Enhancement of neutrophil function by granulocyte-macrophage colony- stimulating factor involves recruitment of a less responsive subpopulation

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances numerous functions of mature neutrophils (PMN) including phagocytosis, superoxide responses to chemotaxins, antibody-dependent cellular cytotoxicity, and expression of complement receptors. A central question concerns whether the mechanism of enhancement involves quantitative increases in the response of all cells v subpopulation recruitment. The effects of GM-CSF on individual cell light scatter changes, membrane potential, and oxidant responses induced by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP) were assessed by flow cytometry and by scoring individual cells for nitroblue tetrazolium dye (NBT) reduction. GM-CSF produced a dose- and time-dependent shift in forward light scatter that was very similar in character to that seen with FMLP or leukotriene B4 stimulation. Although not capable of depolarizing the cells directly, GM-CSF primed PMNs for enhanced membrane potential responses to FMLP by significantly increasing the proportion of depolarizing cells when compared with diluent-treated controls after a 60-minute incubation at 37 degrees C (79.4% +/- 3.4% v 29.5% +/- 4.7% GM-CSF v diluent, mean +/- SE, P less than .005, n = 11). Subpopulation recruitment by GM-CSF treatment was also demonstrated by the FMLP-elicited NBT test. Taken together, these results indicate that GM-CSF can modulate the function of mature PMN by enhancing the proportion of responsive cells.     

Instantaneous pressure gradient: a simultaneous Doppler and dual catheter correlative study

To more precisely measure the beat to beat and instantaneous pressure gradients across outflow stenotic lesions, simultaneous Doppler and dual catheter pressure gradient measurements were performed in 95 patients (mean age 42 years, range 1.5 to 85). There were 38 right ventricular and 62 left ventricular outflow obstructive lesions. Forty-nine patients also had a nonsimultaneous Doppler study performed within 7 days before catheterization. The simultaneous pressure waveforms and Doppler spectral velocity profiles were digitized at 10 ms intervals deriving maximal, mean and instantaneous gradients (mm Hg). For simultaneous maximal Doppler and catheter gradient measurements, the correlation coefficient (r) was 0.95 (SEE = 10 mm Hg), for Doppler and catheter mean gradients it was 0.94 (SEE = 8 mm Hg) and for maximal Doppler and peak to peak catheter gradients it was 0.92 (SEE = 13 mm Hg). The correlation of maximal and mean Doppler gradients with the respective catheter gradients was similarly high when the right and left ventricular outflow lesions were analyzed separately. However, the maximal Doppler gradient was significantly higher than the peak to peak catheter gradient. This was more evident with left ventricular outflow stenotic lesions. The correlation of the outpatient maximal Doppler and catheter gradients (r = 0.80, SEE = 17 mm Hg) was significantly lower than the simultaneous correlation (r = 0.96, SEE = 10 mm Hg) in the 49 patients with two Doppler studies. Continuous wave Doppler echocardiography accurately measures the instantaneous pressure gradient across both left and right ventricular outflow obstructive lesions. The maximal Doppler gradient should not be equated with the peak to peak catheter gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells

Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy- 1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200-10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony- forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.     

Patent foramen ovale in cryptogenic stroke: current understanding and management options

There is increasing interest in the association between patent foramen ovale (PFO) and documented stroke of unknown cause, commonly referred to as cryptogenic stroke. We reviewed the literature and, on the basis of the available data, designed a diagnostic and treatment algorithm for patients with PFO and cryptogenic stroke. Patent foramen ovale is relatively common in the general population, but its prevalence is higher in patients with cryptogenic stroke. Importantly, paradoxical embolism through a PFO should be strongly considered in young patients with cryptogenic stroke. There is no consensus on the optimal management strategy, but treatment options include antiplatelet agents, warfarin sodium, percutaneous device closure, and surgical closure. High-risk features in the patient's history (ie, temporal association between Valsalva-inducing maneuvers and stroke, coexisting hypercoagulable state, recurrent strokes, and PFO with large opening, large right-to-left shunt, or right-to-left shunting at rest, and a coexisting atrial septal aneurysm) should prompt PFO closure.     

Analysis of ASPM in an ethnically diverse cohort of 400 patient samples: perspectives of the molecular diagnostic laboratory

Primary Autosomal Recessive Microcephaly (MCPH) is characterized by congenital microcephaly usually without additional clinical findings. The most common gene implicated in MCPH is ASPM and a large percentage of mutations described have been homozygous and in consanguineous families primarily of East Asian and Middle Eastern origin. ASPM sequencing was performed on 400 patients between the years 2009 and 2012. Seventy of the patient samples were also analyzed for copy number changes in the ASPM gene. Forty protein truncating mutations, including 29 novel mutations, were identified in 39 patients with MCPH. Approximately one third of patients were compound heterozygotes, indicative of non‐consanguinity in these patients. In addition, 46 non‐synonymous variants were identified and interpreted as variants of uncertain significance. No deletion/duplication in ASPM was identified in the patients analyzed. A wide ethnic distribution was observed, including the first reported patients with ASPM‐related MCPH of Hispanic descent. Clinical information was collected for 26 of the ASPM‐positive patients and 41 of the ASPM‐negative patients. As more individuals are identified with MCPH, we anticipate that we will continue to identify ASPM mutation‐positive patients from all ethnic origins supporting the occurrence of this genetic condition beyond that of consanguineous families of certain ethnic populations.