Gene- and Gender-Related Decrease in Serum BDNF Levels in Alzheimer's Disease.

Brain-derived neurotrophic factor (BDNF) has a protective role in Alzheimer's disease (AD). Oxidative stress and inflammatory cytokines are potentially implicated in AD risk. In this study, BDNF was detected in serum of AD and mild cognitive impairment (MCI) patients and investigated in association with gene polymorphisms of BDNF (Val66Met and C270T), of some oxidative stress-related genes (FOXO3A, SIRT3, GLO1, and SOD2), and of interleukin-1 family genes (IL-1α, IL-1ß, and IL-38). The APOE status and mini-mental state examination (MMSE) score were also evaluated. Serum BDNF was significantly lower in AD (p = 0.029), especially when comparing the female subsets (p = 0.005). Patients with BDNFVal/Val homozygous also had significantly lower circulating BDNF compared with controls (p = 0.010). Moreover, lower BDNF was associated with the presence of the T mutant allele of IL-1α(rs1800587) in AD (p = 0.040). These results were even more significant in the female subsets (BDNFVal/Val, p = 0.001; IL-1α, p = 0.013; males: ns). In conclusion, reduced serum levels of BDNF were found in AD; polymorphisms of the IL-1α and BDNF genes appear to be involved in changes in serum BDNF, particularly in female patients, while no effects of other gene variants affecting oxidative stress have been found. These findings add another step in identifying gender-related susceptibility to AD.

Breast Cancer Is Associated with Increased HLA-DRB1*11:01 and HLA-DRB1*10:01 Allele Frequency in a Population of Patients from Central Italy.

The relationship between HLA-DRB1 allele polymorphism and breast cancer (BC) development is still unclear and needs further investigation. To address this issue, we analyzed HLA-DRB1 allele frequency (AF) by sequence-based typing (SBT) in 47 patients from central Italy with BC and 156 sex and age-matched healthy controls. Two hundred ninety-seven individuals from the same region were utilized as historical controls. Pearson's chi-square analysis with Yate's correction or Fisher's Exact test with Bonferroni's correction, as appropriate, were used to compare HLA-DRB1 AF differences in patients and controls. A total of 36 HLA-DRB1 alleles were identified. A detailed study showed that HLA-DRB1*11:01 and HLA-DRB1*10:01 alleles are significantly associated with increased BC risk. In particular, HLA-DRB1*11:01 AF was significantly higher in patients with BC than in healthy females and historical controls, even following Bonferroni's correction (stage I-II BC patients vs historical controls p<0.00; stage III-IV BC patients vs female healthy controls p=0.025 and historical controls p<0.00). The HLA-DRB1*10:01 allele was also positively associated with BC as evidenced by a significantly higher AF in patients with BC than in healthy controls (BC patients stage I-II vs historical controls corrected p =0.01). These results suggest that both HLA-DRB1*11:01 and HLA-DRB1*10:01 AF could represent interesting markers in patients at risk of developing BC.

Role of KIR and CD16A genotypes in colorectal carcinoma genetic risk and clinical stage.

BACKGROUND: NK cell cytotoxicity is regulated by the types of the interaction between killer immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) class I ligands on target cells and the different binding affinity of the Fcγ receptor IIIA (CD16A) for IgG-coated tumor cells. Thus, it is conceivable that KIR and CD16A gene contents may contribute to the function of NK cells by modulating an immune response in the colorectal carcinoma (CRC) microenvironment. This hypothesis is supported by recent evidence suggesting that NK cells improve the clinical course of CRC patients by enhancing the anti-CRC effect of CD8 + T cells. This information provides the rationale to test the hypothesis whether the independent KIR segregation and specificity, as well as CD16A gene polymorphisms, have an impact on CRC. METHODS: Using polymerase chain reaction-sequence-specific primers (PCR-SSP) and sequence-based typing (SBT), we investigated KIR/HLA-C complex and CD16A (48H/R/L,158V/F) gene polymorphisms in 52 CRC patients and 61 local healthy controls (LCTRs). RESULTS: The allele frequency (AF) of at least five activating KIR (aKIRs) of the B haplotype (p = 0.036, OR 0.204), KIR2DL2 (p = 0.047, OR 0.2616), and KIR2DS2 genes (5.8 vs LCTR 13.8 % and vs. Fasano's CTR 16.3 %, p = 0.05, OR 0.3145), in the absence of their cognate HLA-C1 ligands, were significantly associated with a reduced genetic risk of CRC. In contrast, CD16A-48H polymorphism was positively associated with an increased genetic risk of CRC (p = 0.05, OR 2.761). The latter was also found to be correlated with advanced stages of disease [III and IV (p = 0.03, OR 3.625)]. CONCLUSIONS: Our data suggest that the analysis of aKIRs and KIR2DL2 gene and CD16A-48H may be of interest for the identification of individuals at reduced and increased genetic risk of CRC, respectively.

