Combinatorial G-CSF/AMD3100 Treatment in Cardiac Repair after Myocardial Infarction

Aims

Several studies suggest that circulating bone marrow derived stem cells promote the regeneration of ischemic tissues. For hematopoietic stem cell transplantation combinatorial granulocyte-colony stimulating factor (G-CSF)/Plerixafor (AMD3100) administration was shown to enhance mobilization of bone marrow derived stem cells compared to G-CSF monotherapy. Here we tested the hypothesis whether combinatorial G-CSF/AMD3100 therapy has beneficial effects in cardiac recovery in a mouse model of myocardial infarction.

Methods

We analyzed the effect of single G-CSF (250 µg/kg/day) and combinatorial G-CSF/AMD3100 (100 µg/kg/day) treatment on cardiac morphology, vascularization, and hemodynamics 28 days after permanent ligation of the left anterior descending artery (LAD). G-CSF treatment started directly after induction of myocardial infarction (MI) for 3 consecutive days followed by a single AMD3100 application on day three after MI in the G-CSF/AMD3100 group. Cell mobilization was assessed by flow cytometry of blood samples drawn from tail vein on day 0, 7, and 14.

Results

Peripheral blood analysis 7 days after MI showed enhanced mobilization of white blood cells (WBC) and endothelial progenitor cells (EPC) upon G-CSF and combinatorial G-CSF/AMD3100 treatment. However, single or combinatorial treatment showed no improvement in survival, left ventricular function, and infarction size compared to the saline treated control group 28 days after MI. Furthermore, no differences in histology and vascularization of infarcted hearts could be observed.

Conclusion

Although the implemented treatment regimen caused no adverse effects, our data show that combinatorial G-CSF/AMD therapy does not promote myocardial regeneration after permanent LAD occlusion.     

Human Cytomegalovirus-Induces Cytokine Changes in the Placenta with Implications for Adverse Pregnancy Outcomes

Human cytomegalovirus (CMV) infection of the developing fetus can result in adverse pregnancy outcomes including death in utero. Fetal injury results from direct viral cytopathic damage to the CMV-infected fetus, although evidence suggests CMV placental infection may indirectly cause injury to the fetus, possibly via immune dysregulation with placental dysfunction. This study investigated the effects of CMV infection on expression of the chemokine MCP-1 (CCL2) and cytokine TNF-α in placentae from naturally infected stillborn babies, and compared these changes with those found in placental villous explant histocultures acutely infected with CMV ex vivo. Tissue cytokine protein levels were assessed using quantitative immunohistochemistry. CMV-infected placentae from stillborn babies had significantly elevated MCP-1 and TNF-α levels compared with uninfected placentae (p = 0.001 and p = 0.007), which was not observed in placentae infected with other microorganisms (p = 0.62 and p = 0.71) (n = 7 per group). Modelling acute clinical infection using ex vivo placental explant histocultures showed infection with CMV laboratory strain AD169 (0.2 pfu/ml) caused significantly elevated expression of MCP-1 and TNF-α compared with uninfected explants (p = 0.0003 and p<0.0001) (n = 25 per group). Explant infection with wild-type Merlin at a tenfold lower multiplicity of infection (0.02 pfu/ml), caused a significant positive correlation between increased explant infection and upregulation of MCP-1 and TNF-α expression (p = 0.0001 and p = 0.017). Cytokine dysregulation has been associated with adverse outcomes of pregnancy, and can negatively affect placental development and function. These novel findings demonstrate CMV infection modulates the placental immune environment in vivo and in a multicellular ex vivo model, suggesting CMV-induced cytokine modulation as a potential initiator and/or exacerbator of placental and fetal injury.     

Methylation changes at NR3C1 in newborns associate with maternal prenatal stress exposure and newborn birth weight

Early life experiences, including those in utero, have been linked to increased risk for adult-onset chronic disease. The underlying assumption is that there is a critical period of developmental plasticity in utero when selection of the fetal phenotype that is best adapted to the intrauterine environment occurs. The current study is the first to test the idea that extreme maternal psychosocial stressors, as observed in the Democratic Republic of Congo, may modify locus-specific epigenetic marks in the newborn resulting in altered health outcomes. Here we show a significant correlation between culturally relevant measures of maternal prenatal stress, newborn birth weight and newborn methylation in the promoter of the glucocorticoid receptor NR3C1. Increased methylation may constrain plasticity in subsequent gene expression and restrict the range of stress adaptation responses possible in affected individuals, thus increasing their risk for adult-onset diseases.     

A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.     

