Iron-Driven Alterations on Red Blood Cell-Derived Microvesicles Amplify Coagulation during Hemolysis via the Intrinsic Tenase Complex.

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl3) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl3 and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.

Legumain is a predictor of all-cause mortality and potential therapeutic target in acute myocardial infarction.

The prognostic impact of extracellular matrix (ECM) modulation and its regulatory mechanism post-acute myocardial infarction (AMI), require further clarification. Herein, we explore the predictive role of legumain-which showed the ability in ECM degradation-in an AMI patient cohort and investigate the underlying mechanisms. A total of 212 AMI patients and 323 healthy controls were enrolled in the study. Moreover, AMI was induced in mice by permanent ligation of the left anterior descending artery and fibroblasts were adopted for mechanism analysis. Based on the cut-off value for the receiver-operating characteristics curve, AMI patients were stratified into low (n = 168) and high (n = 44) plasma legumain concentration (PLG) groups. However, PLG was significantly higher in AMI patients than that in the healthy controls (median 5.9 µg/L [interquartile range: 4.2-9.3 µg/L] vs. median 4.4 µg/L [interquartile range: 3.2-6.1 µg/L], P < 0.001). All-cause mortality was significantly higher in the high PLG group compared to that in the low PLG group (median follow-up period, 39.2 months; 31.8% vs. 12.5%; P = 0.002). Multivariate Cox regression analysis showed that high PLG was associated with increased all-cause mortality after adjusting for clinical confounders (HR = 3.1, 95% confidence interval (CI) = 1.4-7.0, P = 0.005). In accordance with the clinical observations, legumain concentration was also increased in peripheral blood, and infarcted cardiac tissue from experimental AMI mice. Pharmacological blockade of legumain with RR-11a, improved cardiac function, decreased cardiac rupture rate, and attenuated left chamber dilation and wall thinning post-AMI. Hence, plasma legumain concentration is of prognostic value in AMI patients. Moreover, legumain aggravates cardiac remodelling through promoting ECM degradation which occurs, at least partially, via activation of the MMP-2 pathway.

Early-life inhalant allergen exposure, filaggrin genotype, and the development of sensitization from infancy to adolescence.

BACKGROUND: We hypothesized that filaggrin (FLG) loss-of-function mutations modify the effect of allergen exposure on the development of allergic sensitization. OBJECTIVE: We sought to determine whether early-life exposure to inhalant allergens increases the risk of specific sensitization and whether FLG mutations modulate these odds. METHODS: In a population-based birth cohort we measured mite, cat, and dog allergen levels in dust samples collected from homes within the first year of life. Sensitization was assessed at 6 time points between infancy and age 16 years. Genotyping was performed for 6 FLG mutations. RESULTS: In the longitudinal multivariable model (age 1-16 years), we observed a significant interaction between FLG and Fel d 1 exposure on cat sensitization, with the effect of exposure being significantly greater among children with FLG mutations compared with those without (odds ratio, 1.36; 95% CI, 1.02-1.80; P = .035). The increase in risk of mite sensitization with increasing Der p 1 exposure was consistently greater among children with FLG mutations, but the interaction did not reach statistical significance. Different associations were observed for dogs: there was a significant interaction between FLG and dog ownership, but the risk of sensitization to any allergen was significantly lower among children with FLG mutations who were exposed to a dog in infancy (odds ratio, 0.16; 95% CI, 0.03-0.86; P = .03). CONCLUSIONS: FLG loss-of-function mutations modify the relationship between allergen exposure and sensitization, but effects differ at different ages and between different allergens.

Detection of IgE Reactivity to a Handful of Allergen Molecules in Early Childhood Predicts Respiratory Allergy in Adolescence.

