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Beall, Francis A. (U.S. Army Chemical Corps, Frederick, Md.), Martha J. Taylor, and Curtis B. Thorne. Rapid lethal effect in rats of a third component found upon fractionating the toxin of Bacillus anthracis. J. Bacteriol. 83:1274–1280. 1962.—Rats were found to be more susceptible to the lethal effect of toxin produced by Bacillus anthracis in vitro than were several species considered less resistant to anthrax. Rats were killed much faster by less toxin per gram of body weight than were mice. Guinea pigs survived doses of toxin that killed rats. Intravenous injection of Fischer 344 rats is a rapid test for lethal activity, which facilitates the demonstration of two components, different from protective antigen, in toxin. One of these, a lethal factor, was separated from the other component, which causes cutaneous edema in the guinea pig. The latter component was not necessary for lethal effect. Neither of these factors was active unless combined with protective antigen. Although the guinea pig skin reaction has been used routinely to assay the toxicity of samples, the present results show that this test does not assay the lethal component.

The OxyR regulon is known to mediate protection against oxidizing agents in Salmonella typhimurium. We reported previously that ahp, one of the OxyR-regulated loci, is induced during macrophage interaction (K. P. Francis, P. D. Taylor, C. J. Inchley, and M. P. Gallagher, J. Bacteriol. 179:4046–4048, 1997). We now report on the effects of disrupting ahp or oxyR on virulence in a BALB/c mouse model. Surprisingly, insertion of a Mudlux derivative within ahpC was found to result in attenuation, while irreversible inactivation of the locus through insertion of a cml cassette did not. An SL1344 derivative carrying an oxyR::kan disruption was also found to be as virulent as the parental strain. Moreover, both cell-mediated and humoral responses to AhpC were found to develop during the course of infection, probably through T-helper-cell (type I) activation. These results indicate that, although not essential for virulence, AhpC is expressed by S. typhimurium during infection of BALB/c mice and constitutes a target for the immune system.

Background

The transfer and implementation of acceptable and effective health services, programs and innovations across settings provides an important and potentially cost-effective strategy for reducing Indigenous Australians' high burden of disease. This study reports a systematic review of Indigenous health services, programs and innovations to examine the extent to which studies considered processes of transfer and implementation within and across Indigenous communities and healthcare settings.

Methods

Medline, Informit, Infotrac, Blackwells Publishing, Proquest, Taylor and Francis, JStor, and the Indigenous HealthInfoNet were searched using terms: Aborigin* OR Indigen* OR Torres AND health AND service OR program* OR intervention AND Australia to locate publications from 1992–2011. The reference lists of 19 reviews were also checked. Data from peer reviewed journals, reports, and websites were included. The 95% confidence intervals (95% CI) for proportions that referred to and focussed on transfer were calculated as exact binomial confidence intervals. Test comparisons between proportions were calculated using Fisher's exact test with an alpha level of 5%.

Results

Of 1311 publications identified, 119 (9.1%; 95% CI: 7.6% - 10.8%) referred to the transfer and implementation of Indigenous Australian health services or programs, but only 21 studies (1.6%; 95% CI: 1.0% - 2.4%) actually focused on transfer and implementation. Of the 119 transfer studies, 37 (31.1%; 95% CI: 22.9 - 40.2%) evaluated the impact of a service or program, 28 (23.5%; 95% CI: 16.2% - 32.2%) reported only process measures and 54 were descriptive. Of the 37 impact evaluation studies, 28 (75.7%; 95% CI: 58.8% - 88.2%) appeared in peer reviewed journals but none included experimental designs.

Conclusion

While services and programs are being transferred and implemented, few studies focus on the process by which this occurred or the effectiveness of the service or program in the new setting. Findings highlight a need for partnerships between researchers and health services to evaluate the transfer and implementation of Indigenous health services and programs using rigorous designs, and publish such efforts in peer-reviewed journals as a quality assurance mechanism.

