Age-at-death assessed with Lamendin's original and population-specific models in a modern Brazilian osteological collection.

BACKGROUND: Estimating the age at death is a common procedure in the fields of forensic human identification and anthropological/archaeological investigations. Root translucency and periodontosis are regressive parameters used to estimate the age of adults, more specifically in Lamendin's method - established in 1992 in a French population. This study aimed to test the applicability and validity of Lamendin's method in a Brazilian osteological collection. METHODS: The sample consisted of 74 single-rooted teeth obtained from 50 skeletal remains (mean age: 53.20 ± 16.17 years) from Southeast Brazil. Lamendin's method was applied to enable a comparison between chronological (CA) and estimated ages (EA). A new population-specific equation was designed for the studied sample and the outcomes were compared with those obtained with Lamendin's original equation. RESULTS: The original methods led to a general underestimation of 11.32 years (8.83 years in males and 15.91 years in females). The method had a better performance among individuals between 40 and 59 years (mean differences between CA and EA: 4.8 years). The population-specific equation led to a mean overestimation of -2.04 years in males, and a mean underestimation of 3.77 years in females. Underestimations were considerably higher in other age groups. CONCLUSION: Despite the apparent improvements, both the original and the population-specific equations revealed coefficients of concordance that were constantly low between CA and EA. These outcomes suggest restrictions to the application of Lamendin's method in the forensic field, especially for human identification. The method, however, seems to be applicable for anthropological/archaeological applications.

Deep venous thrombosis and abortion: an unusual clinical manifestation of severe form of pectus excavatum.

Pectus excavatum is a chest wall malformation with a strong psychological and aesthetic impact. Rarely, pectus excavatum patients can show respiratory or cardiac symptoms occurring mainly during physical exertion. We report a case of a 34-year-old pregnant woman with a severe degree of pectus excavatum who developed serious cardiovascular disease resulting in spontaneous twin abortion at the twenty-first week of gestation. Cardiovascular disease was resolved after open surgical correction of pectus excavatum. This case shows how a tardive diagnosis and a delayed surgical approach for pectus excavatum can lead to severe consequences.

Dirofilaria repens Infection Mimicking Lung Melanoma Metastasis.

We describe a rare case of Dirofilaria repens infection presenting as peripheral lung nodules and mimicking a metastatic focus from a previously diagnosed cutaneous melanoma. To avoid invasive investigations before arriving at the correct diagnosis, dirofilariasis should be included as a part of the diagnostic process in subjects with lung nodules who live in (or have traveled to) endemic regions.

Colistin-based Treatment of Multidrug-resistant Gram-negative Bacterial Pulmonary Infections After Lung Transplantation.

BACKGROUND: Lung transplantation (LT) is a viable option for a select group of patients with end-stage lung disease. However, infections are a major complication after LT, accounting for significant morbidity and mortality. Several germs may be responsible; multidrug-resistant Gram-negative (MDR-GN) bacteria are emerging. Colistin is widely used in the treatment of these infections and is administered by inhalation and/or parenterally. At our institution, in patients with tracheostomy, colistin is administered by direct instillation in the airway during bronchoscopy. We reviewed a series of patients who underwent LT complicated by postoperative MDR-GN bacterial pulmonary infection. METHODS: From January 2015 to May 2017, 26 lung transplants were performed. In the postoperative course, 14 (54%) developed MDR-GN bacterial infection; respiratory specimen culture, blood tests, and chest X-ray were considered. Colistin was the only antibiotic usable. Thirteen patients received intravenous (IV) colistin; in the subgroup of patients with tracheostomy, colistin was instilled directly in the airway, and 6 patients received inhaled colistin. RESULTS: Seven patients needed tracheostomy. Pseudomonas aeruginosa was the predominant infection (86%), with Acinetobacter baumanii seen in 2 cases (14%). An early clinical-laboratory response was observed in 9 patients (64%). White blood cell count and C-reactive protein values improved (P = .02 and P = .001, respectively). A significant reduction in bacterial load was observed on microbiologic bronchoalveolar lavage specimens. CONCLUSION: Colistin instilled directly in the airway did not show side effects. The combination of IV and inhaled/instilled colistin could be a useful treatment option for MDR-GN infections after LT.

