Neutrophil Extracellular Trap-Related Extracellular Histones Cause Vascular Necrosis in Severe GN

Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti–glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN.     

Influence of different trigger techniques on twitch mouth pressure during bilateral anterior magnetic phrenic nerve stimulation

BACKGROUND: The trigger has a key role when assessing the twitch mouth pressure (Tw Pmo), since a "gentle" inspiratory or expiratory effort is needed for triggering to ensure an open glottis during twitch, but which also guarantees only very mild changes of transdiaphragmatic pressure following changes in lung volume. STUDY OBJECTIVES: To test if different trigger mechanisms cause different Tw Pmo values, if the predefined trigger criteria were accomplished, and if the breathing maneuver during triggering can influence the Tw Pmo. DESIGN: Experimental study. SETTING: Respiratory muscle and lung function laboratory of a university hospital. PARTICIPANTS: Twenty healthy men (mean age, 25.6 +/- 1.2 years [+/- SD]; mean FEV(1), 105.9 +/- 11.5% of predicted). MEASUREMENTS: Tw Pmo produced by bilateral anterior magnetic phrenic nerve stimulation was measured using an inspiratory flow trigger (40 mL/s), an inspiratory pressure trigger, and an expiratory pressure trigger (3.75 mm Hg). All trigger criteria were controlled. RESULTS: Unusable pressure-time curves occurred in 40% during expiratory triggering, but not during inspiratory triggering. For inspiratory pressure (flow) triggering, 10% (30%) of the predefined trigger criteria were exceeded by 50%, indicating that a "gentle" inspiratory effort was not warranted. The Tw Pmo was higher during inspiratory compared to expiratory triggering (analysis of variance, p < 0.05). The Tw Pmo during inspiratory pressure and flow triggering were comparable and significantly correlated (r = 0.70, p < 0.0001). The time between start of inspiration and trigger release, and the pressure-time product during that period ranged widely, but this could not predict the Tw Pmo (multiple linear regression). CONCLUSIONS: The trigger technique influences the Tw Pmo with higher values during inspiratory compared to expiratory triggering. Expiratory triggering more often produced unusable pressure-time curves. Inspiratory flow and pressure triggering is comparably useful in healthy subjects, but this might be different in patients. The trigger criteria need to be controlled to warrant a gentle breathing effort.     

Using Vascular Closure Devices Following Out-Of-Hospital Cardiac Arrest?

Objectives and Background: Despite a generally broad use of vascular closure devices (VCDs), it remains unclear whether they can also be used in victims from out-of-hospital cardiac arrest (OHCA) treated with mild therapeutic hypothermia (MTH).Methods: All victims from OHCA who received immediate coronary angiography after OHCA between January 1^st 2008 and December 31^st 2013 were included in this study. The operator decided to either use a VCD (Angio-Seal™) or manual compression for femoral artery puncture. The decision to induce MTH was based on the clinical circumstances.Results: 76 patients were included in this study, 46 (60.5%) men and 30 (39.5%) women with a mean age of 64.2 ± 12.8 years. VCDs were used in 26 patients (34.2%), and 48 patients (63.2%) were treated with MTH. While there were significantly more overall vascular complications in the group of patients treated with MTH (12.5% versus 0.0%; p=0.05), vascular complications were similar between patients with VCD or manual compression, regardless of whether or not they were treated with MTH.Conclusion: In our study, the overall rate of vascular complications related to coronary angiography was higher in patients treated with mild therapeutic hypothermia, but was not affected by the application of a vascular closure device. Therefore, our data suggest that the use of VCDs in victims from OHCA might be feasible and safe in patients treated with MTH as well, at least if the decision to use them is individually carefully determined.     

