Use of Head Guards in AIBA Boxing Tournaments-A Cross-Sectional Observational Study.

OBJECTIVE: This study looks at the changes in injuries after the implementation of a new rule by the International Boxing Association (AIBA) to remove head guards from its competitions. DESIGN: A cross-sectional observational study performed prospectively. This brief report examines the removal of head guards in 2 different ways. The first was to examine the stoppages due to blows to the head by comparing World Series Boxing (WSB), without head guards, to other AIBA competitions with head guards. Secondly, we examined the last 3 world championships: 2009 and 2011 (with head guards) and 2013 (without head guards). SETTING: World Series Boxing and AIBA world championship boxing. PARTICIPANTS: Boxers from WSB and AIBA world championships. INTERVENTIONS: The information was recorded by ringside medical physicians. MAIN OUTCOME MEASURES: Stoppages per 10 000 rounds; stoppages per 1000 hours. RESULTS: Both studies show that the number of stoppages due to head blows was significantly decreased without head guards. The studies also showed that there was a notable increase in cuts. CONCLUSIONS: Removing head guards may reduce the already small risk of acute brain injury in amateur boxing.

Apoptotic cells activate AMP-activated protein kinase (AMPK) and inhibit epithelial cell growth without change in intracellular energy stores.

Apoptosis plays an indispensable role in the maintenance and development of tissues. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits the proliferation and survival of PTECs. Here, we examined the effect of apoptotic targets on PTEC cell growth (cell size during G1 phase of the cell cycle). Using a cell culture model, we show that apoptotic cells potently activate AMP-activated protein kinase (AMPK), a highly sensitive sensor of intracellular energy stores. AMPK activation leads to decreased activity of its downstream target, ribosomal protein p70 S6 kinase (p70S6K), and concomitant inhibition of cell growth. Importantly, these events occur without detectable change in intracellular levels of AMP, ADP, or ATP. Inhibition of AMPK, either pharmacologically by compound C or molecularly by shRNA, diminishes the effects of apoptotic targets and largely restores p70S6K activity and cell size to normal levels. Apoptotic targets also inhibit Akt, a second signaling pathway regulating cell growth. Expression of a constitutively active Akt construct partially relieved cell growth inhibition but was less effective than inhibition of AMPK. Inhibition of cell growth by apoptotic targets is dependent on physical interaction between apoptotic targets and PTECs but independent of phagocytosis. We conclude that receptor-mediated recognition of apoptotic targets mimics the effects of intracellular energy depletion, activating AMPK and inhibiting cell growth. By acting as sentinels of environmental change, apoptotic death may enable nearby viable cells, especially nonmigratory epithelial cells, to monitor and adapt to local stresses.

Recognition-dependent signaling events in response to apoptotic targets inhibit epithelial cell viability by multiple mechanisms: implications for non-immune tissue homeostasis.

Apoptosis allows for the removal of damaged, aged, and/or excess cells without harm to surrounding tissue. To accomplish this, cells undergoing apoptosis acquire new activities that enable them to modulate the fate and function of nearby cells. We have shown that receptor-mediated recognition of apoptotic versus necrotic target cells by viable kidney proximal tubular epithelial cells (PTEC) modulates the activity of several signaling pathways critically involved in regulation of proliferation and survival. Generally, apoptotic and necrotic targets have opposite effects with apoptotic targets inhibiting and necrotic targets stimulating the activity of these pathways. Here we explore the consequences of these signaling differences. We show that recognition of apoptotic targets induces a profound decrease in PTEC viability through increased responder cell death and decreased proliferation. In contrast, necrotic targets promote viability through decreased death and increased proliferation. Both target types mediate their effects through a network of Akt-dependent and -independent events. Apoptotic targets modulate Akt-dependent viability in part through a reduction in cellular ß-catenin and decreased inactivation of Bad. In contrast, Akt-independent modulation of viability occurs through activation of caspase-8, suggesting that death receptor-dependent pathways are involved. Apoptotic targets also activate p38, which partially protects responders from target-induced death. The response of epithelial cells varies depending on their tissue origin. Some cell lines, like PTEC, demonstrate decreased viability, whereas others (e.g. breast-derived) show increased viability. By acting as sentinels of environmental change, apoptotic targets allow neighboring cells, especially non-migratory epithelial cells, to monitor and potentially adapt to local stresses.