HLA-DRB1*13:01 allele in the genetic susceptibility to colorectal carcinoma.

Increasing evidence suggests that HLA-DRB1 alleles reduce or increase the risk of developing ulcerative colitis-associated colorectal carcinoma (CRC) tumors. However, the role of HLA-DRB1 locus on the susceptibility to develop CRC tumor, in the absence of a history of inflammatory bowel diseases (IBDs), is unclear. The aim of our study was to determine whether HLA-DRB1 alleles are associated with IBD-independent CRC tumor. HLA-DRB1 allele polymorphisms were identified by sequence-based typing method in 53 CRC patients and 57 sex- and age-matched healthy Caucasian controls. Pearson's chi-squared analysis with Yate's correction or Fisher's exact test with Bonferroni's correction, as appropriate, were used to compare the allele frequency (AF) differences of HLA-DRB1 in patients and controls. A total of 29 HLA-DRB1 alleles were recognized. A detailed study of these alleles allowed to identify DRB1*13:01 and DRB1*11:01 alleles that were significantly associated with an increased and reduced risk to develop CRC tumor, respectively. AF of DRB1*13:01, in CRC patients, was significantly higher than that of healthy controls, even following Bonferroni's correction (p = 0.029). In contrast, the presence of the DRB1*11:01 allele was negatively associated with CRC tumor as evidenced by the significantly lower AF in CRC patients than that of healthy controls (p = 0.005). However, following Bonferroni's correction, the AF of DRB*11:01 lost its statistical significance. These results suggest that HLA-DRB1*13:01 allele could be a potential marker for predicting genetic susceptibility to CRC tumor. In contrast, the protective role of DRB1*11:01 remains unclear.

Aggressiveness in Italian Children with ADHD: MAOA Gene Polymorphism Involvement.

ADHD is a neurodevelopmental disorder that children and adults can develop. A complex interplay of genetic and environmental factors may underlie interindividual variability in ADHD and potentially related aggressive behavior. Using high-resolution molecular biology techniques, we investigated the impact of some MAOA and SLC6A4 variations on ADHD and aggressive behavior in a group of 80 Italian children with ADHD and in 80 healthy controls. We found that homozygous genotypes of MAOA rs6323 and rs1137070 were associated with an increased risk of ADHD (p = 0.02 and p = 0.03, respectively), whereas the heterozygous genotypes (GT of rs6323 and CT of rs1137030) (p = 0.0002 and p = 0.0006) were strongly linked to a lower risk of developing this disorder. In patients with aggressive behavior, we highlighted only a weak negative association of both MAOA polymorphisms (heterozygous genotypes) with aggressiveness, suggesting that these genotypes may be protective towards specific changes in behavior (p = 0.05). Interestingly, an increase in the GG genotype of rs6323 (p = 0.01) and a decrease in GT genotype (p = 0.0005) was also found in patients without aggressive behavior compared to controls. Regarding 5HTT gene genotyping, no allele and genotype differences have been detected among patients and controls. Our work shows that defining a genetic profile of ADHD may help in the early detection of patients who are more vulnerable to ADHD and/or antisocial and aggressive behavior and to design precision-targeted therapies.

Acute Lymphoblastic Leukemia Immunotherapy Treatment: Now, Next, and Beyond.