BACKGROUND: Sensitization in early childhood may precede respiratory allergy in adolescence. METHODS: IgE reactivity against 132 allergen molecules was evaluated using the MeDALL microarray in sera obtained from a random sample of 786 children at the age of 4, 8 and 16years in a population based birth cohort (BAMSE). Symptoms were analyzed by questionnaire at ages 4, 8 and 16years. Clinically and independent relevant allergen molecules accounting for ≥90% of IgE reactivities in sensitized individuals and at all time-points were identified as risk molecules and used to predict respiratory allergy. The data was replicated in the Manchester Asthma and Allergy Study (MAAS) birth cohort by studying IgE reactivity with the use of a commercial IgE microarray. Sera were obtained from children at the ages of 3, 5, 8 and 11years (N=248) and the outcome was studied at 11years. FINDINGS: In the BAMSE cohort 4 risk molecules could be identified, i.e.: Ara h 1 (peanut), Bet v 1 (birch), Fel d 1 (cat), Phl p 1 (grass). For MAAS the corresponding number of molecules was 5: Der p 1 (dust mite), Der f 2 (dust mite), Phl p 1 (grass), Phl p 5 (grass), Fel d 1 (cat). In BAMSE, early IgE reactivity to ≥3 of 4 allergen molecules at four years predicted incident and persistent asthma and/or rhinitis at 16years (87% and 95%, respectively). The corresponding proportions in the MAAS cohort at 16years were 100% and 100%, respectively, for IgE reactivity to ≥3 of 5 risk molecules. INTERPRETATIONS: IgE reactivity to a few allergen molecules early in life identifies children with a high risk of asthma and/or rhinitis at 16years. These findings will be of importance for developing preventive strategies for asthma and rhinitis in children.

IgG Endopeptidase in Highly Sensitized Patients Undergoing Transplantation.

BACKGROUND: Donor-specific antibodies create an immunologic barrier to transplantation. Current therapies to modify donor-specific antibodies are limited and ineffective in the most highly HLA-sensitized patients. The IgG-degrading enzyme derived from Streptococcus pyogenes (IdeS), an endopeptidase, cleaves human IgG into F(ab')2 and Fc fragments inhibiting complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity, which suggests that IdeS might be useful for desensitization. We report on the combined experience of two independently performed open-label, phase 1-2 trials (conducted in Sweden and the United States) that assessed the efficacy of IdeS with regard to desensitization and transplantation of a kidney from an HLA-incompatible donor. METHODS: We administered IdeS to 25 highly HLA-sensitized patients (11 patients in Uppsala or Stockholm, Sweden, and 14 in Los Angeles) before the transplantation of a kidney from an HLA-incompatible donor. Frequent monitoring for adverse events, outcomes, donor-specific antibodies, and renal function was performed, as were renal biopsies. Immunosuppression after transplantation consisted of tacrolimus, mycophenolate mofetil, and glucocorticoids. Patients in the U.S. study also received intravenous immune globulin and rituximab after transplantation to prevent antibody rebound. RESULTS: Recipients in the U.S. study had a significantly longer cold ischemia time (the time elapsed between procurement of the organ and transplantation), a significantly higher rate of delayed graft function, and significantly higher levels of class I donor-specific antibodies than those in the Swedish study. A total of 38 serious adverse events occurred in 15 patients (5 events were adjudicated as being possibly related to IdeS). At transplantation, total IgG and HLA antibodies were eliminated. A total of 24 of 25 patients had perfusion of allografts after transplantation. Antibody-mediated rejection occurred in 10 patients (7 patients in the U.S. study and 3 in the Swedish study) at 2 weeks to 5 months after transplantation; all these patients had a response to treatment. One graft loss, mediated by non-HLA IgM and IgA antibodies, occurred. CONCLUSIONS: IdeS reduced or eliminated donor-specific antibodies and permitted HLA-incompatible transplantation in 24 of 25 patients. (Funded by Hansa Medical; ClinicalTrials.gov numbers, NCT02224820 , NCT02426684 , and NCT02475551 .).

Sensitization to 10 mites in a tropic area. Der p and Der f are important risk factor for sensitization to other mites from Pyroglyphidae, Acaridae, Chortoglyphidae, and Glyciphagidae families.

BACKGROUND: Much is known about the frequency of sensitization to Blomia tropicalis, Dermatophagoides pteronyssinus and Dermatophagoides farinae, although less is known about sensitization to other species and their possible interactions. OBJECTIVE: In patients with allergic manifestations, to evaluate the frequency of sensitization to 10 species of mites in a tropical area and their possible interactions. METHODS: Cross-sectional study. Sensitization was evaluated by skin tests. A generalized linear Poisson regression model with robust variance was used. Based on the sensitization probability reasons and social networking analysis, explorations of relationship for 10 mites were performed. RESULTS: 147 patients were included. The highest sensitization was found to mites' family Pyroglyphidae (> 70 %) and less frequently was the Glycyphagidae family (< 50 %). Sensitization to any mites significantly increased the likelihood of sensitization to others. Sensitization to Der f or Der p increased, more than 20 times the likelihood of sensitization to other mites of the Pyroglyphidae family and more than 10 times to mites from other families. Sensitization to mites from Glycyphagidae, Chortoglyphidae or Acaridae family also increased the risk of sensitization to other mites but less than 5 times. CONCLUSION: Sensitization to mites is frequent in tropical area. Pyroglyphidae sensitization is the main risk factor for polysensitization with other mites from Glycyphagidae, Chortoglyphidae or Acaridae. These results must be considered at diagnosis and treatment of allergy diseases.