David A. Boas, Constantinos Pitris, and Nimmi Ramanujam, Eds.:

Handbook of Biomedical Optics

CRC Press, Taylor and Francis Group, Boca Raton, London, New York, 2011

ISBN: 978-1-4200-9036-9 (Hardback), 787 pages

Previously, we tagged a macrophage-induced Salmonella typhimurium locus with Mudlux (K. P. Francis and M. P. Gallagher, Infect. Immun. 61:640-649, 1993). The insertion lies within the OxyR-regulated ahpC locus and conveys alkyl peroxide sensitivity. Plasmid-encoded ahp reverses sensitivity but reduces luminescence. This suggests that OxyR is titrated by the multicopy ahp promoter.

Objective

Depression and anxiety are associated with autonomic nervous system dysfunction, which may promote the risk of malignant cardiac arrhythmias. This study investigates whether depression and anxiety symptoms are associated with measures of autonomic nervous system dysfunction in patients with implantable cardioverter defibrillators who are at high risk of cardiac rhythm disturbances.

Methods

Patients with an implantable cardioverter defibrillator (ICD) underwent ambulatory electrocardiographic monitoring (n=44, mean age 62.1 ± 9.3 yrs). Depression was assessed using the Beck Depression Inventory and anxiety using the Taylor Manifest Anxiety Scale. Heart rate variability (HRV) was assessed using time (RMSSD, pNN50 and SDNN) and frequency domain measures derived from 24 hour R-R intervals. Multivariate models were adjusted for age, sex, hypertension, diabetes and smoking status.

Results

Defibrillator patients with elevated depression symptoms (n=12) had significantly lower RMSSD (15.25 ± 1.66 ms, vs. 24.97 ± 2.44, p = 0.002) and pNN50 (1.83 ± 0.77 vs. 5.61 ± 1.04, p = 0.006) than defibrillator patients with low depression symptoms (n=32). These associations remained significant after multivariate adjustment for covariates. ICD patients with high anxiety levels (n=10) displayed lower RMSSD (p = 0.013), which became marginally significant when adjusting for covariates (p = 0.069).

Conclusion

Depression and anxiety in defibrillator patients are associated with autonomic nervous system dysfunction indices of reduced parasympathetic control. Autonomic nervous system dysfunction may partially explain the association between depression and anxiety with life-threatening cardiac outcomes in vulnerable patients.

BACKGROUND & AIMS

MicroRNAs (miRNAs) are a class of small noncoding RNAs that can regulate gene expression by translation repression or mRNA degradation. Our aim was to evaluate the role of aberrantly expressed miRNAs in hepatocellular cancer (HCC).

METHODS

miRNA expression in HCC tissues and cells was evaluated by qPCR array and Taqman miRNA assay. Cell proliferation, motility, invasion and angiogenesis index were quantitated using commercial assays. DNA methylation status, matrix metalloproteinases (MMPs) mRNA expression was quantitated by real-time PCR analysis.

RESULTS

miRNA profiling identified a decrease in miR-125b expression in HCC tumor tissues and cell lines. The expression of miR-125b was significantly increased by the methylation inhibitor 5-aza-2'-deoxycytidine in HCC cells but not in normal controls, suggesting that the expression of miR-125b could be epigenetically modulated. Methylation-specific PCR revealed hypermethylation status of miR-125b in HCC cells instead of non-malignant controls. Cell proliferation, anchorage-independent growth, cell migration, invasion and angiogenesis were significantly decreased by the introduction of miR-125b precursor in HCC cell lines. Placenta growth factor was identified as a target of miR-125b by bioinformatics analysis and experimentally verified using luciferase reporter constructs. Overexpression of miR-125b in HCC cells decreased PIGF expression, and altered angiogenesis index. Furthermore, modulation of miR-125b also distorted expression of MMP-2 and -9, the mediators of enzymatic degradation of extracellular matrix.