Acute lymphoblastic leukemia (ALL) is a blood cancer that primarily affects children but also adults. It is due to the malignant proliferation of lymphoid precursor cells that invade the bone marrow and can spread to extramedullary sites. ALL is divided into B cell (85%) and T cell lineages (10 to 15%); rare cases are associated with the natural killer (NK) cell lineage (<1%). To date, the survival rate in children with ALL is excellent while in adults continues to be poor. Despite the therapeutic progress, there are subsets of patients that still have high relapse rates after chemotherapy or hematopoietic stem cell transplantation (HSCT) and an unsatisfactory cure rate. Hence, the identification of more effective and safer therapy choices represents a primary issue. In this review, we will discuss novel therapeutic options including bispecific antibodies, antibody-drug conjugates, chimeric antigen receptor (CAR)-based therapies, and other promising treatments for both pediatric and adult patients.

The Role of CD1 Gene Polymorphism in the Genetic Susceptibility to Spondyloarthropathies in the Moroccan Population and the Possible Cross-Link with Celiac Disease.

Spondyloarthropathies (SpA) are a group of chronic inflammatory disorders usually affecting the axial spine and asymmetrical peripheral joints. Strong evidence links genetic and environmental factors to SpA pathogenesis. The HLA-B27 is the most important genetic factor associated with SpA. Nevertheless, the involvement of other HLA and non-HLA loci has been also reported. Some patients with SpA may also manifest features of celiac disease (CeD), thus suggesting a genetic overlap across these autoimmune diseases. Recently, CD1 glycoproteins, a class of molecules able to bind and present non peptidic antigens to T cells, aroused interest for their contribution to the pathogenesis of CeD. Therefore, to evaluate whether functional polymorphisms of CD1A and E genes also influence susceptibility to SpA, we analyzed 86 patients from Morocco affected by SpA and 51 healthy controls, using direct sequencing analysis. An increase of CD1E*01/01 homozygous genotype (p = 0.046) was found in SpA, compared with controls. CD1E*01/01 genotype was associated particularly to patients with sacroiliac joints/spine/peripheral joints pain (p = 0.0068), while a decrease of CD1E*01/02 genotype was evidenced compared to controls (p = 0.0065). Results from haplotypes analysis demonstrated that CD1A*02-E*02 decreased the risk of SpA, while CD1A*02-E*01 increased risk to develop disease. Our data indicate a relationship between CD1 genes and susceptibility to SpA in the Moroccan population and suggest the existence of shared genetic risk loci across SpA and CeD that might be useful to explain common pathogenetic features and define novel therapeutic strategies.

Impact of HLA Class I Antigen, Killer Inhibitory Receptor, and FCGR3A Genotypes on Breast Cancer Susceptibility and Tumor Stage.

BACKGROUND: The identification in breast cancer (BC) of novel genetic biomarkers regulating natural killer (NK) cell function, including the HLA, KIR, and CD16A (FCGR3A), may be still a challenge. OBJECTIVE: We aimed to evaluate whether the combined effect of these polymorphisms has an impact on BC susceptibility and progression. METHODS: 47 BC Italian patients and healthy individuals (39 females and 66 males/females) were genotyped by Sanger sequencing (HLA-C exon 2-4 and FCGR3A-158V/F, 48L/R/H) and PCR-SSP typing (KIR genes). RESULTS: HLA-C gene allele analysis showed the group C1, with HLA-C*07:02:01 allele, to be significantly associated with tumor progression (16.7% vs. 4.0%, p=0.04, OR=4.867), and instead, group C2, with HLA-C*05:01:01, was protective against disease susceptibility (0.0% vs. 7.2%, p=0.019, OR=0.087). In addition, we highlighted a significant reduction of the KIR2DS4ins in BC patients (pcorr.=0.022) and an increased combined presence of KIR2DL1 and KIR2DS1 genes in advanced BC patients compared to earlier stages (66.7% vs. 19.2%, p=0.002). The concurrent lack of KIR2DL2 and KIR2DS4 genes in the presence of HLA-C2 alleles was significantly associated with increased susceptibility to BC (p=0.012, OR=5.020) or with lymph node involvement (p=0.008, OR=6.375). Lastly, we identified different combinations of the FCGR3A-48/158 variants and KIR genes in BC patients compared to controls. CONCLUSION: Our findings suggest that in the development of BC probably exists a disorder of the NK innate immunity influenced by KIR/HLA-C gene content and FCGR3A-158 polymorphisms and that the combined analysis of these biomarkers might help predict genetic risk scores for tailored screening of BC patients in therapy.

