Smad5 is dispensable for adult murine hematopoiesis

Smad5 is known to transduce intracellular signals from bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-beta (TGF-beta) superfamily and are involved in the regulation of hematopoiesis. Recent findings suggest that BMP4 stimulates proliferation of human primitive hematopoietic progenitors in vitro, while early progenitors from mice deficient in Smad5 display increased self-renewal capacity in murine embryonic hematopoiesis. Here, we evaluate the role of Smad5 in the regulation of hematopoietic stem cell (HSC) fate decisions in adult mice by using an inducible MxCre-mediated conditional knockout model. Surprisingly, analysis of induced animals revealed unperturbed cell numbers and lineage distribution in peripheral blood (PB), bone marrow (BM), and the spleen. Furthermore, phenotypic characterization of the stem cell compartment revealed normal numbers of primitive lin(-)Sca-1(+)c-Kit(+) (LSK) cells in Smad5(-)(/)(-) BM. When transplanted in a competitive fashion into lethally irradiated primary and secondary recipients, Smad5-deficient BM cells competed normally with wild-type (wt) cells, were able to provide long-term reconstitution for the hosts, and displayed normal lineage distribution. Taken together, Smad5-deficient HSCs from adult mice show unaltered differentiation, proliferation, and repopulating capacity. Therefore, in contrast to its role in embryonic hematopoiesis, Smad5 is dispensable for hematopoiesis in the adult mouse.     

<Emphasis Type="Italic">Vegf-A</Emphasis> mRNA transfection as a novel approach to improve mouse and human islet graft revascularisation

Aims/hypothesis

The initial avascular period following islet transplantation seriously compromises graft function and survival. Enhancing graft revascularisation to improve engraftment has been attempted through virus-based delivery of angiogenic triggers, but risks associated with viral vectors have hampered clinical translation. In vitro transcribed mRNA transfection circumvents these risks and may be used for improving islet engraftment.

Methods

Mouse and human pancreatic islet cells were transfected with mRNA encoding the angiogenic growth factor vascular endothelial growth factor A (VEGF-A) before transplantation under the kidney capsule in mice.

Results

At day 7 post transplantation, revascularisation of grafts transfected with Vegf-A (also known as Vegfa) mRNA was significantly higher compared with non-transfected or Gfp mRNA-transfected controls in mouse islet grafts (2.11- and 1.87-fold, respectively) (vessel area/graft area, mean?±?SEM: 0.118?±?0.01 [n?=?3] in Vegf-A mRNA transfected group (VEGF) vs 0.056?±?0.01 [n?=?3] in no RNA [p?<?0.05] vs 0.063?±?0.02 [n?=?4] in Gfp mRNA transfected group (GFP) [p?<?0.05]); EndoC-bH3 grafts (2.85- and 2.48-fold. respectively) (0.085?±?0.02 [n?=?4] in VEGF vs 0.030?±?0.004 [n?=?4] in no RNA [p?<?0.05] vs 0.034?±?0.01 [n?=?5] in GFP [p?<?0.05]); and human islet grafts (3.17- and 3.80-fold, respectively) (0.048?±?0.013 [n?=?3] in VEGF vs 0.015?±?0.0051 [n?=?4] in no RNA [p?<?0.01] vs 0.013?±?0.0046 [n?=?4] in GFP [p?<?0.01]). At day 30 post transplantation, human islet grafts maintained a vascularisation benefit (1.70- and 1.82-fold, respectively) (0.049?±?0.0042 [n?=?8] in VEGF vs 0.029?±?0.0052 [n?=?5] in no RNA [p?<?0.05] vs 0.027?±?0.0056 [n?=?4] in GFP [p?<?0.05]) and a higher beta cell volume (1.64- and 2.26-fold, respectively) (0.0292?±?0.0032 μl [n?=?7] in VEGF vs 0.0178?±?0.0021 μl [n?=?5] in no RNA [p?<?0.01] vs 0.0129?±?0.0012 μl [n?=?4] in GFP [p?<?0.001]).

Conclusions/interpretation

Vegf-A mRNA transfection before transplantation provides a promising and safe strategy to improve engraftment of islets and other cell-based implants.