Cost-efficient selection of a marker panel in genetic studies

Genetic techniques are frequently used to sample and monitor wildlife populations. The goal of these studies is to maximize the ability to distinguish individuals for various genetic inference applications, a process which is often complicated by genotyping error. However, wildlife studies usually have fixed budgets, which limit the number of genetic markers available for inclusion in a study marker panel. Prior to our study, a formal algorithm for selecting a marker panel that included genotyping error, laboratory costs, and ability to distinguish individuals did not exist. We developed a constrained nonlinear programming optimization algorithm to determine the optimal number of markers for a marker panel, initially applied to a pilot study designed to estimate black bear abundance in central Georgia. We extend the algorithm to other genetic applications (e.g., parentage or population assignment) and incorporate possible null alleles. Our algorithm can be used in wildlife pilot studies to assess the feasibility of genetic sampling for multiple genetic inference applications. © 2011 The Wildlife Society.     

The marine sponge Ianthella basta can recover from stress-induced tissue regression

Sponges often exhibit tissue regression in response to stressful conditions. This study investigated whether handling stress invoked tissue regression in Ianthella basta and assessed whether sponges could recover from this regressed tissue state. Six necrotic specimens and 12 healthy explants were collected at Orpheus Is. Australia and transported to aquarium facilities. Sponges were photographed daily and an integrated density (ID) measurement was used to quantify tissue regression. Histological samples were taken from sponge explants to compare cellular organization. Bacterial communities of regressed and recovered tissue were compared using Denaturing Gradient Gel Electrophoresis (DGGE). After 12 h both necrotic and healthy sponges displayed substantial tissue regression. However, within 72 h all sponges recovered to their original condition. The ID of the sponge tissue doubled, confirming tissue recovery in I. basta. Sponges affected by tissue regression had significantly fewer choanocyte chambers and more densely packed granulated cells than recovered sponges. DGGE revealed the same microbial symbionts in both regressed and recovered sponges. Handling stress associated with collection and transportation is sufficient to invoke tissue regression in this species, but sponges can rapidly recover. This study contributes to our understanding of how sponges respond to environmental pressures, influencing population resilience and persistence.     

Cryopreserved human haematopoietic stem cells retain engraftment potential after extended (5-14 years) cryostorage

Harvesting of stem cells during the early phases of treatment with no immediate intention to perform a stem cell transplant is becoming an increasingly common practice. Such "insurance" harvests are often stored for many years before being needed for transplant in a subsequent relapse. The effect of long-term cryostorage (5-14 years) on the viability and functional capacity of haematopoietic stem cells (HSCs) was investigated in 40 bone marrow and peripheral blood harvests using standard in vitro methods, the colony forming unit-granulocyte/macrophage (CFU-GM) assay and a single platform viable CD34(+) cell absolute count by flow cytometry. Forty percent of harvests had CD34(+) HSC counts of at least 0.7 x 10(6)/kg bodyweight and 85% had CFU-GM counts of at least 1.0 x 10(5)/kg bodyweight, these values representing our institutional minimum requirements for safe transplantation. Based on these results, it appears that HSC collections can remain adequate for safe transplantation after up to 14 years of cryostorage. However, as deterioration of HSC quality and viability may occur, some precautions may be warranted, namely harvesting higher than normal numbers of HSCs in collections intended for long-term storage and repeating in vitro assays on harvests after long-term storage prior to transplantation.     

Characterization of a novel Obg-like ATPase in the protozoan Trypanosoma cruzi

We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185 bp long and encodes a 44.3 kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes.     

Tryptophan 67 in the human VPAC(1) receptor: crucial role for VIP binding

The human receptor subtype for VIP and PACAP, referred to as VPAC(1) receptor, has a large N-terminal extracellular domain which is critical for VIP binding. We further investigated this domain by mutating 12 amino acid residues which could participate in the formation of a tight bend (W67) or a coiled coil motif. They were changed to alanine (A) and the cDNAs were transiently transfected into Cos cells. All mutants but W67A exhibited K(d) values similar to that of the wild-type receptor. For the W67A mutant, no specific (125)I-VIP binding could be observed. Mutants at the W67 site were further characterized after stable transfection of epitope-tagged VPAC(1) receptor-GFP fusion proteins into CHO cells. W67A, W67E, W67H, and W67K mutants neither bound VIP nor mediated adenylyl cyclase activation by VIP. The W67F mutant mediated stimulation of adenylyl cyclase only at high VIP concentrations. Microscopic analysis and antibody binding experiments showed that all mutants were similarly expressed at the cell surface of CHO cells. Therefore tryptophan 67 in the human VPAC(1) receptor plays a crucial role in VIP binding due, in part, to its aromatic moiety.