Natural killer cell precursors in the CD44neg/dim T-cell receptor population of mouse bone marrow

Natural killer (NK) cells develop from the nonadherent cell component of NK long-term bone marrow (BM) cultures (NK-LTBMC). Because these nonadherent cells are depleted of mature NK cells and T cells, but appear to enriched for NK precursors, they were used as a starting population to begin to define the NK precursors that function in NK- LTBMC. As the stromal cell component of NK-LTBMC has been shown to support interleukin (IL)-2-induced, CD44 dependent, NK cell development from nonadherent NK precursors, NK-LTBMC stroma was used in a limiting dilution assay (LDA) to quantitate the precursors. NK-LTBMC in 96-well plates were irradiated (20 Gy) to kill hematopoietic cells (including the NK precursors), seeded with limiting dilutions of the cells to be quantitated, cultured with 500 U/mL IL-2 for 13 days and assayed for development of NK activity by adding 51Cr-labeled YAC-1 cells to the wells and evaluating the release of 51Cr after 4 hours. Flow cytometric analysis, sorting, and quantitation of the nonadherent cell component of NK-LTBMC showed that NK precursors were concentrated in the CD44neg/dim subset that comprised 10% of the "lymphoid" gated cells. When the CD44neg/dim subset was sorted from BM of mice treated with 5- fluorouracil (5-FU) day before (-1FUBM), there were about 30% T cells, but no NK-1.1+ cells. When the T cells were removed by sorting and the CD44neg/dim, alphabeta, gammadelta T-cell receptorneg (TCR-) subpopulation was seeded onto irradiated stroma with IL-2, they proliferated, developed NK activity, became NK-1.1+ and CD44bright and remained alphabeta, gammadelta TCR-. The frequency of NK precursors in this population as estimated from the LDA was about 1/500.     

Effects of coronary artery bypass surgery and angioplasty on the total ischemic burden: a study of exercise testing and ambulatory ST segment monitoring.

To assess the effects of standard therapeutic interventions on the total ischemic burden, 86 patients with stable angina underwent 48 hours of ambulatory ST segment monitoring and treadmill exercise testing before and at a mean of 10 weeks after coronary artery bypass surgery (CABG) (group 1, N = 46) or percutaneous transluminal coronary angioplasty (PTCA) (group 2, N = 40). There were 72 male and 14 female patients with a mean age of 56.4 years. All patients had documented coronary artery disease (24, single-vessel; 28, two-vessel; 34, three-vessel disease). Both groups were characteristically similar apart from more severe coronary artery disease (p less than 0.001) and more previous myocardial infarctions (p less than 0.05) in group 1. Groups with CABG and PTCA had significant prolongation of exercise time after intervention (group 1: 7.6 to 9.8 minutes, p less than 0.0001; group 2: 8.1 to 10.0 minutes, p less than 0.001), and both interventions led to a significant reduction in ischemic responses (group 1: 33 to 4, p less than 0.001; group 2: 20 to 13, p less than 0.05) to exercise. During a total of 7643 hours of ST segment monitoring, 253 episodes of ischemia were recorded in 3768 hours before and 44 ischemic episodes in 3875 hours after intervention (group 1, 113 episodes in 24 patients and 21 episodes in 10 patients; group 2, 140 episodes in 13 patients and 23 episodes in six patients). Both interventions reduced the mean frequency of ischemia per 24 hours (group 1: 1.24 to 0.22 episodes per 24 hours; p less than 0.01; group 2: 1.9 to 0.3 episodes per 24 hours; p less than 0.05). Almost 28% (N = 24) of resting electrocardiographic findings were altered as a result of intervention.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assembly of contact-phase factors on the surface of the human neutrophil membrane

H-kininogen (HK), a major factor involved in contact-phase activation, was recently immunolocalized on the external surface of human neutrophils. Experiments were, therefore, designed to consider the question of whether the complete assembly of contact factors occurs on the outer surface of the neutrophil membrane. By immunolocalization techniques, and using specific antibodies directed against the various contact factors, we now demonstrate that plasma prekallikrein (PK), factor XI (FXI), and factor XII (FXII) are present on the exterior face of the human neutrophil. Failure to localize HK, PK, or FXI by monoclonal antibodies directed to their reciprocal binding sites, and displacement of PK/FXI by peptide HK31, which mimics the relevant binding site(s) of HK, suggested that prekallikrein and FXI are anchored to the neutrophil membrane through attachment to the kininogen molecule. Probing of the kinin moiety by a specific antibody showed that kininogen molecules bound to the neutrophil cell membrane contain the kinin sequence, which can be released by plasma kallikrein or by tissue kallikrein. Our results led us to the novel conclusion that neutrophils provide a circulating platform for the components of the contact-phase system.     

