Homing and long-term engraftment of long- and short-term renewal hematopoietic stem cells

Long-term hematopoietic stem cells (LT-HSC) and short-term hematopoietic stem cells (ST-HSC) have been characterized as having markedly different in vivo repopulation, but similar in vitro growth in liquid culture. These differences could be due to differences in marrow homing. We evaluated this by comparing results when purified ST-HSC and LT-HSC were administered to irradiated mice by three different routes: intravenous, intraperitoneal, and directly into the femur. Purified stem cells derived from B6.SJL mice were competed with marrow cells from C57BL/6J mice into lethally irradiated C57BL/6J mice. Serial transplants into secondary recipients were also carried out. We found no advantage for ST-HSC engraftment when the cells were administered intraperitoneally or directly into femur. However, to our surprise, we found that the purified ST-HSC were not short-term in nature but rather gave long-term multilineage engraftment out to 387 days, albeit at a lower level than the LT-HSC. The ST-HSC also gave secondary engraftment. These observations challenge current models of the stem cell hierarchy and suggest that stem cells are in a continuum of change.     

Protein kinase C phosphorylates the carboxyl terminus of the plasma membrane Ca(2+)-ATPase from human erythrocytes.

Purified Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase) from human erythrocytes was phosphorylated with a stoichiometry of about 1 mol of phosphate/mol of ATPase at both threonine and serine residues by purified rat brain type III protein kinase C. In the presence of calmodulin, the phosphorylation was markedly reduced. Labeled phosphate from [gamma-32P]ATP was retained on an 86-kDa calmodulin-binding tryptic fragment of Ca(2+)-ATPase but not on 82- and 77-kDa non-calmodulin-binding fragments. Similarly, fragmentation of the phosphorylated Ca(2+)-ATPase by calpain I revealed that calmodulin-binding fragments (127 and 125 kDa) retained phosphate label whereas a non-calmodulin-binding fragment (124 kDa) did not. The calmodulin-binding domain, located about 12 kDa from the carboxyl terminus of the Ca(2+)-ATPase, was thus located as a site of protein kinase C phosphorylation. A synthetic peptide corresponding to a segment of the calmodulin-binding domain (H2 N-R-G-L-N-R-I-Q-T-Q-I-K-V-V-N-COOH) was indeed phosphorylated at the single threonine residue within this sequence. The additional serine phosphorylation site was carboxyl terminal to the calmodulin domain. Phosphorylation by purified type III protein kinase C (canine heart) antagonized the calmodulin activation of the Ca(2+)-ATPase, particularly at lower Ca2+ concentrations (0.2-1.0 microM). By contrast, a purified but unresolved protein kinase C isoenzyme mixture from rat brain stimulated the activity of Ca(2+)-ATPase prepared in asolectin, but not glycerol, by more than 2-fold in the presence of the ionophore A23187, without increasing its Ca2+ sensitivity. The results clearly indicate that human erythrocyte Ca(2+)-ATPase is a substrate of protein kinase C, but the effect of phosphorylation on the activity of the enzyme depends on the isoenzyme form of protein kinase C used and on the lipid associated with the Ca(2+)-ATPase.     

Seasonality of MRSA infections

Using MRSA isolates submitted to our hospital microbiology laboratory January 2001–March 2010 and the number of our emergency department (ED) visits, quarterly community-associated (CA) and hospital-associated (HA) MRSA infections were modeled using Poisson regressions. For pediatric patients, approximately 1.85x (95% CI 1.45x–2.36x, adj. p<0.0001) as many CA-MRSA infections per ED visit occurred in the second two quarters as occurred in the first two quarters. For adult patients, 1.14x (95% CI 1.01x–1.29x, adj.p = 0.03) as many infections per ED visit occurred in the second two quarters as in the first two quarters. Approximately 2.94x (95% CI 1.39x–6.21x, adj.p = 0.015) as many HA-MRSA infections per hospital admission occurred in the second two quarters as occurred in the first two quarters for pediatric patients. No seasonal variation was observed among adult HA-MRSA infections per hospital admission. We demonstrated seasonality of MRSA infections and provide a summary table of similar observations in other studies.     

Distinguishing characteristics between pandemic 2009-2010 influenza A (H1N1) and other viruses in patients hospitalized with respiratory illness

Background

Differences in clinical presentation and outcomes among patients infected with pandemic 2009 influenza A H1N1 (pH1N1) compared to other respiratory viruses have not been fully elucidated.

