Inhibition of hydrogen sulfide production by gene silencing attenuates inflammatory activity of LPS-activated RAW264.7 cells

Hydrogen sulfide is an inflammatory mediator and is produced by the activity of the enzyme cystathionine γ-lyase (CSE) in macrophages. Previously, pharmacological inhibition of CSE has been reported to have conflicting results, and this may be due to the lack of specificity of the pharmacological agents. Therefore, this study used a very specific approach of small interfering RNA (siRNA) to inhibit the production of the CSE in an in vitro setting. We found that the activation of macrophages by lipopolysaccharide (LPS) resulted in higher levels of CSE mRNA and protein as well as the increased production of proinflammatory cytokines and nitric oxide (NO). We successfully used siRNA to specifically reduce the levels of CSE mRNA and protein in activated macrophages. Furthermore, the levels of proinflammatory cytokines in LPS-activated macrophages were significantly lower in siRNA-transfected cells compared to those in untransfected controls. However, the production levels of NO by the transfected cells were higher, suggesting that CSE activity has an inhibitory effect on NO production. These findings suggest that the CSE enzyme has a crucial role in the activation of macrophages, and its activity has an inhibitory effect on NO production by these cells.     

When growth models are not universal: evidence from marine invertebrates

The accumulation of body mass, as growth, is fundamental to all organisms. Being able to understand which model(s) best describe this growth trajectory, both empirically and ultimately mechanistically, is an important challenge. A variety of equations have been proposed to describe growth during ontogeny. Recently, the West Brown Enquist (WBE) equation, formulated as part of the metabolic theory of ecology, has been proposed as a universal model of growth. This equation has the advantage of having a biological basis, but its ability to describe invertebrate growth patterns has not been well tested against other, more simple models. In this study, we collected data for 58 species of marine invertebrate from 15 different taxa. The data were fitted to three growth models (power, exponential and WBE), and their abilities were examined using an information theoretic approach. Using Akaike information criteria, we found changes in mass through time to fit an exponential equation form best (in approx. 73% of cases). The WBE model predominantly overestimates body size in early ontogeny and underestimates it in later ontogeny; it was the best fit in approximately 14% of cases. The exponential model described growth well in nine taxa, whereas the WBE described growth well in one of the 15 taxa, the Amphipoda. Although the WBE has the advantage of being developed with an underlying proximate mechanism, it provides a poor fit to the majority of marine invertebrates examined here, including species with determinate and indeterminate growth types. In the original formulation of the WBE model, it was tested almost exclusively against vertebrates, to which it fitted well; the model does not however appear to be universal given its poor ability to describe growth in benthic or pelagic marine invertebrates.     

Socioeconomic Status,Functional Recovery,and Long-Term Mortality among Patients Surviving Acute Myocardial Infarction

Objectives

To examine the relationship between socio-economic status (SES), functional recovery and long-term mortality following acute myocardial infarction (AMI).

Background

The extent to which SES mortality disparities are explained by differences in functional recovery following AMI is unclear.

Methods

We prospectively examined 1368 patients who survived at least one-year following an index AMI between 1999 and 2003 in Ontario, Canada. Each patient was linked to administrative data and followed over 9.6 years to track mortality. All patients underwent medical chart abstraction and telephone interviews following AMI to identify individual-level SES, clinical factors, processes of care (i.e., use of, and adherence, to evidence-based medications, physician visits, invasive cardiac procedures, referrals to cardiac rehabilitation), as well as changes in psychosocial stressors, quality of life, and self-reported functional capacity.

Results

As compared with their lower SES counterparts, higher SES patients experienced greater functional recovery (1.80 ml/kg/min average increase in peak V02, P<0.001) after adjusting for all baseline clinical factors. Post-AMI functional recovery was the strongest modifiable predictor of long-term mortality (Adjusted HR for each ml/kg/min increase in functional capacity: 0.91; 95% CI: 0.87–0.94, P<0.001) irrespective of SES (P = 0.51 for interaction between SES, functional recovery, and mortality). SES-mortality associations were attenuated by 27% after adjustments for functional recovery, rendering the residual SES-mortality association no longer statistically significant (Adjusted HR: 0.84; 95% CI:0.70–1.00, P = 0.05). The effects of functional recovery on SES-mortality associations were not explained by access inequities to physician specialists or cardiac rehabilitation.

