Hematopoietic growth factors in autologous transplantation

Hematopoietic growth factors (HGFs) sustain the survival, proliferation and differentiation of hematopoietic stem cells and some functions of mature blood cells. In man several HGFs have been characterised and cloned so far, and this has allowed investigators to confer the rationale for the clinical application of these molecules in hematology and oncology. In particular G-CSF and GM-CSF are currently utilised to abrogate the hematological toxicity of chemotherapy for standard and dose-intensified therapy, neutropenia following bone marrow and peripheral blood stem cell transplantation. Moreover there has recently been great interest in the ex vivo expansion of hematopoietic stem and progenitor cells for a variety of applications, such as in vitro tumor cell purging or for reducing the volume of blood processed by the leukapheresis. Several combinations of HGFs have been described to sustain the ex vivo survival and proliferation of these cells disclosing new opportunities in the field of stem cells transplants.     

Phosphorylation of serine residues in the N-terminus modulates the activity of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana

ACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca(2+)-ATPase of Arabidopsis thaliana. Several phosphopeptides corresponding to portions of the regulatory N-terminus of ACA8 have been identified in phospho-proteomic studies. To mimic phosphorylation of the ACA8 N-terminus, each of the serines found to be phosphorylated in those studies (Ser19, Ser22, Ser27, Ser29, Ser57, and Ser99) has been mutated to aspartate. Mutants have been expressed in Saccharomyces cerevisiae and characterized: mutants S19D and S57D--and to a lesser extent also mutants S22D and S27D--are deregulated, as shown by their low activation by CaM and by tryptic cleavage of the N-terminus. The His-tagged N-termini of wild-type and mutant ACA8 (6His-(1)M-I(116)) were expressed in Escherichia coli, affinity-purified, and used to analyse the kinetics of CaM binding by surface plasmon resonance. All the analysed mutations affect the kinetics of interaction with CaM to some extent: in most cases, the altered kinetics result in marginal changes in affinity, with the exception of mutants S57D (K(D) ≈ 10-fold higher than wild-type ACA8) and S99D (K(D) about half that of wild-type ACA8). The ACA8 N-terminus is phosphorylated in vitro by two isoforms of A. thaliana calcium-dependent protein kinase (CPK1 and CPK16); phosphorylation of mutant 6His-(1)M-I(116) peptides shows that CPK16 is able to phosphorylate the ACA8 N-terminus at Ser19 and at Ser22. The possible physiological implications of the subtle modulation of ACA8 activity by phosphorylation of its N-terminus are discussed.     

Relative burden of large CNVs on a range of neurodevelopmental phenotypes

While numerous studies have implicated copy number variants (CNVs) in a range of neurological phenotypes, the impact relative to disease severity has been difficult to ascertain due to small sample sizes, lack of phenotypic details, and heterogeneity in platforms used for discovery. Using a customized microarray enriched for genomic hotspots, we assayed for large CNVs among 1,227 individuals with various neurological deficits including dyslexia (376), sporadic autism (350), and intellectual disability (ID) (501), as well as 337 controls. We show that the frequency of large CNVs (>1 Mbp) is significantly greater for ID-associated phenotypes compared to autism (p = 9.58 × 10(-11), odds ratio = 4.59), dyslexia (p = 3.81 × 10(-18), odds ratio = 14.45), or controls (p = 2.75 × 10(-17), odds ratio = 13.71). There is a striking difference in the frequency of rare CNVs (>50 kbp) in autism (10%, p = 2.4 × 10(-6), odds ratio = 6) or ID (16%, p = 3.55 × 10(-12), odds ratio = 10) compared to dyslexia (2%) with essentially no difference in large CNV burden among dyslexia patients compared to controls. Rare CNVs were more likely to arise de novo (64%) in ID when compared to autism (40%) or dyslexia (0%). We observed a significantly increased large CNV burden in individuals with ID and multiple congenital anomalies (MCA) compared to ID alone (p = 0.001, odds ratio = 2.54). Our data suggest that large CNV burden positively correlates with the severity of childhood disability: ID with MCA being most severely affected and dyslexics being indistinguishable from controls. When autism without ID was considered separately, the increase in CNV burden was modest compared to controls (p = 0.07, odds ratio = 2.33).     