Bmi1 enhances skeletal muscle regeneration through MT1-mediated oxidative stress protection in a mouse model of dystrophinopathy

The Polycomb group (PcG) protein Bmi1 is an essential epigenetic regulator of stem cell function during normal development and in adult organ systems. We show that mild up-regulation of Bmi1 expression in the adult stem cells of the skeletal muscle leads to a remarkable improvement of muscle function in a mouse model of Duchenne muscular dystrophy. The molecular mechanism underlying enhanced physiological function of Bmi1 depends on the injury context and it is mediated by metallothionein 1 (MT1)–driven modulation of resistance to oxidative stress in the satellite cell population. These results lay the basis for developing Bmi1 pharmacological activators, which either alone or in combination with MT1 agonists could be a powerful novel therapeutic approach to improve regeneration in muscle wasting conditions.Skeletal muscle is characterized by a remarkable capacity to regenerate after injury, mainly due to the function of satellite cells, the main skeletal muscle stem cells (Brack and Rando, 2012; Wang and Rudnicki, 2012). Polycomb group (PcG) proteins are essential regulators of stem cell function during normal development and in adult organs. They form multi-protein chromatin-associated complexes that play an essential role in the genome-wide epigenetic-mediated remodeling of gene expression during myogenic differentiation of satellite cells, mainly through posttranslational modifications of histones (Asp et al., 2011). Ezh2 and Bmi1 are required for adult satellite cell homeostasis and proliferation in response to muscle injury, an effect mediated at least in part by repression of the ink4a locus (Juan et al., 2011; Robson et al., 2011). Importantly, although Bmi1 is expressed in several types of cancer and its mechanism of action may be similar in a non-neoplastic and neoplastic context, its overexpression does not initiate tumorigenesis (He et al., 2009; Yadirgi et al., 2011).An emerging role for PcG proteins is their involvement in DNA repair (Liu et al., 2009; Facchino et al., 2010; Ismail et al., 2010; Ginjala et al., 2011; Pan et al., 2011). Bmi1^−/−-derived cells show significant mitochondrial dysfunction accompanied by sustained increase in reactive oxygen species (ROS) production that are sufficient to engage the DNA repair pathway (Liu et al., 2009), which is in turn impaired, thus leading to a magnified cellular damage. The balance between intracellular ROS and antioxidant molecules is vital in determining the rate of oxidative damage accumulation and the impaired function of satellite cells in aging and in myopathies, in which decreased anti-oxidative capacity has been documented (Fulle et al., 2005; Whitehead et al., 2006; Tidball and Wehling-Henricks, 2007). X-linked Duchenne muscular dystrophy (DMD) is the most common primary myopathy caused by the loss of the dystrophin protein from the plasma membrane, which causes loss of its integrity and fiber damage during repeated cycles of muscle degeneration and regeneration (Duncan, 1989). The proliferative capacity of myogenic cells was reported to be rapidly exhausted in dystrophin-deficient muscle, also because they are more sensitive to oxidative stress injury, leading to reduced and defective regeneration of the muscle as the disease progresses (Blau et al., 1983, 1985; Disatnik et al., 1998). Moreover, enzymatic adaptations to exercise-induced production of ROS and free radical damage are significantly decreased in dystrophic compared with normal muscles (Faist et al., 1998, 2001). Overall, an impaired protection against ROS in dystrophic muscle appears to contribute to disease progression as also indicated by the beneficial, albeit transient, effect of antioxidants in ameliorating the skeletal muscle pathophysiology of DMD patients (Whitehead et al., 2008).Metallothionein 1 (MT1) and MT2 are ubiquitously expressed (Kägi and Hunziker, 1989) low molecular weight, cysteine–rich zinc binding proteins. Although the role of MT1 in promoting cell proliferation is controversial (Smith et al., 2008), studies on MT-null liver cells showed their failure to regenerate after oxidative stress injury (Oliver et al., 2006).Here, we show that overexpression of Bmi1 in the satellite cells significantly improves muscle strength through enhanced MT1-mediated protection of these cells from oxidative stress in a mouse model of dystrophinopathies but not after acute traumatic injury.     

Assessment of burn wound sepsis by swab, full thickness biopsy culture and blood culture--a comparative study

Fifty patients with burns ranging from 30 to 50 per cent of their body surface area were monitored for sepsis throughout their hospital stay using swab, blood and full thickness biopsy culture techniques. The relative merits of these techniques in the diagnosis of burn wound sepsis were evaluated. Only 62.5 per cent of the patients with a positive surface culture showed signs of clinical sepsis, while 87.5 per cent of the patients with significant bacterial count on biopsy culture showed signs of clinical sepsis. A decrease in bacterial count on follow up correlated with clinical improvement while a count of 10(8) orgs/gm indicated a bad prognosis. Wound surface cultures, though the simplest method gave poor indication of the organisms invading into the burn wound. Blood cultures were of only prognostic value. Full thickness biopsy culture and quantification of the number of bacteria in the burn wound was felt to be the best method for rapid diagnosis and for assessing the progress of burn wound infection.