Methodology/Principal Findings

A retrospective study was performed of all hospitalized patients at the peak of the pH1N1 season in whom a single respiratory virus was detected by a molecular assay targeting 18 viruses/subtypes (RVP, Luminex xTAG). Fifty-two percent (615/1192) of patients from October, 2009 to December, 2009 had a single respiratory virus (291 pH1N1; 207 rhinovirus; 45 RSV A/B; 37 parainfluenza; 27 adenovirus; 6 coronavirus; and 2 metapneumovirus). No seasonal influenza A or B was detected. Individuals with pH1N1, compared to other viruses, were more likely to present with fever (92% & 70%), cough (92% & 86%), sore throat (32% & 16%), nausea (31% & 8%), vomiting (39% & 30%), abdominal pain (14% & 7%), and a lower white blood count (8,500/L & 13,600/L, all p-values<0.05). In patients with cough and gastrointestinal complaints, the presence of subjective fever/chills independently raised the likelihood of pH1N1 (OR 10). Fifty-five percent (336/615) of our cohort received antibacterial agents, 63% (385/615) received oseltamivir, and 41% (252/615) received steroids. The mortality rate of our cohort was 1% (7/615) and was higher in individuals with pH1N1 compared to other viruses (2.1% & 0.3%, respectively; p = 0.04).

Conclusions/Significance

During the peak pandemic 2009–2010 influenza season in Rhode Island, nearly half of patients admitted with influenza-like symptoms had respiratory viruses other than influenza A. A high proportion of patients were treated with antibiotics and pH1N1 infection had higher mortality compared to other respiratory viruses.     

Orientation Dependent MR Signal Decay Differentiates between People with MS,Their Asymptomatic Siblings and Unrelated Healthy Controls

R2* relaxometry of the brain is a quantitative magnetic resonance technique which is influenced by iron and myelin content across different brain regions. Multiple sclerosis (MS) is a common inflammatory, demyelinating disease affecting both white and grey matter regions of the CNS. Using R2*, increased iron deposition has been described in deep gray matter of MS patients. Iron accumulation might promote oxidative stress in the brain, which can lead to cell death and neurodegeneration. However, recent histological work indicates that iron may be reduced within the normal appearing white matter (WM) in MS. In the present study we analyzed the R2* signal across the white matter in 39 patients with MS, 31 asymptomatic age matched siblings of patients and 30 age-matched controls. The measurement of R2* in white matter is affected by the signal''s dependency on white matter fibre orientation with respect to the main magnetic field which can be accounted using diffusion tensor imaging. We observed a clear separation of the three study groups in R2*. The values in the MS group were significantly lower compared to the siblings and controls, while the siblings group presented with significantly higher R2* values than both unrelated healthy controls and patients. Furthermore, we found significantly decreased normal-appearing white matter R2* values in patients with more severe disease course. Angle resolved analysis of R2* improves the sensitivity for detecting subtle differences in WM R2* compared to standard histogram based analyses. Our findings suggest that the decreased R2* values in MS are due to diffuse tissue damage and decreased myelin in the normal appearing and diffusely abnormal WM. The increased R2* in unaffected siblings may identify a predisposition to increased iron and the potential for oxidative stress as a risk factor for developing MS.     

Persistent Ca2(+)-induced activation of erythrocyte membrane Ca2(+)-ATPase unrelated to calpain proteolysis

Preincubation of human erythrocyte membranes with calcium in the submillimolar to millimolar concentration range resulted in an increase of the Ca2+ affinity and apparent maximum velocity of the Ca2(+)-stimulated Mg2(+)-dependent ATPase (Ca2(+)-ATPase). The activation was persistent, as it was not reversed when the Ca2(+)-preincubated membranes were washed with ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid-containing buffers. Magnesium was not required for the activation, whereas greater than 2 mM Mg2+ partially antagonized the activation by Ca2+. In some membrane preparations ATP was required in addition to Ca2+ for activation of the Ca2(+)-ATPase, but nonhydrolyzable analogs of ATP had the same effect. Calmodulin prevented the activation by Ca2+ over the same concentration range in which it interacts with the Ca2(+)-ATPase. Taken together the results obtained provided strong evidence that the Ca2+ activation of the enzyme was not due to proteolytic cleavage by endogenous calpain. Thus, activation by Ca2+ was not blocked by leupeptin (100-200 microM), did not require dithiothreitol, and occurred at Ca2+ concentrations greater than those required for activation of calpain I. Furthermore, Ca2+ activation did not result in change in the mobility the native 136-kDa species of the Ca2(+)-ATPase on SDS-gel electrophoresis. Moreover, solubilization of the Ca2(+)-pretreated membranes with Triton X-100 reversed the Ca2+ activation of the Ca2(+)-ATPase. On the other hand, Ca2(+)-pretreatment of the membranes modified the susceptibility of the Ca2(+)-ATPase to both cleavage and activation by exogenously added calpain I. We conclude that pretreatment of Ca2(+)-ATPase in erythrocyte membranes with millimolar Ca2+ activates the enzyme by inducing a persistent conformational change of the enzyme which is, however, subsequently reversed by detergent solubilization.