Peroxisome proliferator-activated receptor gamma ligands attenuate immunological symptoms of experimental allergic asthma

Asthma is characterized by a predominant T(H)2 type immune response to airborne allergens. Controlling T(H)2 cell function has been proposed as therapy for this disease. We show here that ligands for the nuclear receptor peroxisome proliferator activated receptor (PPAR)gamma significantly reduced the immunological symptoms of allergic asthma in a murine model of this disease. A PPARgamma ligand, 15-deoxy-delta(12,14)-prostaglandin J(2), significantly inhibited production of the T(H)2 type cytokine IL-5 from T cells activated in vitro. More importantly, in a murine model of allergic asthma, mice treated orally with ciglitazone, a potent synthetic PPARgamma ligand, had significantly reduced lung inflammation and mucous production following induction of allergic asthma. T cells from these ciglitazone treated mice also produced less IFNgamma, IL-4, and IL-2 upon rechallenge in vitro with the model allergen. Our results suggest that ligands for PPARgamma may be effective treatments for asthmatic patients.     

Nuclear localization and DNA interaction of protein disulfide isomerase ERp57 in mammalian cells

Protein disulfide isomerase ERp57 is localized predominantly in the endoplasmic reticulum, but is also present in the cytosol and, according to preliminary evidence, in the nucleus of avian cells. Conclusive evidence of its nuclear localization and of its interaction with DNA in vivo in mammalian cells is provided here on the basis of DNA-protein cross-linking experiments performed with two different cross-linking agents on viable HeLa and 3T3 cells. Nuclear ERp57 could also be detected by immunofluorescence in HeLa cells, where it showed an intracellular distribution clearly different from that of an homologous protein, located exclusively in the endoplasmic reticulum. Mammalian ERp57 resembles the avian protein in its recognition of S/MAR-like DNA sequences and in its association with the nuclear matrix. It can be hypothesized that ERp57, which is known to associate with other proteins, in particular STAT3 and calreticulin, may contribute to their nuclear import, DNA binding, or other functions that they fulfil inside the nucleus.     

Oxidative damage of the gastric mucosa in Helicobacter pylori positive chronic atrophic and nonatrophic gastritis, before and after eradication

Background. Helicobacter pylori is the main cause of gastritis and a primary carcinogen. The aim of this study was to assess oxidative damage in mucosal compartments of gastric mucosa in H. pylori positive and negative atrophic and nonatrophic gastritis. Materials and methods. Five groups of 10 patients each were identified according to H. pylori positive or negative chronic atrophic (Hp‐CAG and CAG, respectively) and nonatrophic gastritis (Hp‐CG and CG, respectively), and H. pylori negative normal mucosa (controls). Oxidative damage was evaluated by nitrotyrosine immunohistochemistry in the whole mucosa and in each compartment at baseline and at 2 and 12 months after eradication. Types of intestinal metaplasia were classified by histochemistry. Results. Total nitrotyrosine levels appeared significantly higher in H. pylori positive than in negative patients, and in Hp‐CAG than in Hp‐CG (p < .001); no differences were found between H. pylori negative gastritis and normal mucosa. Nitrotyrosine were found in foveolae and intestinal metaplasia only in Hp‐CAG. At 12 months after H. pylori eradication, total nitrotyrosine levels showed a trend toward a decrease in Hp‐CG and decreased significantly in Hp‐CAG (p = .002), disappearing from the foveolae (p = .002), but remaining unchanged in intestinal metaplasia. Type I and II of intestinal metaplasia were present with the same prevalence in Hp‐CAG and CAG, and did not change after H. pylori eradication. Conclusions. Oxidative damage of the gastric mucosa increases from Hp‐CG to Hp‐CAG, involving the foveolae and intestinal metaplasia. H. pylori eradication induces a complete healing of foveolae but not of intestinal metaplasia, reducing the overall oxidative damage in the mucosa.