Recognition of apoptotic cells by epithelial cells: conserved versus tissue-specific signaling responses.

During apoptosis, cells acquire new activities that enable them to modulate the fate and function of interacting phagocytes, particularly macrophages (m). Although the best known of these activities is anti-inflammatory, apoptotic targets also influence m survival and proliferation by modulating proximal signaling events, such as MAPK modules and Akt. We asked whether modulation of these same signaling events extends to epithelial cells, a minimally phagocytic cell type. We used BU.MPT cells, a mouse kidney epithelial cell line, as our primary model, but we also evaluated several epithelial cell lines of distinct tissue origins. Like m, mouse kidney epithelial cells recognized apoptotic and necrotic targets through distinct non-competing receptors, albeit with lower binding capacity and markedly reduced phagocytosis. Also, modulation of inflammatory activity and MAPK-dependent signaling by apoptotic and necrotic targets was indistinguishable in kidney epithelial cells and m. In contrast, modulation of Akt-dependent signaling differed dramatically between kidney epithelial cells and m. In kidney epithelial cells, modulation of Akt was linked to target cell recognition, independently of phagocytosis, whereas in m, modulation was linked to phagocytosis. Moreover, recognition of apoptotic and necrotic targets by kidney epithelial cells elicited opposite responses; apoptotic targets inhibited whereas necrotic targets stimulated Akt activity. These data confirm that nonprofessional phagocytes recognize and respond to dying cells, albeit in a manner partially distinct from m. By acting as sentinels of environmental change, apoptotic and necrotic targets may permit neighboring viable cells, especially non-migratory epithelial cells, to monitor and adapt to local stresses.

AMPK protects proximal tubular cells from stress-induced apoptosis by an ATP-independent mechanism: potential role of Akt activation.

We examined the role of AMP-activated protein kinase (AMPK) in modulating the viability of cultured kidney proximal tubular cells subjected to metabolic stress induced by either dextrose deprivation, inhibition of glycolysis, or inhibition of mitochondrial respiration. We used BU.MPT cells, a conditionally immortalized kidney epithelial cell line derived from the proximal tubules of transgenic mice bearing a temperature-sensitive mutation of the simian virus 40 large-tumor antigen. All three forms of metabolic stress increased the phosphorylation and activity of AMPK. Activation of AMPK led to changes in the phosphorylation of two downstream targets of AMPK, acetyl coenzyme A carboxylase and p70 S6 kinase. Inhibition of AMPK, either pharmacologically with compound C (CC) or by gene silencing, significantly increased the amount of apoptosis in response to all three forms of metabolic stress. Although the amount of apoptosis was directly related to the severity of ATP depletion, inhibition of AMPK had no effect on cellular ATP levels. Notably, metabolic stress increased the phosphorylation and activity of Akt. Furthermore, inhibition of AMPK, with CC or gene silencing, abrogated the ability of metabolic stress to activate Akt. The augmentation of apoptosis induced by inhibition of AMPK was comparable to that induced by inhibition of Akt. We conclude that activation of AMPK following acute metabolic stress plays a major role in promoting the viability of cultured proximal tubular cells. Protection by AMPK appears to be due not to AMPK-mediated conservation of cell energy stores, but rather, at least in part, to AMPK-mediated activation of Akt.

Macrophages from lupus-prone MRL mice have a conditional signaling abnormality that leads to dysregulated expression of numerous genes.