Conclusions

Functional recovery may play an important role in explaining SES-mortality gradients following AMI.     

Effect of Tenofovir on Nucleotidases and Cytokines in HIV-1 Target Cells

Tenofovir (TFV) has been widely used for pre-exposure prophylaxis of HIV-1 infection with mixed results. While the use of TFV in uninfected individuals for prevention of HIV-1 acquisition is actively being investigated, the possible consequences of TFV exposure for the HIV-target cells and the mucosal microenvironment are unknown. In the current study, we evaluated the effects of TFV treatment on blood-derived CD4⁺ T cells, monocyte-derived macrophages and dendritic cells (DC). Purified HIV-target cells were treated with different concentrations of TFV (0.001-1.0 mg/ml) for 2 to 24hr. RNA was isolated and RT-PCR was performed to compare the levels of mRNA expression of nucleotidases and pro-inflammatory cytokine genes (MIP3α, IL-8 and TNFα) in the presence or absence of TFV. We found that TFV increases 5’-ecto-nucleotidase (NT5E) and inhibits mitochondrial nucleotidase (NT5M) gene expression and increases 5’ nucleotidase activity in macrophages. We also observed that TFV stimulates the expression and secretion of IL-8 by macrophages, DC, and activated CD4⁺ T cells and increases the expression and secretion of MIP3α by macrophages. In contrast, TFV had no effect on TNFα secretion from macrophages, DC and CD4⁺ T cells. Our results demonstrate that TFV alters innate immune responses in HIV-target cells with potential implications for increased inflammation at mucosal surfaces. As new preventive trials are designed, these findings should provide a foundation for understanding the effects of TFV on HIV-target cells in microbicide trials.     

Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

Previous studies have shown that the small molecule iron transport inhibitor ferristatin (NSC30611) acts by down-regulating transferrin receptor-1 (TfR1) via receptor degradation. In this investigation, we show that another small molecule, ferristatin II (NSC8679), acts in a similar manner to degrade the receptor through a nystatin-sensitive lipid raft pathway. Structural domains of the receptor necessary for interactions with the clathrin pathway do not appear to be necessary for ferristatin II induced degradation of TfR1. While TfR1 constitutively traffics through clathrin-mediated endocytosis, with or without ligand, the presence of Tf blocked ferristatin II induced degradation of TfR1. This effect of Tf was lost in a ligand binding receptor mutant G647A TfR1, suggesting that Tf binding to its receptor interferes with the drug’s activity. Rats treated with ferristatin II have lower TfR1 in liver. These effects are associated with reduced intestinal ⁵⁹Fe uptake, lower serum iron and transferrin saturation, but no change in liver non-heme iron stores. The observed hypoferremia promoted by degradation of TfR1 by ferristatin II appears to be due to induced hepcidin gene expression.     

Selection Affects Genes Involved in Replication during Long-Term Evolution in Experimental Populations of the Bacteriophage φX174

Observing organisms that evolve in response to strong selection over very short time scales allows the determination of the molecular mechanisms underlying adaptation. Although dissecting these molecular mechanisms is expensive and time-consuming, general patterns can be detected from repeated experiments, illuminating the biological processes involved in evolutionary adaptation. The bacteriophage φX174 was grown for 50 days in replicate chemostats under two culture conditions: Escherichia coli C as host growing at 37°C and Salmonella typhimurium as host growing at 43.5°C. After 50 days, greater than 20 substitutions per chemostat had risen to detectable levels. Of the 97 substitutions, four occurred in all four chemostats, five arose in both culture conditions, eight arose in only the high temperature S. typhimurium chemostats, and seven arose only in the E. coli chemostats. The remaining substitutions were detected only in a single chemostat, however, almost half of these have been seen in other similar experiments. Our findings support previous studies that host recognition and capsid stability are two biological processes that are modified during adaptation to novel hosts and high temperature. Based upon the substitutions shared across both environments, it is apparent that genome replication and packaging are also affected during adaptation to the chemostat environment, rather than to temperature or host per se. This environment is characterized by a large number of phage and very few hosts, leading to competition among phage within the host. We conclude from these results that adaptation to a high density environment selects for changes in genome replication at both protein and DNA sequence levels.     