Assessing the efficacy of a novel temperature and humidity control machine to minimize house dust mite allergen exposure and clinical symptoms in allergic rhinitis children sensitized to dust mites: a pilot study.

BACKGROUND: House dust mite avoidance is advised in dust mite sensitized patients to decrease the risk to develop allergic symptoms. Maintaining a relative humidity (RH) of less than 50% in households is recommended to prevent dust mite proliferation. OBJECTIVE: To investigate the efficacy of a novel temperature and humidity machine to control the level of dust mite allergens and total nasal symptom score (TNSS) in dust mite sensitized allergic rhinitis children. METHOD: Children (8-15 years) with dust mite sensitized persistent allergic rhinitis (AR) were enrolled. The temperature and humidity control machine was installed in the bedroom where the enrolled children stayed for 6 months. TNSS was assessed before and every month after machine set up and the level of dust mite allergen (Der p 1 and Der f 1) from the mattress were measured before and every 2 months after machine set up using enzyme-linked immunosorbent assay (ELISA). RESULTS: A total of 7 children were enrolled. Noticeable reduction of Der f 1 was observed as early as 2 months after installing the machine, but proper significant differences appeared 4 months after and remained low until the end of the experiment (p <0.05). Although no correlation was observed between TNSS and the level of dust mite allergens, there was a significant reduction in TNSS at 2 and 4 months (p <0.05) and 70% of the patients were able to stop using their intranasal corticosteroids by the end of the experiment. CONCLUSIONS: The level of house dust mite in mattresses was significantly reduced after using the temperature and humidity control machine. This machine may be used as an effective tool to control clinical symptoms of dust mite sensitized AR children.

High expression of the cysteine proteinase legumain in colorectal cancer - implications for therapeutic targeting.

BACKGROUND: The cysteine proteinase legumain is highly expressed in cancer. Legumain is a potential biomarker and has been suggested to be utilised for prodrug activation in cancer therapy. However, to define the suitability of legumain for such purposes, detailed knowledge of cell type-specific and subcellular expression together with proteolytic activity patterns in tumour tissue is necessary. METHODS: Expression of legumain was examined in a panel of 277 primary tumours from colorectal cancer (CRC) patients using immunohistochemistry. Tumour (cytoplasmic diffuse, cytoplasmic granulated, and nuclear) and stromal cell expression of legumain was quantified, and associations with clinicopathological parameters and outcome were analysed. Additionally, normal colon tissue and spontaneous mouse tumours were stained for legumain. RESULTS: Legumain was highly expressed in tumour and stromal cells. Nuclear legumain was detected in 30% of the tumours. In colon cancer patients, high legumain expression was associated with overall and metastasis-free survival (OS; MFS) in uni- and multivariate analysis. Nuclear legumain was associated with poor OS, but not MFS in the colon cancer subgroup. Cytoplasmic granulated or diffuse expression was not associated with OS or MFS. Normal epithelial cells exhibited granulated legumain mainly at the apical pole, and legumain was highly expressed in CD68 positive macrophages. CONCLUSIONS: Legumain is a highly expressed proteinase in CRC and associated with poor outcome in colon cancer. Diversified localisation of legumain expression in tumour and stromal cells suggests multiple functions in CRC, representing both a challenge and an opportunity for use in therapeutic targeting.

Establishing an allergic eczema model employing recombinant house dust mite allergens Der p 1 and Der p 2 in BALB/c mice.

The major house dust mite allergens Der p 1 and Der p 2 are prevalent inducers of eczema. Der p 1 is a cysteine protease disrupting epithelial barriers, whereas Der p 2 functionally mimics the LPS-binding compound MD-2 within the TLR4 complex. In this work, we tested the percutaneous sensitizing capacity of recombinant (r) Der p 1 and Der p 2 in BALB/c mice. Mice were sensitized by percutaneous application of low (10 µg/application) and high dose (100 µg) rDer p 1 or rDer p 2, or with rDer p 1 followed by rDer p 2. Allergen-specific and total IgE antibodies were determined by ELISA. Eczema of BALB/c was classified by the itching score and corresponded to erosions. Infiltrating immune cells were identified by haematoxylin/eosin and Giemsa staining for eosinophils or mast cells, CD3 staining for T lymphocytes. Percutaneous treatments with rDer p 1, but not rDer p 2-induced specific IgG1. However, cotreatment with rDer p 1 led to increase in anti-Der p 2 IgG titres. Both allergens elicited skin erosions because of scratching, thickening of the epidermis, and eosinophil and T-cell infiltration. Our data indicate that recombinant mite allergens in the absence of adjuvant are sufficient for inducing eczema in BALB/c mice. As the enzymatic activity of an allergen might be an important cofactor for specific sensitization via the skin, Der p 1 may act as adjuvant for other allergens too. The presented mouse model is suitable for investigating the mechanisms of allergic eczema.