Macrophages (mϕ) from pre-diseased mice of the major murine inbred models of spontaneous autoimmunity (AI), including multiple lupus-prone strains and the type I diabetes-prone NOD (non-obese diabetic) strain, have identical apoptotic target-dependent abnormalities. This characteristic feature of mϕ from AI-prone mice suggests that abnormal signaling events induced within mϕ following their interaction with apoptotic targets may predispose to AI. Such signaling abnormalities would affect predominantly the processing and presentation of self-antigen (i.e., derived from apoptotic targets), while sparing the processing and presentation of foreign antigen (i.e., derived from non-apoptotic sources). Here, we used DNA microarrays to test the hypothesis that mϕ from AI-prone mice (MRL/MpJ [MRL/+] or MRL/MpJ-Tnfrsf6 ( lpr ) [MRL/lpr]) differentially express multiple genes in comparison to non-AI mϕ (BALB/c), but do so in a largely apoptotic cell-dependent manner. Mϕ were stimulated with lipopolysaccharide, a potent innate stimulus, in the presence or absence of serum (an experimental surrogate for apoptotic targets). In accord with our hypothesis, the number of genes differentially expressed by MRL mϕ was significantly increased in the presence vs. the absence of serum, the apoptotic target surrogate (n = 401 vs. n = 201). Notably, for genes differentially expressed by MRL mϕ in the presence of serum, serum-free culture normalized their expression to a level statistically indistinguishable from that by non-AI mϕ. Comparisons of mϕ from AI-prone NOD and non-AI C57BL/6 mice corroborated these findings. Together, these data support the hypothesis that mϕ from MRL and other AI-prone mice are characterized by a conditional abnormality elicited by serum lipids or apoptotic targets.

The affirmative response of the innate immune system to apoptotic cells.

Growing evidence exists for a new role for apoptotic cell recognition and clearance in immune homeostasis. Apoptotic cells at all stages, irrespective of membrane integrity, elicit a signature set of signaling events in responding phagocytes, both professional and non-professional. These signaling events are initiated by receptor-mediated recognition of apoptotic determinants, independently of species, cell type, or apoptotic stimulus. We propose that the ability of phagocytes to respond to apoptotic targets with a characteristic set of signaling events comprises a second distinct dimension of innate immunity, as opposed to the traditional innate discrimination of self vs. non-self. We further propose that a loss or abnormality of the signaling events elicited by apoptotic cells, as distinct from the actual clearance of those cells, may predispose to autoimmunity.

Abnormal regulation of the cytoskeletal regulator Rho typifies macrophages of the major murine models of spontaneous autoimmunity.

Macrophages (mphi) from prediseased mice of all the major murine models of spontaneous autoimmunity have an identical defect in cytokine expression that is triggered by serum and/or apoptotic cells. We show here that mphi from prediseased mice of the same models of spontaneous autoimmunity share a serum-dependent defect in the activity of Rho, a cytoplasmic G protein and cytoskeletal regulator. Affected strains include those developing lupus (BXSB, LG, MRL/l+, MRL/lpr, NZBWF1) and autoimmune diabetes (nonobese diabetic). No similar defect in Rho activity occurred in seven control strains. In the presence of serum, Rho activity in mphi from all autoimmune-prone strains was reduced to less than 10% of that in control mice. In contrast, under serum-free conditions, Rho activity was completely normal in autoimmune-prone mphi. The activities of Ras, another cytoplasmic G protein, and Rac and Cdc42, two additional G protein regulators of the cytoskeleton, were regulated normally in autoimmune-prone strains. Serum-dependent dysregulation of Rho was associated with multiple abnormalities, including increased adhesion to various surfaces, a more spread dendritic morphology, and an altered actin cytoskeletal organization. Our results suggest that mphi from multiple, genetically diverse, autoimmune-prone strains share a mutation or allelic difference affecting signal transduction within a specific Rho-regulatory pathway.

Macrophages from lupus-prone MRL mice are characterized by abnormalities in Rho activity, cytoskeletal organization, and adhesiveness to extracellular matrix proteins.

