Growth characteristics of circulating hematopoietic progenitor cells from patients with essential thrombocythemia

Peripheral blood mononuclear cells from five patients with essential thrombocythemia (ET) were cultured in vitro to evaluate restricted megakaryocytic (CFU-Meg), myeloid (CFU-GM), and erythroid (BFU-E) progenitor cell development. Varying concentrations of aplastic canine serum served as the source of megakaryocyte colony-stimulating activity, and cultured megakaryocyte ploidy distributions were determined by Feulgen staining and microfluorometry. Megakaryocyte colony growth was strikingly abnormal in all five patients evaluated. Four of the 5 had a marked expansion in the concentration of circulating CFU-Meg and 3 of 4 manifested abnormalities in cultured megakaryocyte colony size (2 unusually large and 1 small). Unstimulated megakaryocyte colony growth was substantially increased in three patients. However, the fraction of megakaryocyte progenitors in cell cycle was near or below normal in all instances. Endomitotic megakaryocyte development was disordered in each of the four ET patients in whom it was evaluable. In normal subjects, mean megakaryocyte ploidy values vary biphasically with aplastic canine serum concentration and peak at 13.2 N following 12 to 15 days of culture. In contrast, day 12 mean ploidy values in cultures from the ET patients remained low at all aplastic canine serum concentrations and reached a maximum averaging only 8.4 N. Three patients were evaluated serially at extended culture durations of up to 21 days. The cultured megakaryocyte ploidy was unchanged during this interval for two of the patients. For the third patient, ploidy increased steadily, reaching abnormally high ploidy values by day 21. Progenitor cell expansion was limited to the megakaryocyte line in three patients. However, two patients had substantial increases in CFU-GM, one of whom also had a marked increase in BFU-E. There was no significant unstimulated colony growth by either CFU-GM or BFU-E. These data indicate that ET is usually characterized by an expansion in the concentration of circulating CFU-Meg in vivo which manifest both disordered replication and endoreduplication in vitro.     

Does histologic acute rejection in lung allografts predict the development of bronchiolitis obliterans?

Clinical acute lung rejection (AR) occurs in lung allografts usually within 50 days after transplantation. While perivascular infiltrates characterize AR, with moderate-to-severe acute rejection small airway injury occurs. We investigated the significance of small airway injury in AR and its relationship to the development of bronchiolitis obliterans (OB) in 11 recipients of combined heart-lung or double-lung allografts. In general, the intensity and persistence of early acute rejection episodes associated with injury to bronchioles correlated with the development of histologic bronchiolitis obliterans. Early AR may "prime" lymphocytes for subsequent respiratory epithelial injury and airway fibrosis late in the postoperative period.     

Separation of bronchoalveolar cells from the guinea pig on continuous gradients of Percoll: functional properties of fractionated lung macrophages

Lung macrophages from normal guinea pig lungs were separated from bronchoalveolar cells into three fractions according to buoyant density by centrifugation on continuous iso-osmotic gradients of Percoll [3]. A reproducible pattern of functional activity distinguished these three macrophage fractions. With increasing density and decreasing cell size, the respective fractions exhibited increased stimulated migration, superoxide anion release and pinocytosis, and increased protein concentration of the cells. These differences, coupled with previous observations that these fractions also exhibited morphological and cytochemical differences [3], support the notion that these fractions of macrophages may represent different stages of maturation (or differentiation) of alveolar macrophages in the lungs of normal guinea pigs.     

Flow cytometric analysis of lymphocyte phenotypes in bronchoalveolar lavage fluid: comparison of a two-color technique with a standard immunoperoxidase assay.

Characterization of lymphocytes in bronchoalveolar fluid has provided insight into the pathogenesis of many pulmonary diseases. Identification of lymphocyte phenotypes has become highly successful due to development of specific monoclonal antibodies and reliable methods for detecting labeled cells such as flow cytometry (FCM) and immunocytochemistry. FCM permits rapid screening of many cells, but this analysis may be confounded by heterogeneity in the size and granularity of the cells being evaluated. Such heterogeneity may lead to exclusion of cells of interest and inclusion of unwanted cells. Often peripheral blood leukocytes are used to define the gate for lung lymphocytes, but this gate may be inappropriate due to considerable variation in size and granularity of cells in bronchoalveolar lavage (BAL) fluid. Here we report an alternative method for generating a gate which employed fluorescence and side scatter signals to analyze lymphocyte subsets in BAL fluid by FCM. This gating technique avoids the pitfalls inherent in using the conventional lymphocyte gate to analyze lung cells. To validate this approach, we compared the results generated by this gate and those from the conventional forward/side light scatter gate to results derived from an immunocytochemical technique (ABC) that has been extensively employed in our laboratory to identify lymphocyte subsets in blood and lavage fluid. FCM tended to underestimate the proportions of T-cell subsets compared with ABC when the conventional gate was used. Counting only cells that stained with fluorescein-conjugated anti-CD45 antibody and that had side scatter properties of lymphocytes, however, resulted in excellent agreement between FCM and ABC. It appears that the CD45+/side scatter gate includes the vast majority of lymphocytes in BAL fluid while excluding most of the nonlymphoid cells that contaminate the conventional gate. It was this latter group of cells, and erythrocytes in particular, that led to the artificially low values for lymphocyte phenotypes in BAL fluid by FCM when the conventional lymphocyte gate was used. Although erythrocytes in BAL fluid may be eliminated by hypotonic lysis, this may also result in contamination of the conventional lymphocyte gate with nuclear debris and particulates from macrophages. Despite these advantages, the fluorescence/side scatter gate may not always be optimal for the evaluation of T lymphocytes if BAL fluid contains CD45+, nonlymphoid cells with low side light scatter. In these instances, additional antibodies such as anti-CD14 and anti-CD11 may be employed to determine the size of contaminant monocytic cells and neutrophils.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heterozygous Mutations in Natriuretic Peptide Receptor‐B (NPR2) Gene as a Cause of Short Stature