Efficacy of Extracorporeal Photopheresis in Patients With Bronchiolitis Obliterans Syndrome After Lung Transplantation.

BACKGROUND: Lung transplantation (LT) is only therapeutic option for patients affected by chronic respiratory failure. Chronic rejection, also known as bronchiolitis obliterans syndrome (BOS), is still the main cause of death and the most important factor that influences post-transplantation quality of life. Currently available therapies have not been proven to result in significant benefit in the prevention or treatment of BOS. Extracorporeal photopheresis (ECP) seems to reduce the rate of lung function decline in transplant recipients with progressive BOS. METHODS: From 1991 until now, 239 LTs were performed at our center. Fifty-four patients (22.5%) developed BOS; 15 of these (27.7%) were treated with ECP. At the beginning of the treatment, all patients showed a mean decline of forced expiratory volume in 1 second (FEV1) from baseline values of 45.8% ± 17.2%; 2 patients were in long-term oxygen therapy. RESULTS: Mean follow-up from November 2013 to June 2016 was 11.6 ± 7 months. Twelve patients (80%) showed lung function stabilization with an FEV1 range after treatment between -6% to +8% from the pre-treatment values. We did not report any adverse effects or increase of infections incidence. DISCUSSION: ECP seems to be an effective and well-tolerated therapeutic option for LT patients with BOS in terms of stabilization of lung function and increased survival.

Evaluation of Renal Function in Patients Undergoing Lung Transplantation.

BACKGROUND: Acute kidney injury and chronic kidney failure are serious complications after lung transplantation. Glomerular filtration rate (GFR) is the primary indicator of renal function. Several equations have been proposed to evaluate the estimated GFR (eGFR). We compared three different equations to determine which has the better correlation with the development of acute and chronic renal failure in lung recipients. METHODS: Twenty-two patients with a mean age of 54.4 ± 8.5 years underwent lung transplantation from 2010 to 2015. Thirteen (59%) had pulmonary fibrosis, 7 (32%) emphysema, 1 (4.5%) bronchiectasis, and 1 (4.5%) lymphangioleiomyomatosis. In all patients, eGFR was measured preoperatively using Cockcroft-Gault (CG), Modification of Diet in Renal Disease (MDRD), and Levey's Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations. In 20 patients (90%) eGFR was calculated at 1, 3, and 6 months. RESULTS: According to CKD-EPI and MDRD, eight patients (36.3%) had preoperative reduction in eGFR, whereas 6 patients (27.2%) had preoperative reduction according to the CG (P = .04). The mean values were higher for the CG (103.2 vs. 102 vs. 94.4). Five patients (22.7%) developed perioperative acute renal failure requesting a dialysis treatment; four of these showed a preoperative eGFR to the highest CG (P = .05). At 1 and 6 months after lung transplantation, the CG, MDRD and CKD-EPI eGFR values were, respectively, 86.6, 84.1 and 76.6 mL/min/1.73m2 and 75.8, 72.7, and 72.3 mL/min/1.73m2. CKD-EPI eGFR values are more predictable than the other equations of AKI. CONCLUSIONS: Preoperative assessment of eGFR using the MDRD and CKD-EPI seems to correlate better than the CG to the prediction of acute renal failure, whereas for the chronic form the three equations seem equivalent.

Evolution of Socioeconomic Conditions and Its Relation to Spatial-Temporal Changes of Giardiasis and Helminthiasis in Amazonian Children.

This study analyzed the evolution of socioeconomic, sanitary, and personal factors as well as spatiotemporal changes in the prevalence of helminthiasis and giardiasis in urban Amazonian children between 2003 and 2011. Child age, lack of sanitation, and lack of access to bottled water were identified as significant associated factors for helminthiasis and giardiasis. There was an overall improvement in socioeconomic and sanitary conditions in the city resulting in decreased helminth prevalences from 12.42 to 9.63% between 2003 and 2010, but the prevalence increased to 15.03% in 2011 due to migratory movement and unstable sanitary conditions. As for Giardiasis, socioeconomic and environmental changes were not enough to reduce prevalence (16% in 2003 and 23% in 2011). Spatial analysis identified a significant cluster for helminthiasis in an area of poor housing conditions. Control programs in the Amazon need to target high-risk areas focusing changes in sanitation, water usage, and health education.