CONCLUSIONS

Our studies showing epigenetic silencing of miR-125b contributes to an invasive phenotype provide novel mechanistic insights and identify a potential target mechanism that could be manipulated for therapeutic benefit in HCC.

Background

In several tumours the endogenous activity of histidine decarboxylase (HDC), the enzyme stimulating histamine synthesis, sustains the autocrine trophic effect of histamine on cancer progression. Cholangiocarcinoma is a biliary cancer with limited treatment options. Histamine interacts with four G-protein coupled receptors, H1–H4 histamine receptors (HRs).

Objective

To determine the effects of histamine stimulation and inhibition of histamine synthesis (by modulation of HDC) on cholangiocarcinoma growth.

Methods

In vitro studies were performed using multiple human cholangiocarcinoma lines. The expression levels of the histamine synthetic machinery and HRs were evaluated along with the effects of histamine stimulation and inhibition on cholangiocarcinoma proliferation. A xenograft tumour model was used to measure tumour volume after treatment with histamine or inhibition of histamine synthesis by manipulation of HDC. Vascular endothelial growth factor (VEGF) expression was measured in cholangiocarcinoma cells concomitant with the evaluation of the expression of CD31 in endothelial cells in the tumour microenvironment.

Results

Cholangiocarcinoma cells display (1) enhanced HDC and decreased monoamine oxidase B expression resulting in increased histamine secretion; and (2) increased expression of H1–H4 HRs. Inhibition of HDC and antagonising H1HR decreased histamine secretion in Mz-ChA-1 cells. Long-term treatment with histamine increased proliferation and VEGF expression in cholangiocarcinoma that was blocked by HDC inhibitor and the H1HR antagonist. In nude mice, histamine increased tumour growth (up to 25%) and VEGF expression whereas inhibition of histamine synthesis (by reduction of HDC) ablated the autocrine stimulation of histamine on tumour growth (~80%) and VEGF expression. No changes in angiogenesis (evaluated by changes in CD31 immunoreactivity) were detected in the in vivo treatment groups.

Conclusion

The novel concept that an autocrine loop (consisting of enhanced histamine synthesis by HDC) sustains cholangiocarcinoma growth is proposed. Drug targeting of HDC may be important for treatment of patients with cholangiocarcinoma.

Although large cholangiocytes exert their functions by activation of cyclic adenosine 3′,5′-monophosphate (cAMP), Ca2+-dependent signaling regulates the function of small cholangiocytes. Histamine interacts with four receptors, H1–H4HRs. H1HR acts by Gαq activating IP3/Ca2+, whereas H2HR activates Gαs stimulating cAMP. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1–H4HRs was evaluated in liver sections, isolated and cultured (normal rat intrahepatic cholangiocyte culture (NRIC)) cholangiocytes. In vivo, normal rats were treated with histamine or H1–H4HR agonists for 1 week. We evaluated: (1) intrahepatic bile duct mass (IBDM); (2) the effects of histamine, H1HR or H2HR agonists on NRIC proliferation, IP3 and cAMP levels and PKCα and protein kinase A (PKA) phosphorylation; and (3) PKCα silencing on H1HR-stimulated NRIC proliferation. Small and large cholangiocytes express H1–H4HRs. Histamine and the H1HR agonist increased small IBDM, whereas histamine and the H2HR agonist increased large IBDM. H1HR agonists stimulated IP3 levels, as well as PKCα phosphorylation and NRIC proliferation, whereas H2HR agonists increased cAMP levels, as well as PKA phosphorylation and NRIC proliferation. The H1HR agonist did not increase proliferation in PKCα siRNA-transfected NRICs. The activation of differential signaling mechanisms targeting small and large cholangiocytes is important for repopulation of the biliary epithelium during pathologies affecting different-sized bile ducts.

Objective

Patients with end-stage lung disease experience significant decrements in quality of life (QOL). Although coping strategies are related to QOL in patients with end-stage lung disease, the extent to which specific native lung disease moderates this relationship is unknown.