     

Genetic diversity of sapovirus infections in Danish children 2005–2007

BackgroundSapoviruses are known to cause gastroenteritis mainly in young children.ObjectivesTo establish a collection of sapoviruses and to gain knowledge about the genetic diversity and epidemiology of sapoviruses circulating in children in Denmark.Study designDuring a 24-month period in 2005–2007 samples from 1104 children, aged 0–3 years, submitted for acute gastroenteritis diagnostics, were analysed for sapoviruses by real-time PCR. Genotyping of positive findings was carried out by sequencing part of the capsid gene, and in several cases also part of the polymerase gene.ResultsSapoviruses were detected in stool samples from 97 children (9%), with the highest prevalence in the 7–18 months age group. Viruses from three genogroups and seven genotypes were found. Genotype I.1 was demonstrated in half of the positive samples and was observed throughout the study period. The less common types seemed to appear during shorter periods, often in succession of each other. Positive samples were detected throughout the study period. The only months, in both years studied, with high proportions of positive samples were September, November and February.ConclusionsSapoviruses were commonly found in children with gastroenteritis in Denmark. No clear seasonal pattern could be seen. Genotype I.1 was clearly the most common genotype found, but several other genotypes circulated during shorter periods.     

Trait inferences in goal-directed behavior: ERP timing and localization under spontaneous and intentional processing

This study measured event-related potentials (ERPs) during multiple goal and trait inferences, under spontaneous or intentional instructions. Participants read sentences describing several goal-implying behaviors of a target person from which also a strong trait could be inferred or not. The last word of each sentence determined the consistency with the inference induced during preceding sentences. In comparison with behaviors that implied only a goal, stronger waveforms beginning at ∼150 ms were obtained when the behaviors additionally implied a trait. These ERPs showed considerable parallels between spontaneous and intentional inferences. This suggests that traits embedded in a stream of goal-directed behaviors were detected more rapidly and automatically than mere goals, irrespective of the participants’ spontaneous or intentional instructions. In line with this, source localization (LORETA) of the ERPs show predominantly activation in the temporo-parietal junction (TPJ) during 150–200 ms, suggesting that goals were detected at that time interval. During 200–300 ms, activation was stronger at the medial prefrontal cortex (mPFC) for multiple goals and traits as opposed to goals only, suggesting that traits were inferred during this time window. A cued recall measure taken after the presentation of the stimulus material support the occurrence of goal and trait inferences and shows significant correlations with the neural components, indicating that these components are valid neural indices of spontaneous and intentional social inferences. The early detection of multiple goal and trait inferences is explained in terms of their greater social relevance, leading to privileged attention allocation and processing in the brain.     

How can antiepileptic drugs affect bone mass, structure and metabolism? Lessons from animal studies.

Patients with epilepsy, treated with antiepileptic drugs (AEDs) are at increased risk of fractures. Although several commonly used AEDs reduce bone mass in patients, the mechanisms are only scarcely known. In this review, we focus on the usefulness of animal models to explore the skeletal effects of AEDs. Moreover, we report our findings from a recent study comparing the effect of levetiracetam (LEV), phenytoin (PHT) and valproate (VPA) on various aspects of bone health in actively growing female rats. Our data indicate that these AEDs act differently on bone mass, structure and metabolism. A novel finding is that LEV reduces bone strength and bone formation without altering bone mass. Based on these results we propose that epidemiological fracture studies of patients treated with LEV are needed, and that these patients should be evaluated regularly to identify possible bone-related side effects.     

Endemic and emerging acute virus infections in Indonesia: an overview of the past decade and implications for the future

Being the largest archipelago country in the world, with a tropical climate and a unique flora and fauna, Indonesia habitats one of the most diverse biome in the world. These characteristics make Indonesia a popular travel destination, with tourism numbers increasing yearly. These characteristics also facilitate the transmission of zoonosis and provide ideal living and breading circumstances for arthropods, known vectors for viral diseases. A review of the past 10 years of literature, reports of the Ministry of Health, Republic of Indonesia and ProMED-mail shows a significant increase in dengue infection incidence. Furthermore, chikungunya, Japanese encephalitis and rabies are proven to be endemic in Indonesia. The combination of cohort studies, governmental data and ProMED-mail reveals an integrated overview for those working in travel medicine and public health, focusing on both endemic and emerging acute virus infections. This review summarizes the epidemiology of acute virus infections in Indonesia, including outbreak reports, as well as public health response measurements and their potential or efficacy. Knowledge about human behaviour, animal reservoirs, climate factors, environment and their role in emerging virus infection are discussed. We aim to support public health authorities and health care policy makers in a One Health approach.     