Altered expression of metabolic proteins and adipokines in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the loss of upper cortical and lower motor neurons. ALS causes death within 2-5years of diagnosis. Diet and body mass index influence the clinical course of disease, however there is limited information about the expression of metabolic proteins and fat-derived cytokines (adipokines) in ALS. In healthy controls and subjects with ALS, we have measured levels of proteins and adipokines that influence metabolism. We find altered levels of active ghrelin, gastric inhibitory peptide (GIP), pancreatic polypeptide (PP), lipocalin-2, plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6) and 8 (IL-8), and tumor necrosis factor alpha (TNFα) in the plasma of ALS patients relative to controls. We also observe a positive correlation between the expression of plasma nerve growth factor (NGF) relative to disease duration, and an inverse correlation between plasma glucagon and the ALS functional rating scale-revised (ALSFRS-R). Further studies are required to determine whether altered expression of metabolic proteins and adipokines contribute to motor neuron vulnerability and how these factors act to modify the course of disease.

Skewed B cells in chronic hepatitis C virus infection maintain their ability to respond to virus-induced activation.

Chronic hepatitis C virus (HCV) infection is characterized by persistent B-cell activation, with enhanced differentiation and reduced proliferative ability. To assess the possible role of HCV in altering B-cell subset distribution, we examined ex vivo frequencies and B-cell inhibitory receptor expression in 37 chronic HCV-infected patients and 25 healthy donors (HD). In addition, we determined whether short-term exposure to culture-derived HCV (HCVcc) resulted in B-cell subset skewing and/or activation. There was a statistically significant increase in the frequencies of immature transitional, activated memory and tissue-like memory (TLM) B cells in HCV-infected patients compared with HD. We also found that the frequency of memory B cells correlated with serum HCV RNA levels. The proportion of B cells expressing the marker of exhaustion Fc receptor-like 4 (FcRL4) was generally low even though significantly higher in the patients' memory B-cell compartment compared with HD, and a positive correlation was found between the frequencies of the patients' TLM FcRL4+ B cells and serum alanine aminotransferase and histological activity index at liver biopsy. Exposure to cell-free HCVcc in vitro did not result in B-cell skewing but induced significant activation of naïve, TLM and resting memory B cells in HCV-infected patients but not in HD, in whom cell-associated virus was an absolute requirement for activation of memory B cells. These findings provide corroborative evidence in favour of significant B-cell subset skewing in chronic HCV infection and in addition show that expression of exhaustion markers in selected B-cell subsets does not impair virus-induced B-cell activation.

Elevation of the terminal complement activation products C5a and C5b-9 in ALS patient blood.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterized by the progressive loss of motor neurons within the central nervous system. Neural degeneration and inflammatory processes, including activation of the complement system are hallmarks of this pathology. Our past work in ALS animal models (hSOD1 G93A rodents) has revealed that blockade of the receptor for complement activation fragment C5a (C5aR), improves ALS-like symptoms and extends survival. We now show that the levels of C5a and C5b-9, but not C3a nor C4a, are significantly elevated in plasma from ALS patients compared to healthy controls. C5a was also elevated within leukocytes from ALS patients suggesting heightened C5a receptor interaction. Overall, these findings indicate that there is enhanced peripheral immune complement terminal pathway activation in ALS, which may have relevance in the disease process.

Extracorporeal membrane oxygenation as bridge to lung transplantation.

Lung transplantation (OLT) is a viable option for end-stage pulmonary diseases in selected patients with satisfactory long-term results. However, the paucity of available donors engenders a prolonged stay on the waiting list with progressive decline of lung function. In cases of sudden respiratory failure, admission to an intensive care unit with institution of extracorporeal membrane oxygenation (ECMO) may be an option while a waiting an emergency OLT. In 12 OLT candidates we started ECMO because of acute decline of lung function. Eleven patients had cystic fibrosis and the other subject, histiocytosis X. In 7 patients bilateral OLT was performed after a mean waiting time of 6 days from ECMO institution; 5 patients died on ECMO at a mean time of 11.6 days. After OLT 2 patients required reoperation for hemothorax; renal failure and acute leg ischemia occurred in 2 patients. The mean weaning time from ECMO after OLT was 2.14 days. No patient died in the perioperative period and 1-year survival was 85.7%. ECMO represents a valid option as a bridge to urgent OLT for selected candidates.