Methods

We investigated the relationship between coping, native lung disease, and QOL among 187 patients awaiting lung transplantation, including 139 patients with chronic obstructive pulmonary disease (COPD) and 48 patients with cystic fibrosis (CF). Participants completed a psychosocial battery assessing psychological QOL, physical QOL, and coping strategies.

Results

For both COPD and CF patients, higher levels of Active Coping (P < .0001) and lower levels of Disengagement (P < .0001) were associated with better psychological QOL. For physical QOL, we observed a native disease by coping interaction (P = .01) such that Active Coping was associated with better physical QOL in patients with COPD but not in patients with CF.

Conclusion

The relationship between coping and QOL may vary as a function of native lung disease. In order to develop effective interventions to help patients cope successfully with end stage lung disease, patients’ native disease may need to be considered.

The clinical manifestations of infection in cystic fibrosis (CF) are restricted to the lung, and involve a limited number of pathogens, suggesting a specific defect in mucosal immunity. We postulated that cystic fibrosis transmembrane conductance regulator (CTFR) mutations could affect the activation of type I interferon signaling in airway epithelial cells, which function in immune surveillance and initiate the recruitment and activation of immune cells. In response to infection with Pseudomonas aeruginosa, Ifnb was induced more than 100-fold in the murine lung, and the phosphorylation of STAT1 was similarly induced by the expected TLR4/TRIF/MD2/TBK1 cascade. The stimulation by P. aeruginosa of CF (IB3) cells and control (C-38) human cell lines similarly resulted in the induction of IFN-β, but to a significantly lower extent in CF airway cells. The potential consequences of diminished type I IFN signaling were demonstrated in a murine model of P. aeruginosa pneumonia, pretreatment with polyinosinic:polycytidylic acid significantly enhanced bacterial clearance and correlated with increased numbers of mature CD11c+/CD86+ dendritic cells (DCs) in the lung. Using culture supernatants from CF or control cell lines stimulated with P. aeruginosa, we similarly demonstrated the diminished activation of human monocyte–derived DCs by incubation with CF compared with normal epithelial cell culture supernatants, which was dependent on IFN-β. These observations suggest that dysfunction of the CFTR in airway epithelial cells may contribute to impaired immune surveillance in the CF airway and resultant colonization by P. aeruginosa.

Human Papillomavirus-16 (HPV-16) associated squamous carcinoma of the oropharynx has a favorable prognosis. Patients with HPV-16 positive cancers have elevated peripheral blood CD8+ T lymphocyte levels that correlate with response to chemotherapy and survival. Tumor infiltrating lymphocyte subpopulations (TIL) were assessed in pretreatment biopsies from a prospective patient cohort to determine if TIL subsets differed by HPV status, clinical factors, patient outcome or correlated with peripheral blood T cell levels.

Methods

Measured were CD8, CD4, CD68 and Treg (FoxP3) lymphocytes by immunohistochemistry in a tissue microarray created from patients (n=46) with advanced oropharynx cancer. Correlations with peripheral blood levels, HPV status, expression of EGFR, clinical tumor and patient characteristics and outcome were determined. Patients were treated with a single course of neoadjuvant chemotherapy (cisplatin, 5-fluorouracil) followed by either surgery (non-responders) or chemoradiation (cisplatin 100 mg/m2 every 3 weeks × 3; 70 Gy, 2 Gy daily × 7 wks) for responders. Median follow up was 6.6 years.

Results

HPV-16 positive patients had improved survival (p=0.016). Degree of T cell infiltration did not differ by HPV status but was significantly related to disease specific (DSS) and overall survival (OS). Higher infiltration by CD8, CD4 and FoxP3 subsets was significantly associated with lower T stage and survival. Even after adjusting for HPV status, CD8, FoxP3 and total T cells were significantly associated with DSS (p=0.0236; 0.0040; 0.0197) and OS (p=0.0137; 0.0158; 0.0115, respectively). Less T cell infiltration (p=0.0130) and CD4 cells in particular (p=0.0792) were associated with higher EGFR expression. FoxP3 infiltration correlated significantly and directly with CD4 and CD8 infiltration but not with peripheral blood levels.