Dissecting the causes of atopic dermatitis in children: less foods, more mites.

Atopic dermatitis (AD) is a common, chronic or chronically relapsing, multifactorial skin disease that mainly occurs in children but affects also adults. AD usually begins early in life and often concerns people with a personal or family history of asthma and allergic rhinitis. AD is characterized by eczematous changes in the epidermis and originates from a late, T-cell mediated reaction associated to the formation and production of memory T-cell of TH2 type, occurrence of homing receptor at skin level and cutaneous lymphocyte-associated (CLA) antigens. Extrinsic or allergic AD, but not intrinsic AD, shows high total serum IgE levels and the presence of specific IgE for environmental and food allergens. A pivotal role in the pathogenesis of AD is played by filaggrin, a protein contained in the granular layer of the epidermis regulating the aggregation of keratin filaments. Mutation in the filaggrin gene causes decreased barrier function of the corny layers of the epidermis. This favours the enter through the skin of environmental allergens, especially the house dust mite, that further facilitates such entering by the proteolytic activity of its major allergen Der p 1. In fact, recent advances suggest that the dust mite, more than foods, is the major cause of allergic AD. As far as the causal diagnosis of AD is concerned, there is notable evidence supporting the capacity of the atopy patch test (APT) to reproduce the pathophysiologic events of AD. This makes APT a valuable diagnostic tool for AD.

Nonproteasomal targets of the proteasome inhibitors bortezomib and carfilzomib: a link to clinical adverse events.

PURPOSE: Bortezomib (Velcade), a dipeptide boronate 20S proteasome inhibitor and an approved treatment option for multiple myeloma, is associated with a treatment-emergent, painful peripheral neuropathy (PN) in more than 30% of patients. Carfilzomib, a tetrapeptide epoxyketone proteasome inhibitor, currently in clinical investigation in myeloma, is associated with low rates of PN. We sought to determine whether PN represents a target-mediated adverse drug reaction (ADR). EXPERIMENTAL DESIGN: Neurodegenerative effects of proteasome inhibitors were assessed in an in vitro model utilizing a differentiated neuronal cell line. Secondary targets of both inhibitors were identified by a multifaceted approach involving candidate screening, profiling with an activity-based probe, and database mining. Secondary target activity was measured in rats and patients receiving both inhibitors. RESULTS: Despite equivalent levels of proteasome inhibition, only bortezomib reduced neurite length, suggesting a nonproteasomal mechanism. In cell lysates, bortezomib, but not carfilzomib, significantly inhibited the serine proteases cathepsin G (CatG), cathepsin A, chymase, dipeptidyl peptidase II, and HtrA2/Omi at potencies near or equivalent to that for the proteasome. Inhibition of CatG was detected in splenocytes of rats receiving bortezomib and in peripheral blood mononuclear cells derived from bortezomib-treated patients. Levels of HtrA2/Omi, which is known to be involved in neuronal survival, were upregulated in neuronal cells exposed to both proteasome inhibitors but was inhibited only by bortezomib exposure. CONCLUSION: These data show that bortezomib-induced neurodegeneration in vitro occurs via a proteasome-independent mechanism and that bortezomib inhibits several nonproteasomal targets in vitro and in vivo, which may play a role in its clinical ADR profile.

Gingipain-dependent interactions with the host are important for survival of Porphyromonas gingivalis.

Porphyromonas gingivalis, a major periodontal pathogen, must acquire nutrients from host derived substrates, overcome oxidative stress and subvert the immune system. These activities can be coordinated via the gingipains which represent the most significant virulence factor produced by this organism. In the context of our contribution to this field, we will review the current understanding of gingipain biogenesis, glycosylation, and regulation, as well as discuss their role in oxidative stress resistance and apoptosis. We can postulate a model, in which gingipains may be part of the mechanism for P. gingivalis virulence.