Highlights on the Role of Galectin-3 in Colorectal Cancer and the Preventive/Therapeutic Potential of Food-Derived Inhibitors.

Colorectal cancer (CRC) is a leading cause of death worldwide. Despite advances in surgical and therapeutic management, tumor metastases and resistance to therapy still represent major hurdles. CRC risk is highly modifiable by lifestyle factors, including diet, which strongly influences both cancer incidence and related mortality. Galectin-3 (Gal-3) is a multifaceted protein involved in multiple pathophysiological pathways underlying chronic inflammation and cancer. Its versatility is given by the ability to participate in a wide range of tumor-promoting processes, including cell-cell/cell-matrix interactions, cell growth regulation and apoptosis, and the immunosuppressive tumor microenvironment. This review provides an updated summary of preclinical and observational human studies investigating the pathogenetic role of Gal-3 in intestinal inflammation and CRC, as well as the potential of Gal-3 activity inhibition by plant-source food-derived bioactive compounds to control CRC onset/growth. These studies highlight both direct and immuno-mediated effects of Gal-3 on tumor growth and invasiveness and its potential role as a CRC prognostic biomarker. Substantial evidence indicates natural food-derived Gal-3 inhibitors as promising candidates for CRC prevention and therapy. However, critical issues, such as their bioavailability and efficacy, in controlled human studies need to be addressed to translate research progress into clinical applications.

Direct CD32 T-cell cytotoxicity: implications for breast cancer prognosis and treatment.

The FcγRII (CD32) ligands are IgFc fragments and pentraxins. The existence of additional ligands is unknown. We engineered T cells with human chimeric receptors resulting from the fusion between CD32 extracellular portion and transmembrane CD8α linked to CD28/ζ chain intracellular moiety (CD32-CR). Transduced T cells recognized three breast cancer (BC) and one colon cancer cell line among 15 tested in the absence of targeting antibodies. Sensitive BC cell conjugation with CD32-CR T cells induced CD32 polarization and down-regulation, CD107a release, mutual elimination, and proinflammatory cytokine production unaffected by human IgGs but enhanced by cetuximab. CD32-CR T cells protected immunodeficient mice from subcutaneous growth of MDA-MB-468 BC cells. RNAseq analysis identified a 42 gene fingerprint predicting BC cell sensitivity and favorable outcomes in advanced BC. ICAM1 was a major regulator of CD32-CR T cell-mediated cytotoxicity. CD32-CR T cells may help identify cell surface CD32 ligand(s) and novel prognostically relevant transcriptomic signatures and develop innovative BC treatments.

Immunotherapy as a Turning Point in the Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a malignant disease of hematopoietic precursors at the earliest stage of maturation, resulting in a clonalproliferation of myoblasts replacing normal hematopoiesis. AML represents one of the most common types of leukemia, mostly affecting elderly patients. To date, standard chemotherapy protocols are only effective in patients at low risk of relapse and therapy-related mortality. The average 5-year overall survival (OS) is approximately 28%. Allogeneic hematopoietic stem cell transplantation (HSCT) improves prognosis but is limited by donor availability, a relatively young age of patients, and absence of significant comorbidities. Moreover, it is associated with significant morbidity and mortality. However, increasing understanding of AML immunobiology is leading to the development of innovative therapeutic strategies. Immunotherapy is considered an attractive strategy for controlling and eliminating the disease. It can be a real breakthrough in the treatment of leukemia, especially in patients who are not eligible forintensive chemotherapy. In this review, we focused on the progress of immunotherapy in the field of AML by discussing monoclonal antibodies (mAbs), immune checkpoint inhibitors, chimeric antigen receptor T cells (CAR-T cells), and vaccine therapeutic choices.

CD1 gene polymorphism and susceptibility to celiac disease: Association of CD1E*02/02 in Moroccans.