Conclusions

Improved outcomes are associated with increased TILs independent of HPV status and suggest the local immune response to HPV-16 may be related in part to factors such as tumor size, EGFR expression, smoking history, performance status or innate immunity. Assessment of TILs in tissue microarrays is difficult due to small core sample size and variation in tumor representation in tissue cores. Further study of larger numbers of patients and infiltrates in whole tumor sections combined with functional analysis of individual subsets may be necessary to detect differences in local immunity in HPV-16 related cancers.

The mammalian suprachiasmatic nuclei (SCN) contain thousands of neurons capable of generating near 24-h rhythms. When isolated from their network, SCN neurons exhibit a range of oscillatory phenotypes: sustained or damping oscillations, or arrhythmic patterns. The implications of this variability are unknown. Experimentally, we found that cells within SCN explants recover from pharmacologically-induced desynchrony by re-establishing rhythmicity and synchrony in waves, independent of their intrinsic circadian period We therefore hypothesized that a cell's location within the network may also critically determine its resynchronization. To test this, we employed a deterministic, mechanistic model of circadian oscillators where we could independently control cell-intrinsic and network-connectivity parameters. We found that small changes in key parameters produced the full range of oscillatory phenotypes seen in biological cells, including similar distributions of period, amplitude and ability to cycle. The model also predicted that weaker oscillators could adjust their phase more readily than stronger oscillators. Using these model cells we explored potential biological consequences of their number and placement within the network. We found that the population synchronized to a higher degree when weak oscillators were at highly connected nodes within the network. A mathematically independent phase-amplitude model reproduced these findings. Thus, small differences in cell-intrinsic parameters contribute to large changes in the oscillatory ability of a cell, but the location of weak oscillators within the network also critically shapes the degree of synchronization for the population.

Author Summary

Circadian rhythms are daily, near 24-h oscillations in biological processes that nearly all organisms on Earth experience. Single cells contain a molecular clock that drives circadian rhythms in physiology and, when many cells synchronize in a population, daily behaviors. We hypothesized that small differences in intrinsic cellular properties allow for a diversity of circadian periods and amplitudes across cells. We observed circadian cells and their synchrony before, during, and after limiting communication between cells and then compared their intrinsic properties to their resynchronization behavior. We found that arrhythmic, weakly oscillating, and self-sustained circadian cells rejoined the rhythmic population independent of their cell-intrinsic oscillations. Using a mechanistic computational model of circadian cells, we found that resynchronization could be enhanced by including more weak oscillators or by placing weak oscillators at more connected nodes in the network. We conclude that intrinsic properties (e.g. oscillator weakness and responsiveness) and network structure (e.g. positions of weak oscillators) can independently buffer tissue rhythms from perturbations. This reveals how cellular and network properties impose rules on systems of circadian cells that must achieve synchrony from a desynchronized state, for example during perinatal development or when forced to overcome societal constraints on sleep-wake behavior, such as working early or late shifts.

Background

Developments in DNA resequencing microarrays include mitochondrial DNA (mtDNA) sequencing and mutation detection. Failure by the microarray to identify a base, compared to the reference sequence, is designated an 'N-call.' This study re-examined the N-call distribution of mtDNA samples sequenced by the Affymetrix MitoChip v.2.0, based on the hypothesis that N-calls may represent insertions or deletions (indels) in mtDNA.

Findings

We analysed 16 patient mtDNA samples using MitoChip. N-calls by the proprietary GSEQ software were significantly reduced when either of the freeware on-line algorithms ResqMi or sPROFILER was utilized. With sPROFILER, this decrease in N-calls had no effect on the homoplasmic or heteroplasmic mutation levels compared to GSEQ software, but ResqMi produced a significant change in mutation load, as well as producing longer N-cell stretches. For these reasons, further analysis using ResqMi was not attempted. Conventional DNA sequencing of the longer N-calls stretches from sPROFILER revealed 7 insertions and 12 point mutations. Moreover, analysis of single-base N-calls of one mtDNA sample found 3 other point mutations.