Evaluation of Epstein-Barr virus-specific immunologic response in solid organ transplant recipients with an enzyme-linked ImmunoSpot assay.

Epstein-Barr virus (EBV) is a γ-herpes virus, responsible for infectious mononucleosis in immunocompetent hosts. Cellular immunity appears rapidly during EBV primary infection, keeping it silent despite long-life persistence in B lymphocytes. Defects of the EBV-specific cellular immunity are supposed to be the basis of post-transplantation lymphoproliferative disorders, promoted by high levels of immunosuppression. We retrospectively reviewed 197 solid organ transplant recipients to investigate EBV-specific lymphocyte responsiveness using Enzyme-linked ImmunoSpot assay (EliSpot), which assesses the EBV-specific interferon (IFN)-γ producing peripheral blood mononuclear cells, and kinetics of EBV infection/reactivation post-transplantation using quantitative real-time polymerase chain reaction (PCR) on whole blood. Overall, 102 of the 197 patients (51.8%) showed EBV responsiveness at the EBV-EliSpot assay: 68 (66.6%) showed a persistently positive EBV response in 3 or more determinations and 34 (33.3%) had transient episodes of nonresponsiveness. Ninety-five (48.2%) patients were persistently EBV nonresponders. EBV-DNAemia data were available for 58 patients: 27.6% presented at least one episode of EBV-DNA occurrence. No differences were found in EBV-EliSpot response stratification between the groups of patients who experienced episodes of EBV reactivation and those without EBV-DNAemia. However, EBV DNAemia peak values tended to be higher in the first year post-transplantation in the group of patients with a persistent positive EBV-specific immune response. EBV viral load quantitation in blood and EliSpot EBV-specific immune response determination may represent a powerful tool for monitoring solid organ transplant recipients, guiding immunosuppression modulation in patients with active EBV replication.

Clinical impact of HSV-1 detection in the lower respiratory tract from hospitalized adult patients.

The occurrence and clinical impact of herpes simplex virus (HSV) were evaluated in 342 bronchoalveolar lavage specimens from 237 patients. HSV-1 and HSV-2 were detected in 32.1% and <1% of patients, respectively. A significant difference of HSV-1 prevalence and load was found in relation to admission to intensive care unit, mechanical ventilation and mortality within 28 days; in particular, a viral load ≥10(5) copies/mL bronchoalveolar lavage fluid was significantly associated with critical features. No association was found with immune status or other characteristics. Nine of 21 (42.9%) cases of ventilator-associated pneumonia were positive for HSV-1, with poor outcome in six.

The lack of cytomegalovirus-specific cellular immune response may contribute to the onset of organ infection and disease in lung transplant recipients.

Cellular immune response has been demonstrated to play a role in the control of human cytomegalovirus (HCMV) replication in organ transplant recipients. Herein, HCMV-specific T-cell response and association to the onset of organ infection/disease were prospectively evaluated by EliSPOT assay in a population of 46 lung transplant (LT) recipients at 1, 3, 6, 9 and 12 months post-transplantation. According to our centre?s practice, a combined prolonged antiviral prophylaxis (HCMV-IG for 12 months and ganciclovir or valganciclovir for 3 weeks from postoperative day 21) was given to all LT recipients. HCMV-DNA was concomitantly detected on bronchoalveolar lavage (BAL) and whole blood by real-time PCR. Approximately one third of patients resulted HCMV persistently non-responder; the rate of HCMV infection, as evaluated by HCMV-DNA positivity, tended to be higher in non-responders. Mean viral load on BAL was significantly higher in non-responders vs other patients (p < 0.001). Temporal profile of infections appeared related to the HCMV responder status with a shorter time to onset of infection post-transplantation and a longer duration in non-responders. The occurrence of organ disease (i.e. pneumonia) tended to be higher in non-responders, with poor prognosis, as death occurred in one of three non-responder patients that developed HCMV pneumonia. The lack of HCMV-specific cellular response can contribute to the onset of organ infection and disease also in patients in which antiviral prophylaxis was adopted; this could be due to the potential occurrence of incomplete control of replication in lungs or a delayed priming of T-cell reconstitution.