CD1 glycoproteins are a class of antigen presenting molecules that bind and present non-peptidic antigens (lipids and glycolipids) for immune recognition. CD1 polymorphisms, although limited, could have a critical role in antimicrobial, anticancer, and autoimmune responses and disease susceptibility. Ethnic differences and interactions between genetic and environmental factors make it attractive the study of these molecules in autoimmune inflammatory disorders, such as celiac disease (CD), in which a strong genetic predisposition (HLA-DQ2/DQ8) and pressure of environmental factors have a central role. CD1A, CD1D and CD1E polymorphisms in exon 2 were assessed in patients from Morocco affected by CD, using direct sequencing analysis, in order to investigate possible associations with the disease in a North African population. Differences in genotype and haplotype distribution of CD1E between celiac patients and controls were found: in particular, an increase of CD1E*02/02 homozygous (OR 2.93, CI 1.30-6.59, p = 0.007) and CD1A*02-E*02 estimated haplotypes in CD, compared with controls. Frequencies of CD1A and CD1D genotypes/alleles were not different between groups. CD1E*02/02, previously suggested as a potential immune protective genotype to malaria susceptibility, could be an additional gene involved in celiac risk in this geographic area.

Redox, immune and genetic biomarker system for personalized treatments in colorectal cancer.

BACKGROUND: Identifying biomarkers for the risk of developing degenerative processes linked to aging and colorectal cancer (CRC) onset that could improve clinical strategies. AIM: To determine valid targets and a predictive biomarker's system of chronicization of inflammation for cancer treatment. METHODS: A group of 147 CRC patients was studied. Clinical diagnosis was confirmed histopathologically, and patients were sub-typed using the pathological tumor-node-metastasis classification. Thirteen colon adenoma patients and 219 healthy subjects were also studied. A system biology study on Thioredoxin1/CD30 redox-immune systems (Trx1/CD30), T helper cytokines and polymorphisms of killer immunoglobulin-like receptors, FcγRIIa-131H/R and FcγRIIIa-158V/F was carried out. Enzyme-linked immunosorbent assay was performed to analyze sera. Genetic study was executed by polymerase chain reaction sequence-specific primers and sequence-based typing method. Statistical analysis was performed by using the "Statgraphics software systems". RESULTS: We found a positive increase between Trx1/RTrx1 levels and sCD30 level and increased age. With respect to the gender relationships, there were distinct differences. Females showed a primary relationship between transforming growth factor beta (TGFß) with Trx1, whereas males had one with TGFß and RTrx1. Trx1/CD30 controls the redox immune homeostasis, and an imbalance in the relationship between the Trx1/RTrx1 and sCD30 levels is linked to the onset and progression of tumor. This event happens through different gender-specific cytokine pathways. Our study demonstrated that the serum levels of Trx1/RTrx1, TGFß/interleukin (IL)6 and TGFß/IL4 combinations and the sCD30, IFNγ and IL2 combination constitute a predictive gender specific biomarker system. This is relevant for clinical screening to detect the risk of the potential development or progression of a tumor. CONCLUSION: Oxidative stress on Trx1/CD30 is a trigger of cancer disease, and the selected oxidation and immune products are a biomarker system for aging and cancer.

Susceptibility to Guillain-Barré syndrome is associated to polymorphisms of CD1 genes.

Guillain-Barré syndrome (GBS) is the prototype of a postinfectious autoimmune neuropathy. Molecular mimicry between glycolipid antigens expressed by an infective antigen such as Campylobacter jejuni and the human peripheral nerve has been hypothesized to be the causative mechanism of GBS. However, only 1/1000 of C. jejuni enteritis develops GBS. This emphasizes the importance of host-related factors in the development of the disease. HLA studies in GBS failed to show an association or gave conflicting results but MHC class I and II process and present peptides to T lymphocytes making unlikely that the HLA system plays a role in GBS with autoantibodies against self-gangliosides. CD1 molecules are MCH-like glycoproteins specialized in capturing and presenting a variety of glycolipid to antigen-specific T cells. There are five closely linked CD1 genes in humans located in chromosome 1 (named CD1A, B, C, D, and E) all showing limited polymorphism in exon 2 which codifies for the alpha1 domain of CD1 molecules. The nucleotide substitutions in CD1B and CD1C are rare and reported to be silent. In 100 controls and 65 GBS patients (21 with a recent C. jejuni infection and 35 with anti-glycolipid antibodies) we used direct sequencing by polymerase chain reaction to genotype exon 2 of CD1A, CD1D and CD1E genes. CD1D is monomorphic in both controls and patients whereas CD1A and CD1E are biallelic in exon 2. Subjects with CD1E*01/01 genotype are 2.5 times more likely to develop GBS, whereas subjects with CD1A*01/02 or CD1E*01/02 have a reduced relative risk by 3.6 and 2.3 times respectively. The combination of CD1A*01/02 and CD1E*01/02 reduces by 5 times the risk of developing GBS. Although a correlation between CD1E*01/01 genotype and recent C. jejuni infection or presence of antiganglioside antibodies was not found the overall findings indicate that susceptibility to develop GBS is associated with polymorphisms of CD1E and CD1A genes.