Macrophages (mphi) from prediseased mice of the major murine models of lupus have an identical defect in cytokine expression that is triggered by serum and/or apoptotic cells. It is striking that cytokine expression in the absence of serum and apoptotic cells is equivalent to that of nonautoimmune mice. Here, we show that mphi from prediseased lupus-prone MRL/MpJ (MRL/+) or MRL/MpJ-Tnfrsf6(lpr) (MRL/lpr) mice also have reversible abnormalities in morphology, cytoskeletal organization, and adhesive properties. In the presence of serum, MRL mphi adhered in increased numbers to a variety of extracellular matrix proteins compared with mphi from two nonautoimmune strains. However, in the absence of serum, adhesion by MRL mphi was similar to that of nonautoimmune mphi. Increased adhesion by MRL mphi was also observed in the presence of apoptotic, but not necrotic, cells. The morphology and actin-staining pattern of adherent MRL mphi were consistent with reduced activity of Rho, a cytoskeletal regulator. Indeed, MRL mphi cultured in the presence of serum had markedly decreased levels of active Rho compared with nonautoimmune mphi. It is remarkable that when cultured in the absence of serum, MRL mphi displayed normal Rho activity and cytoskeletal morphology. Addition of a Rho inhibitor to normal mphi reproduced the morphologic and cytoskeletal abnormalities observed in MRL mphi. Taken together, our findings support the hypothesis that mphi from MRL and other systemic lupus erythematosus-prone mice have an apoptotic, cell-dependent, autoimmune phenotype that affects a broad range of mphi functions, including cytokine gene expression and Rho-dependent cytoskeletal regulation.

Apoptotic and necrotic cells as sentinels of local tissue stress and inflammation: response pathways initiated in nearby viable cells.

Virtually all cells in the body have the capacity to recognize and respond to dead cells. Viable cells discriminate apo from nec targets via distinct cell surface receptors. Engagement of these receptors induces "recognition-dependent" signaling events in viable responding cells that differ for apo vs. nec targets. Although "engulfment-dependent" signaling events also contribute to the response by viable cells, these events do not differ for apo vs. nec targets. While many signaling events are conserved across diverse cell lineages, other signaling events, especially those involving Akt, demonstrate lineage-specific variation. Whereas apo targets activate Akt in MPhi, they inhibit Akt in kidney epithelial cells. Differences in the responses to dead targets by viable migratory cells, such as MPhi, and viable fixed cells, such as kidney epithelial cells, permit cell-specific adaptations to local environmental change or stress. We propose that dead cells (apo and nec) act as sentinels to alert nearby viable cells to local environmental change or stress.

Cyclooxygenase-2 rescues rat mesangial cells from apoptosis induced by adriamycin via upregulation of multidrug resistance protein 1 (P-glycoprotein).

Cyclooxygenase-2 (COX-2) is constitutively expressed in restricted subpopulations of kidney cells, where it presumably acts as an antiapoptotic factor. In conditions that are characterized by inflammation, COX-2 expression also has been described in glomerular mesangial cells (GMC), where COX-2 is not expressed constitutively. It was shown previously that adenovirus-mediated gene transfer of COX-2 into rat GMC led to increased expression and activity of multidrug resistance protein 1 (MDR-1), a membrane transporter that functions as an efflux pump for chemotherapeutic drugs, including Adriamycin (ADR). In ADR nephrotoxicity, a pathologic change in glomeruli could be partially explained by ADR-mediated changes in GMC. Here it is demonstrated that ADR (also known as doxorubicin; 1 microg/ml) induced apoptosis in 15.3 +/- 2.2% of GMC, whereas after adenovirus-mediated COX-2 expression, only 6.6 +/- 0.4% of ADR-treated cells underwent apoptosis. This protective effect was nullified by treatment with NS398, specific COX-2 inhibitor. ADR efflux is greater in COX-2-overexpressing cells, when compared with control, which is attributed to the increased MDR-1 expression. Addition of PSC833, the specific MDR-1 inhibitor, completely abolished the protective effect of COX-2 overexpression and increased the level of apoptosis in GMC that were exposed to ADR. These data suggest that COX-2 protects GMC from ADR-mediated apoptosis via transcriptional upregulation of MDR-1 and that induced COX-2 expression would lessen ADR nephrotoxicity.

Apoptotic cells, at all stages of the death process, trigger characteristic signaling events that are divergent from and dominant over those triggered by necrotic cells: Implications for the delayed clearance model of autoimmunity.