Based on the observation of reduced stature in relatives of patients with acromesomelic dysplasia, Maroteaux type (AMDM), caused by homozygous or compound heterozygous mutations in natriuretic peptide receptor‐B gene (NPR2), it has been suggested that heterozygous mutations in this gene could be responsible for the growth impairment observed in some cases of idiopathic short stature (ISS). We enrolled 192 unrelated patients with short stature and 192 controls of normal height and identified seven heterozygous NPR2 missense or splice site mutations all in the short stature patients, including one de novo splice site variant. Three of the six inherited variants segregated with short stature in the family. Nine additional rare nonsynonymous NPR2 variants were found in three additional cohorts. Functional studies identified eight loss‐of‐function mutations in short individuals and one gain‐of‐function mutation in tall individuals. With these data, we were able to rigorously verify that NPR2 functional haploinsufficiency contributes to short stature. We estimate a prevalence of NPR2 haploinsufficiency of between 0 and 1/26 in people with ISS. We suggest that NPR2 gain of function may be a more common cause of tall stature than previously recognized.     

The 2013 Model of the Clinical Practice of Emergency Medicine

In 2001, “The Model of the Clinical Practice of Emergency Medicine” was first published. This document, the first of its kind, was the result of an extensive practice analysis of emergency department (ED) visits and several expert panels, overseen by representatives from six collaborating professional organizations (the American Board of Emergency Medicine, the American College of Emergency Physicians, the Society for Academic Emergency Medicine, the Residency Review Committee for Emergency Medicine, the Council of Emergency Medicine Residency Directors, and the Emergency Medicine Residents' Association). Every 2 years, the document is reviewed by these organizations to identify practice changes, incorporate new evidence, and identify perceived deficiencies. For this revision, a seventh organization was included, the American Academy of Emergency Medicine.     

Physical and functional interaction between CB1 cannabinoid receptors and ��2-adrenoceptors

Background and purpose:

The CB₁ cannabinoid receptor and the β₂-adrenoceptor are G protein-coupled receptors (GPCRs) co-expressed in many tissues. The present study examined physical and functional interactions between these receptors in a heterologous expression system and in primary human ocular cells.

Experimental approach:

Physical interactions between CB₁ receptors and β₂-adrenoceptors were assessed using bioluminescence resonance energy transfer (BRET). Functional interactions between these receptors were evaluated by examining receptor trafficking, as well as extracellular signal-regulated kinase (ERK) and cyclic AMP response element binding protein (CREB) signalling.

Key results:

Physical interactions between CB₁ receptors and β₂-adrenoceptors were demonstrated using BRET. In human embryonic kidney (HEK) 293H cells, co-expression of β₂-adrenoceptors tempered the constitutive activity and increased cell surface expression of CB₁ receptors. Co-expression altered the signalling properties of CB₁receptors, resulting in increased Gα_i-dependent ERK phosphorylation, but decreased non-Gα_i-mediated CREB phosphorylation. The CB₁ receptor inverse agonist AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) attenuated β₂-adrenoceptor-pERK signalling in cells expressing both receptors, while the CB₁ receptor neutral antagonist O-2050 ((6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) did not. The actions of AM251 and O-2050 were further examined in primary human trabecular meshwork (HTM) cells, which are ocular cells endogenously co-expressing CB₁ receptors and β₂-adrenoceptors. In HTM cells, as in HEK 293H cells, AM251 but not O-2050, altered the β₂-adrenoceptor–pERK response.

Conclusion and implications:

A complex interaction was demonstrated between CB₁ receptors and β₂-adrenoceptors in HEK 293H cells. As similar functional interactions were also observed in HTM cells, such interactions may affect the pharmacology of these receptors in tissues where they are endogenously co-expressed.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x