Lower respiratory tract viral infections in hospitalized adult patients.

AIM: The epidemiology of lower respiratory tract (LRT) viral infections in adults is probably underestimated and the high frequency of multiple viral infections complicates the evaluation of the possible role of the single viruses. The aim of this study was to investigate the clinical epidemiology and impact of respiratory viral pathogens, in particular of those singularly detected, in bronchoalveolar lavage (BAL) specimens from hospitalized adult patients. METHODS: A panel for the detection of 16 respiratory viruses was used to prospectively evaluate 324 consecutive specimens obtained from 219 patients over a full-year period. RESULTS: Two-hundred-twenty-one specimens (68.2%) were positive for at least one virus, 119/324 (36.7%) to a single viral agent. The most commonly detected viruses were herpesviruses HHV-7 (26.2%), human cytomegalo-virus (HCMV, 22.2%), HHV-6 (19.8%), EBV (12.7%), enteroviruses and rhinoviruses (both 11.7%), parainfluenza viruses (4.9 %), and metapneumovirus (4.0%). Human cytomegalo-virus was significantly more prevalent as single viral pathogen with a viral load >105 copies/ml associated to pneumonia in solid organ transplant recipients. Other viral pathogens might account for some cases of pneumonia or respiratory insufficiency, although multiple infections were common. CONCLUSIONS: The use of a comprehensive diagnostic panel for respiratory viral infections may be useful to clarify the epidemiology and clinical impact of viral pathogens in hospitalized adult patients. The occurrence of multiple infections is a common finding and results should be interpreted taking into account the clinical context as well as viral load and the biological characteristics of each virus.

Induction of B1 bradykinin receptors in the kindled hippocampus increases extracellular glutamate levels: a microdialysis study.

A link between temporal lobe epilepsy (the most common epileptic syndrome in adults) and neuropeptides has been established. Among neuropeptides, the possible involvement of bradykinin has recently received attention. An autoradiographic analysis has shown that B1 receptors, which are physiologically absent, are expressed at high levels in the rat brain after completion of kindling, a model of temporal lobe epilepsy. Thus, the present work aimed at investigating the functional implications of this observation, by studying the effect of B1 receptor activation on extracellular glutamate levels in the kindled hippocampus. Microdialysis experiments have been performed in two groups of rats, control and kindled. Glutamate outflow has been measured under basal conditions and after chemical stimulation with high K+ (100 mM in the dialysis solution). Basal glutamate outflow in kindled animals was significantly higher than in controls. High K+-evoked glutamate outflow was also more pronounced in kindled animals, consistent with the latent hyperexcitability of the epileptic tissue. The B1 receptor agonist Lys-des-Arg9-BK induced an increase of basal and high K+-evoked glutamate outflow in kindled but not in control rats, and the selective B1 receptor antagonist R-715 prevented both these effects. Furthermore, R-715 significantly reduced high K+-evoked glutamate outflow when applied alone. These data suggest that the bradykinin system contributes to the modulation of epileptic neuronal excitability through B1 receptors.

Cytotoxic T-lymphocyte responses in melanoma through in vitro stimulation with the Melan-A peptide analogue A27L: a qualitative analysis.

Modifications in tumour antigen-derived epitopes that stabilize the major histocompatibility complex (MHC)-peptide complex result in enhanced stimulatory capacity and improved immunogenicity of the altered peptide. These epitope analogues are attractive candidates for the development of peptide-based vaccine trials. Any modification, however, in tumour antigens may induce T-cell responses that could either fail to react against the naturally occurring peptides or represent only a subset of the total antigen-specific repertoire. In the present study, we performed a critical analysis of the ability of cytotoxic T-lymphocyte (CTL) clones, derived from two melanoma patients through stimulation with the A27L peptide analogue, to cross-react with the naturally processed Melan-A/MART-1 (Melan-A) peptides in terms of T-cell receptor (TCR) affinity, functional avidity and fine antigen specificity. We found that all the A27L-specific clones analysed possessed a very low avidity for the natural Melan-A peptides, and that their binding affinity for human leukocyte antigen (HLA) tetramers complexed with both the modified and the natural Melan-A peptides did not strictly correlate with their functional avidity. We also observed that these clones were able to cross-recognize both natural Melan-A peptides in one patient, but only one peptide in the second patient. We discuss the capability of the A27L peptide analogue to stimulate all the available Melan-A-specific repertoire.