Conclusions

Our study is the first to analyse N-calls produced from GSEQ software for the MitoChipv2.0. By narrowing the focus to longer stretches of N-calls revealed by sPROFILER, conventional sequencing was able to identify unique insertions and point mutations. Shorter N-calls also harboured point mutations, but the absence of deletions among N-calls suggests that probe confirmation affects binding and thus N-calling. This study supports the contention that the GSEQ is more capable of assigning bases when used in conjunction with sPROFILER.

Background: Although high doses of ionizing radiation have long been linked to circulatory disease, evidence for an association at lower exposures remains controversial. However, recent analyses suggest excess relative risks at occupational exposure levels.

Objectives: We performed a systematic review and meta-analysis to summarize information on circulatory disease risks associated with moderate- and low-level whole-body ionizing radiation exposures.

Methods: We conducted PubMed/ISI Thomson searches of peer-reviewed papers published since 1990 using the terms “radiation” AND “heart” AND “disease,” OR “radiation” AND “stroke,” OR “radiation” AND “circulatory” AND “disease.” Radiation exposures had to be whole-body, with a cumulative mean dose of < 0.5 Sv, or at a low dose rate (< 10 mSv/day). We estimated population risks of circulatory disease from low-level radiation exposure using excess relative risk estimates from this meta-analysis and current mortality rates for nine major developed countries.

Results: Estimated excess population risks for all circulatory diseases combined ranged from 2.5%/Sv [95% confidence interval (CI): 0.8, 4.2] for France to 8.5%/Sv (95% CI: 4.0, 13.0) for Russia.

Conclusions: Our review supports an association between circulatory disease mortality and low and moderate doses of ionizing radiation. Our analysis was limited by heterogeneity among studies (particularly for noncardiac end points), the possibility of uncontrolled confounding in some occupational groups by lifestyle factors, and higher dose groups (> 0.5 Sv) generally driving the observed trends. If confirmed, our findings suggest that overall radiation-related mortality is about twice that currently estimated based on estimates for cancer end points alone (which range from 4.2% to 5.6%/Sv for these populations).

Aims

Ovarian cancer has the highest mortality of any gynaecological malignancy; this is due to rapid peritoneal spread of tumour cells and neovascularization. Understanding the mechanisms underlying this is critical to developing early diagnostic or treatment strategies. We devised a pilot study to examine the role of γ-SYNUCLEIN (γ-SYN), oestrogen receptor (ER)α, and the splice variant ERαΔ3.

Methodology

With ethical approval, ovarian tissue was collected from patients (n=24) undergoing oopherectomy for non-ovarian pathology or primary surgery for suspected ovarian cancer. Quantitative gene expression analysis was employed for γ-SYN, ERα, and ERαΔ3. To identify the in situ localization, immunofluorescence for γ-syn was carried out.

Results

Ovarian tumour tissue exhibited an elevated expression of γ-SYN and high-grade tumours had an elevated ERαΔ3:ERα ratio compared with benign tissue. The majority of previous studies point to the γ-syn protein being present in epithelial cells of high-grade disease. Our study supports this, but additionally we conclusively identify its presence in the endothelial cells of vasculature surrounding low-grade disease; immunofluorescence was strongest in the apical cells surrounding the lumen.

Conclusion

Our results demonstrate for the first time that there are readily-expressed levels of γ-SYN and ERαΔ3 in normal ovarian tissue and ovarian tumours. In high-grade disease, γ-syn and an elevated ERαΔ3:ERα ratio might confer metastatic potential to the tumourigenic cells and promote neoangiogenesis. Future in vitro studies might be necessary to delineate such a mechanism, which could potentially be the basis of early intervention.