Inhibition of T-tropic HIV Strains by Selective Antagonization of the Chemokine Receptor CXCR4

Bicyclams are a novel class of antiviral compounds that are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. Surprisingly, however, when the prototype compound AMD3100 was tested against M-tropic virus strains such as BaL, ADA, JR-CSF, and SF-162 in human peripheral blood mononuclear cells, the compound was completely inactive. Because of the specific and potent inhibitory effect of AMD3100 on T-tropic viruses, but not M-tropic viruses, it was verified that AMD3100 interacts with the CXC-chemokine receptor CXCR4, the main coreceptor used by T-tropic viruses. AMD3100 dose dependently inhibited the binding of a specific CXCR4 monoclonal antibody to SUP-T1 cells as measured by flow cytometry. It did not inhibit the binding of the biotinylated CC-chemokine macrophage inflammatory protein (MIP) 1α or MIP-1β, ligands for the chemokine receptor CCR5 (the main coreceptor for M-tropic viruses). In addition, AMD3100 completely blocked (a) the Ca²⁺ flux at 100 ng/ml in lymphocytic SUP-T1 and monocytic THP-1 cells, and (b) the chemotactic responses of THP-1 cells induced by stromal cell–derived factor 1α, the natural ligand for CXCR4. Finally, AMD3100 had no effect on the Ca²⁺ flux induced by the CC-chemokines MIP-1α, regulated on activation normal T cell expressed and secreted (RANTES; also a ligand for CCR5), or monocyte chemoattractant protein 3 (a ligand for CCR1 and CCR2b), nor was it able to induce Ca²⁺ fluxes by itself. The bicyclams are, to our knowledge, the first low molecular weight anti-HIV agents shown to act as potent and selective CXCR4 antagonists.The bicyclam derivatives were described several years ago as potent and selective inhibitors of HIV type 1 and type 2 replication (1, 2). AMD3100, previously called JM3100 (2) or SID791 (3), exhibits anti-HIV potency at concentrations of 1–10 ng/ml, with a selectivity index ⩾100,000 (2). Based on time-of-addition experiments, the compound has been assumed to interact with the HIV fusion-uncoating process, but does not inhibit virus binding to the CD4 receptor (1, 2). AMD3100 blocks syncytium formation at a concentration that is 10–100-fold higher than the concentration required to inhibit virus infection (1). The env glycoprotein (gp)120 has been considered the major target molecule for this class of compounds because, for viruses that were made resistant to the bicyclams, a number of mutations accumulated in the gp120, especially in the V3-V4 region (3, 4).Numerous publications over the last year have demonstrated the importance of chemokine receptors for HIV entry. Chemokines are chemotactic cytokines, which are classified as CC or CXC, depending on the positioning of conserved cysteine residues. Fusin/LESTR, now designated CXC-chemokine receptor 4 (CXCR4), mediates entry of T-tropic viruses (5, 6) which can be inhibited by its natural ligand, the CXC-chemokine stromal cell–derived factor 1α (SDF-1α) (7, 8). The CC-chemokine receptor, CCR5, mediates entry of M-tropic viruses (9–13) and the CC-chemokines regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP) 1α and MIP-1β have been shown to inhibit the replication of M-tropic viruses (14). Moreover, M-tropic env proteins can interact directly with CCR5 (15, 16).In previous studies AMD3100 was shown to inhibit the replication of T-tropic HIV strains or clinical isolates in T cell lines (such as MT-4, MOLT-4, or CEM cells; references 1–4). While verifying whether AMD3100 was active against M-tropic viruses in PBMCs, we found that AMD3100 does not inhibit M-tropic viruses such as BaL, ADA, JR-CSF, and SF-162. Here we show that AMD3100 selectively inhibits the binding of a CXCR4-specific mAb, but not the binding of biotinylated human MIP-1α or MIP-1β. The bicyclam was also found to inhibit the Ca²⁺ flux and the chemotactic response induced by SDF-1α but not such effects induced by RANTES, MIP-1α, or monocyte chemoattractant protein 3 (MCP-3).