Current models of autoimmunity suggest that delayed clearance of apoptotic cells leads to the presentation of apoptotic antigens in the context of inflammatory signals, with resultant autoimmunity. These models implicitly assume that, in contrast to early apoptotic cells (that retain membrane integrity), late apoptotic cells (with compromised membranes) act like necrotic cells (which also lack intact membranes), possibly because of the release of proinflammatory intracellular contents. We showed previously that early apoptotic and necrotic cells induce distinct mitogen-activated protein kinase modules in macrophages with which they interact. Exposure to apoptotic cells led to nearly complete inhibition of both basal and macrophage colony-stimulating factor-induced ERK1/2 by macrophages. In contrast, necrotic cells induced ERK1/2. We show here that apoptotic cells also strongly induced both c-Jun N-terminal kinase and p38, whereas necrotic cells had no detectable effect on c-Jun N-terminal kinase and p38. We also compared the signaling events induced in macrophages by exposure to early apoptotic cells, late apoptotic cells, and necrotic cells. The signaling events induced by late apoptotic cells were identical to and just as potent as those induced by early apoptotic cells. Thus, apoptotic cells are functionally equivalent throughout the cell death process, irrespective of membrane integrity. Moreover, the effects of both early and late apoptotic cells on signaling were dominant over those of necrotic cells. These data show that apoptotic cells do not become proinflammatory upon the loss of membrane integrity and are inconsistent with the notion that delayed clearance alone can lead to autoimmunity.

Regulation of MDR-1 (P-glycoprotein) by cyclooxygenase-2.

Cyclooxygenase-2 (Cox-2), an inducible form of the enzyme that catalyzes the first step in the synthesis of prostanoids, has been shown to be overexpressed in a wide range of tumors and possesses proangiogenic and antiapoptotic properties. To understand the molecular mechanism of Cox-2 action we used adenovirus-mediated transfer of rat Cox-2 cDNA into renal rat mesangial cells and determined the differential gene expression using cDNA microarrays. One of the several genes that were highly up-regulated by over expressed Cox-2 was MDR1. MDR1 or P-glycoprotein (P-gp), the product of the MDR1 gene, is implicated as the primary cause of multidrug resistance (MDR) in tumors where it acts as an efflux pump for chemotherapeutic agents. It is also expressed in normal tissues of the liver and kidney where it functions to actively transport lipophilic xenobiotics. Reverse transcriptase-PCR analysis confirmed the results of the microarray, showing increased mRNA levels for MDR1 in Cox-2 overexpressing cells. This increase in mRNA translated to an increase in MDR1 protein expression, which was dose-dependent on Cox-2 expression. Furthermore, using rhodamine 123 efflux assay we observed a significant increase in P-gp activity in Cox-2 overexpressing renal mesangial cells. The specific Cox-2 inhibitor NS398 was able to block the Cox-2-mediated increase in MDR1 expression and activity, suggesting that Cox-2 products may be implicated in this response. These results prove the existence of a causal link between Cox-2 and P-gp activity, which would have implications for kidney function and multidrug resistance in tumors where Cox-2 is overexpressed.

Isolation and characterization of human thyroid endothelial cells.

From collagenase digests of human thyroid, endothelial cells were separated from follicular cells by their greater adherence to gelatin-coated plates. Endothelial cells were further purified using fluorescence-activated cell sorting, selecting for cells expressing factor VIII-related antigen. Isolated cells were negative for thyroglobulin and calcitonin when examined by immunostaining. The receptor for the angiopoietins, Tie-2, was expressed by the cells, and expression was increased by agents that elevate cAMP. Nitric oxide synthase (NOS) 3, the endothelial form of NOS, was expressed by the cells and similarly regulated. Cells responded strongly to the mitogen fibroblast growth factor (FGF)-2 in growth assays but only weakly to vascular endothelial growth factor (VEGF). VEGF was, however, able to stimulate nitric oxide release from the cells consistent with their endothelial origin. The FGF receptor (FGFR1) was full length (120 kDa) and immunolocalized to the cytosol and nucleus. Thyrotropin (TSH) did not regulate FGFR1, but its expression was increased by VEGF. Thrombospondin, a product of follicular cells, was a growth inhibitor, but neither TSH nor 3,5,3'-triiodothyronine had direct mitogenic effects. Thyroid follicular cell conditioned medium contained plasminogen activator activity and stimulated the growth of the endothelial cells, but when treated with plasminogen to produce the endothelial-specific inhibitor, angiostatin, growth was inhibited. Human thyroid endothelial cell cultures will be invaluable in determining the cross talk between endothelial and follicular cells during goitrogenesis.