Regulatory T Cell–Derived IL-10 Ameliorates Crescentic GN

Regulatory T cells (Tregs) exert their immunosuppressive activity through several immunoregulatory mechanisms, including the production of anti-inflammatory cytokines such as IL-10. Although several studies suggest a role for Tregs in modulating crescentic GN, the underlying mechanisms are not well understood. Here, using IL-10 reporter mice, we detected IL-10–producing Foxp3⁺ T cells in the kidney, blood, and secondary lymphoid tissue in a mouse model of crescentic GN. Specific inactivation of Il10 in Foxp3⁺ Tregs eliminated the ability of these cells to suppress renal and systemic production of IFNγ and IL-17; these IL-10–deficient Tregs lost their capacity to attenuate renal tissue injury. These data highlight the suppressive functions of Tregs in crescentic GN and suggest the importance of Treg-derived IL-10 in ameliorating disease severity and in modulating both the Th1 and most notably Th17 immune response.The discovery of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in the 1990s and their indispensable role in (self) tolerance and autoimmunity marked the beginning of a new era in immunology.¹ Since then, different suppressive mechanisms mediated by various Treg cell subsets were identified,^2,3 particularly in well studied models of autoimmune diseases such as Crohn''s disease,⁴ multiple sclerosis,⁵ or rheumatoid arthritis.^6,7 Until now, only limited numbers of studies have assessed the function of regulatory T cells in crescentic GN. Adoptive cell transfer experiments in mice showed the beneficial role of exogenous wild-type (wt) CD4⁺CD25⁺ Tregs in attenuation of crescentic GN,⁸ whereas CCR6- and CCR7-deficient CD4⁺CD25⁺ Tregs failed to protect mice against GN.^9,10 Recently, our own published data revealed the importance of endogenous Foxp3⁺ Tregs in suppressing the Th1 immune response and consequently ameliorating the disease severity in the T cell–dependent GN model of nephrotoxic nephritis (NTN).¹¹ Concurrently, Ooi and coworkers confirmed the relevance of endogenous Foxp3⁺ Tregs in an accelerated model of experimental crescentic GN.¹² However, the mechanisms of Treg cell-mediated suppression in crescentic GN are still unclear. One important player might be the anti-inflammatory cytokine IL-10, which is known to be released by Tregs in order to suppress immune responses and therefore might protect against autoimmunity.¹³ Indeed, endogenous IL-10 regulates the Th1 immune response in an accelerated model of experimental crescentic GN, as kidney damage is aggravated in IL-10–deficient mice.¹⁴ However, the source of protective IL-10 still needs to be clarified. Because IL-10 detection and tracking in vivo is difficult, most findings are based on studies with IL-10^−/− mice.Therefore, to study the cell-specific function of IL-10, we used a double-knockin reporter mouse model (Foxp3-IRES-mRFP (FIR) x IL-10 ires gfp-enhanced reporter [tiger]), which enables detection of the well-defined and simultaneous expression of IL-10 (green fluorescent protein [GFP]) and Foxp3 (monomeric red fluorescent protein [mRFP]). Indeed, we detected a distinct population of renal mRFP⁺(Foxp3⁺) Tregs expressing GFP (IL-10) upon induction of NTN. Thus, to investigate the role of Treg cell-derived IL-10 in NTN, we first adoptively transferred CD4⁺CD25⁺ Tregs from wt or IL-10^−/− mice into wt mice subsequently challenged with nephrotoxic sheep serum. Adoptively transferred wt Tregs attenuated the course of NTN, whereas IL-10^−/− Tregs did not. Furthermore, to analyze the role of endogenous IL-10 produced by Tregs, we generated Foxp3^YFP-Cre x Il10^flox/flox mice, in which IL-10 is selectively inactivated in Foxp3⁺ Tregs.¹⁵ Indeed, lack of Treg-derived IL-10 resulted in an aggravated course of NTN. In summary, we demonstrated a crucial role of Treg cell-derived IL-10 in regulating the Th1 and most notably the Th17 immune response in NTN. Hence, this study contributes to the understanding of the suppressive mechanisms of Tregs in crescentic GN and will have biologic implications for designing therapeutic approaches.     

13th St. Gallen International Breast Cancer Conference 2013: Primary Therapy of Early Breast Cancer Evidence,Controversies, Consensus – Opinion of a German Team of Experts (Zurich 2013)

Zusammenfassung

Alle zwei Jahre findet in St. Gallen (Schweiz) die internationale Konsensuskonferenz zur Behandlung des primären Mammakarzinoms statt. Da sich das internationale Panel in St. Gallen aus Experten unterschiedlicher Länder zusammensetzt, spiegelt der Konsensus ein internationales Meinungsbild wider. Vor diesem Hintergrund erscheint es aus deutscher Sicht sinnvoll, die Abstimmungsergebnisse für den Therapiealltag in Deutschland zu konkretisieren. Eine deutsche Arbeitsgruppe mit acht Brustkrebsexperten, von denen zwei Mitglieder des internationalen St. Gallen-Panels sind, hat daher die Abstimmungsergebnisse der St. Gallen-Konsensuskonferenz (2013) für den Klinikalltag in Deutschland kommentiert. Inhaltliche Schwerpunkte der diesjährigen St. Gallen-Konferenz waren operative Fragestellungen der Brust und der Axilla, strahlentherapeutische und systemische Therapieoptionen sowie die klinische Relevanz der Tumorbiologie. Intensiv diskutiert wurde der klinische Einsatz von Multigen-Assays, inkl. ihrer Bedeutung für die individuelle Therapieentscheidung.     

Age- and stage-dependent glyoxalase I expression and its activity in normal and Alzheimer's disease brains

The reaction of lysine and arginine residues of proteins with 1,2-dicarbonyl compounds result in the formation of advanced glycation end products (AGEs). Accumulation of AGEs is a characteristic feature of the aging brain and contributes to the development of neurodegenerative diseases such as Alzheimer's disease (AD). Therefore, it is of particular interest to study the cellular defense mechanisms against AGE formation and particularly the detoxification of their precursors. AGE precursor compounds such as methylglyoxal and glyoxal were cellulary detoxified by the glyoxalase system, consisting of glyoxalases I and II. Glyoxalase I levels are diminished in old aged brains but elevated in AD brains. However, it is still unknown how glyoxalase I level of AD brains changes in a disease and in an age-dependent manner. Therefore, we investigated the AD stage- and the age-dependent levels of glyoxalase I in the Brodmann area 22 of AD brains (n=25) and healthy controls (n=10). Our results obtained from RT-PCR reveal reducing glyoxalase I RNA levels with advancing stage of AD and with increasing age. Western Blot analysis indicates that in comparison to healthy controls, glyoxalase I protein amounts are 1.5-fold increased in early AD subjects and continuously decrease in middle and late stages of AD. The glyoxalase I protein amounts of AD patients also decrease with age. Results obtained from glyoxalase I activity measurement show 1.05-1.2-fold diminished levels in AD brains compared to healthy controls and no significant decrease neither with the stage of AD nor with age. The immunohistochemical investigations demonstrate an elevated number of glyoxalase I stained neurons in brains of early and middle but not in late AD subjects compared to age-matched healthy controls. In addition, the stage-dependent immunohistochemical investigation demonstrates that with reduced glyoxalase I staining AGE deposits prevail, specifically in late stage of AD. In conclusion, the decrease of glyoxalase I expression with increasing AD stage might be one reason for methylglyoxal-induced neuronal impairment, apoptosis, and AGE formation in plaques and tangles.