MicroRNA Profiling in Human Neutrophils during Bone Marrow Granulopoiesis and In Vivo Exudation

The purpose of this study was to describe the microRNA (miRNA) expression profiles of neutrophils and their precursors from the initiation of granulopoiesis in the bone marrow to extravasation and accumulation in skin windows. We analyzed three different cell populations from human bone marrow, polymorphonuclear neutrophil (PMNs) from peripheral blood, and extravasated PMNs from skin windows using the Affymetrix 2.0 platform. Our data reveal 135 miRNAs differentially regulated during bone marrow granulopoiesis. The majority is differentially regulated between the myeloblast/promyelocyte (MB/PM) and myelocyte/metamyelocyte (MC/MM) stages of development. These 135 miRNAs were divided into six clusters according to the pattern of their expression. Several miRNAs demonstrate a pronounced increase or reduction at the transition between MB/PM and MC/MM, which is associated with cell cycle arrest and the initiation of terminal differentiation. Seven miRNAs are differentially up-regulated between peripheral blood PMNs and extravasated PMNs and only one of these (miR-132) is also differentially regulated during granulopoiesis. The study indicates that several different miRNAs participate in the regulation of normal granulopoiesis and that miRNAs might also regulate activities of extravasated neutrophils. The data present the miRNA profiles during the development and activation of the neutrophil granulocyte in healthy humans and thus serves as a reference for further research of normal and malignant granulocytic development.     

Complications of Antiretroviral Therapy Initiation in Hospitalised Patients with HIV-Associated Tuberculosis

Background

HIV-associated tuberculosis is a common coinfection in Sub-Saharan Africa, which causes high morbidity and mortality. A sub-set of HIV-associated tuberculosis patients require prolonged hospital admission, during which antiretroviral therapy initiation may be required. The aim of this study was to document the causes of clinical deterioration of hospitalised patients with HIV-associated tuberculosis starting antiretroviral therapy in order to inform healthcare practice in low- to middle-income countries.

Methods

Prospective, observational cohort study of adult inpatients with HIV-associated tuberculosis starting antiretroviral therapy in a dedicated tuberculosis hospital in Cape Town, South Africa. Causes of clinical deterioration and outcome were recorded in the first 12 weeks of antiretroviral therapy. Patients with rifampicin-resistant tuberculosis were excluded.

Results

Between May 2009 and November 2010, 112 patients (60% female), with a median age of 32 years were enrolled. At baseline the median CD4 count was 55 cells/mm³ (IQR 31–106) and HIV viral load 5.6 log copies/mL. All patients had significant comorbidity: 82% were bed-bound, 65% had disseminated tuberculosis and 27% had central nervous system tuberculosis. Seventy six patients (68%) developed 144 clinical events after starting antiretroviral therapy. TB-IRIS, hospital-acquired infections and significant drug toxicities occurred in 42%, 20.5% and 15% of patients respectively. A new opportunistic disease occurred in 15% of patients and a thromboembolic event in 8%. Mortality during the 12 week period was 10.6%.

Conclusions

High rates of TB-IRIS, hospital-acquired infections and drug toxicities complicate the course of patients with HIV-associated tuberculosis starting antiretroviral therapy in hospital. Despite the high morbidity, mortality was relatively low. Careful clinical management and adequate resources are needed in hospitalised HIV-TB patients in the 1^st three months following ART initiation.