Specific cytotoxic T lymphocyte responses against Melan-A/MART1, tyrosinase and gp100 in vitiligo by the use of major histocompatibility complex/peptide tetramers: the role of cellular immunity in the etiopathogenesis of vitiligo.

Vitiligo is a common skin disease characterized by the presence of well circumscribed, depigmented, milky white macules devoid of identifiable melanocytes. Although the detection of circulating anti-melanocytic antibodies and of infiltrating lymphocytes at the margin of lesions supports the view that vitiligo is an autoimmune disorder, its etiology remains unknown. In particular, it is still a matter of debate whether the primary pathogenic role is exerted by humoral or cellular abnormal immune responses. In this study, the presence of specific cytotoxic T lymphocyte responses against the melanocyte differentiation antigens Melan-A/MART1, tyrosinase, and gp100 in vitiligo patients have been investigated by the use of major histocompatibility complex/peptide tetramers. High frequencies of circulating melanocyte-specific CD8+ T cells were found in all vitiligo patients analyzed. These cells exerted anti-melanocytic cytotoxic activity in vitro and expressed skin-homing capacity. In one patient melanocyte-specific cells were characterized by an exceptionally high avidity for their peptide/major histocompatibility complex ligand. These findings strongly suggest a role for cellular immunity in the pathogenesis of vitiligo and impact on the common mechanisms of self tolerance.

Mutation of somatostatin receptor type 5 in an acromegalic patient resistant to somatostatin analog treatment.

Introduction of somatostatin analogs has greatly contributed to improving the prognosis of acromegaly. Although the majority of patients are effectively treated by these agents, resistance occurs in a subset of patients. So far, resistance to somatostatin has never been associated with mutations of the somatostatin receptor subtypes (sst2 and sst5) that inhibit GH secretion. Molecular analysis of genomic DNA from pituitary tumor and peripheral blood obtained from an acromegalic resistant to octreotide showed a somatic activating mutation of Gsalpha (Arg201Cys), no mutation in sst2, and one polymorphism (Pro109Ser) and one germ line mutation (Arg240Trp) in sst5. Wild-type (WT) and mutant sst5 PCR products were cloned and transfected into Chinese hamster ovary K1 cells. In Chinese hamster ovary K1 cells stably expressing mutant sst5, somatostatin-28 was less potent in inhibiting cyclic AMP levels than in WT cells. Proliferation of mutant cells exceeded that of WT by 50%. Moreover, somatostatin reduced cell growth and MAPK activity in WT but not in mutant cells in which the peptide even increased MAPK activity. We suggest that this mutation that abrogates the antiproliferative action of somatostatin and activates mitogenic pathways may be involved in the resistance to somatostatin treatment.

Kinetics of GATA-3 gene expression in early polarizing and committed human T cells.

Different transcription factors have been shown to control the transition of naive T cells into T helper 1 (Th1)/Th2 subsets. The T-cell-specific transcription factor GATA-3 is known to be selectively expressed in murine developing Th2 cells and to exert a positive action on Th2-specific cytokine production. Investigating GATA-3 gene regulation in human T cells we have found that naive T cells highly express GATA-3, and during early T2 or T1 polarization, respectively, they either maintain or quickly down-regulate expression. In developing T2 cells, as well as in committed Th2 cell lines and clones, we found a positive correlation among GATA-3, interleukin (IL)-5 and IL-4 gene expression kinetics, supporting the positive action of GATA-3 on Th2-specific cytokine production. A possible relationship between GATA-3 gene expression and the down-regulation of the IL-12 receptor (beta2-chain; IL-12Rbeta2) gene was evident only in the early phases of T2 polarization (within 24 hr), and not demonstrated at later times. During T-cell commitment the presence of IL-4 in the culture was essential to maintain or enhance GATA-3 transcription, while IL-12 was not necessary for full repression of GATA-3. Finally, we showed selective GATA-3 up-regulation in human Th2 cell lines and clones and the maintainance of a low basal level of GATA-3 expression in Th1 cells upon activation.