CD1A, D and E gene polymorphisms in a North African population from Morocco.

CD1 molecules are specialized in capturing and presenting lipids and glycolipids to distinct subsets of T and NKT cells. Glycolipid presentation could play a significant role in the immune response against microbial infections. There are five closely linked CD1 genes in humans, named CD1A, B, C, D, and E, which all show a limited polymorphism. In this study, exon 2 polymorphisms of CD1A, CD1D and CD1E were investigated and allele, genotype and haplotype frequencies of these loci were reported in a Moroccan population. A comparison with allele, genotype and haplotype frequencies observed in other geographic areas was also performed. Results confirmed the presence of ethnic differences in CD1 polymorphism, mainly in CD1D (in this population two additional CD1D variant alleles, CD1D(∗)03 and CD1D(∗)04, were described) and E genes. These data could be useful to evaluate a possible pathogenetic role of CD1 in diseases. Increasing the knowledge in this field may offer possibilities for the development of new immunotherapeutic approaches.

Sequence-Based analysis of the HLA-DRB1 polymorphism in Metalsa Berber and Chaouya Arabic-speaking groups from Morocco.

To examine the genetic diversity in Morocco, the polymorphism at the HLA-DRB1 locus was investigated in two populations: the Metalsa group consisting of Berbers from north Morocco (who speak the Tarifit language and live in the Nador area), and the Chaouya group who are Arabic-speaking people from west Morocco (Atlantic coast) living in the Settat area. The DRB1 alleles of 197 healthy unrelated individuals were identified by direct DNA sequencing of exon 2 using fluorescently-labeled primers. A total of 28 and 29 alleles at DRB1 locus were identified in the Metalsa and Chaouya groups, respectively. The most frequent alleles in the Metalsa group are DRB1*03011 (20.2%), DRB1*0701 (12.12%), and DRB1*1302 (11.11%). In the Chaouya group, DRB1*0701 (16.33%), DRB1*15011 (12.76%), and DRB1*03011 (11.73%) are most common. Each population exhibits some specific variants and some uncommon alleles. The frequency of the DRB1*03011 allele differs significantly between the two populations (p = 0.0311). The DRB1 frequency distributions in the two groups suggest the effects of balancing selection. The interpopulation analysis highlighted a strong relatedness, based on genetic distances, between the two Moroccan groups and the other north Africans (the Moroccans from El Jadida area, Moroccan Souss Berbers, Algerians, and Tunisians), and to a lesser extent with the Iberians, French, and Ethiopians.

Chimerism in a child with severe combined immunodeficiency: a case report.

Severe combined immunodeficiency (SCID) represents a group of rare, sometimes fatal, congenital disorders in which there is a combined absence of T-lymphocyte and B-lymphocyte function. Children with SCID die within two years of age, if untreated. The effective treatment for SCID is a hematopoietic stem cell transplantation (HSCT). It has been repeatedly described that in peripheral blood of infants with SCID maternal T cells can be found. Here we report a case of blood chimerism in a one-year-old boy with SCID.

Artificial organs as a bridge to transplantation.

Current organ shortage is estimated to keep outpacing demand for years to come. Among the advocated strategies, artificial and bioartificial devices may prove beneficial to a wide category of patients on transplant waiting lists. Bionic organ science allows to reproduce organ architecture and function through a complex interplay of cellular and mechanical elements. Some bioartificial organs may well be used to replace anatomical defects, while others allow to compensate for failing organ functions and to bridge patients to transplantation. Among these latter, bioartificial liver (BAL) systems bear the highest potential for clinical application, even if their use is raising several controversial issues. These latter regard the identification and stratification of patients fit for transplantation, timing and type of transplantation after recovery, appropriateness of double-blind, randomized clinical trials and safety of animal and/or human cell lines. Nonetheless, bionic organ science needs to be regarded as a useful adjunct in the armamentarium of organ replacement therapies for the third millennium.