients with schizophrenia showed similar amygdala activation to the controls during the initial stage of processing, but their activation decreased during the sustained stage. The controls showed increasing amygdala activity across the emotional blocks, whereas activity progressively decreased in the schizophrenia group. The patients with schizophrenia showed increased cortical activity and interconnectivity in the medial frontal and inferior parietal cortex in the initial stage of emotional processing. There was increased activity in the superior temporal cortex and greater connectivity with the inferior parietal cortex in the sustained stage. Performance accuracy was lower in the schizophrenia group in the first part of the block, while their reaction time was longer in the latter part of the block.

Limitations

It was not possible to specify the moment at which the switch in amygdala response occurred.

Conclusion

Our findings suggest that patients with schizophrenia have an initial automatic emotional response but that they need to switch to a compensatory cognitive strategy to solve the task.     

Human intestinal parasitism,potable water availability and methods of sewage disposal among nomadic Fulanis in Kuraje rural settlement of Zamfara state

ObjectiveTo ascertain the level of intestinal parasites vis-à-vis the quality of housing and water supply, and sanitary conditions among the people of Kuraje village in Zamfara state.MethodsThe study was cross sectional in nature. Individual households were selected using systematic random sampling methods and pre-tested questionnaires were administered to all the members of each household. Stool samples were collected and processed using standard laboratory procedures. Housing conditions, sources of water and sanitary conditions of the households were also inspected. Results were analysed using Epi Info 2006 model.ResultsThe prevalence of intestinal parasites was 67.0% (347/519). 72.3% (251/347), 17.0% (59/347), and 10.7% (37/347) had one, two and three or more parasites, respectively. The associated factors with intestinal parasites were poor housing and sanitary conditions, lack of potable water and illiteracy. The commonest parasites encountered were hookworm (22.0%), Ascaris lumbricoides (18.5%), and Strongyloides stercoralis (15.6%) while the least common was Enterobius vermicularis (1.6%). Others were Giardia lamblia (5.7%), Hymenolepsis nana (5.0%), Trichuris trichiura (8.8%), Entamoeba histolytica (14.4%) and Schistosoma mansoni (8.4%).ConclusionsThe infection rate of intestinal parasites in Kuraje village is high. More efforts should be intensified towards improvement in sanitary and housing standards, supply of potable water and institution of a more comprehensive literacy programme for the people of the community.