Isolation and expansion of thymus-derived regulatory T cells for use in pediatric heart transplant patients

Regulatory T-cells (Tregs) are a subset of T cells generated in the thymus with intrinsic immunosuppressive properties. Phase I clinical trials have shown safety and feasibility of Treg infusion to promote immune tolerance and new studies are ongoing to evaluate their efficacy. During heart transplantation, thymic tissue is routinely discarded providing an attractive source of Tregs. In this study, we developed a GMP-compatible protocol for expanding sorted thymus-derived CD3⁺CD4⁺CD25⁺CD127^– (Tregs) as well as CD3⁺CD4⁺CD25⁺CD127^–CD45RA⁺ (RA⁺Tregs) cells. We aimed to understand whether thymic RA⁺Tregs can be isolated and expanded offering an advantage in terms of stability as it has been previously shown for circulating adult CD45RA⁺ Tregs. We show that both Tregs and RA⁺Tregs could be expanded in large numbers and the presence of rapamycin is essential to inhibit the growth of IFN-γ producing cells. High levels of FOXP3, CTLA4, and CD25 expression, demethylation of the FOXP3 promoter, and high suppressive ability were found with no differences between Tregs and RA⁺Tregs. After freezing and thawing, all Treg preparations maintained their suppressive ability, stability, as well as CD25 and FOXP3 expression. The number of thymic Tregs that could be isolated with our protocol, their fold expansion, and functional characteristics allow the clinical application of this cell population to promote tolerance in pediatric heart transplant patients.     

Development and characterization of a new chitosan-based scaffold associated with gelatin,microparticulate dentin and genipin for endodontic regeneration

ObjectiveAn ideal scaffold for endodontic regeneration should allow the predictableness of the new tissue organization and limit the negative impact of residual bacteria. Therefore, composition and functionalization of the scaffold play an important role in tissue bioengineering. The objective of this study was to assess the morphological, physicochemical, biological and antimicrobial properties of a new solid chitosan-based scaffold associated with gelatin, microparticulate dentin and genipin.MethodsScaffolds based on chitosan (Ch); chitosan associated with gelatin and genipin (ChGG); and chitosan associated with gelatin, microparticulate dentin and genipin (ChGDG) were prepared by using the freeze-drying method. The morphology of the scaffolds was analyzed by scanning electron microscopy (SEM). The physicochemical properties were assessed for biodegradation, swelling and total released proteins. The biological aspects of the scaffolds were assessed using human cells from the apical papilla (hCAPs). Cell morphology and adhesion to the scaffolds were evaluated by SEM, cytotoxicity and cell proliferation by MTT reduction-assay. Cell differentiation in scaffolds was assessed by using alizarin red assay. The antimicrobial effect of the scaffolds was evaluated by using the bacterial culture method, and bacterial adhesion to the scaffolds was observed by SEM.ResultsAll the scaffolds presented porous structures. The ChCDG had more protein release, adhesion, proliferation and differentiation of hCAPs, and bacteriostatic effect on Enterococcus faecalis than Ch and ChGG (p < 0.05).SignificanceThe chitosan associated with gelatin, microparticulate dentin and genipin has morphological, physicochemical, biological and antibacterial characteristics suitable for their potential use as scaffold in regenerative endodontics.     

Doubly heterozygous LMNA and TTN mutations revealed by exome sequencing in a severe form of dilated cardiomyopathy

Familial dilated cardiomyopathy (DCM) is a heterogeneous disease; although 30 disease genes have been discovered, they explain only no more than half of all cases; in addition, the causes of intra-familial variability in DCM have remained largely unknown. In this study, we exploited the use of whole-exome sequencing (WES) to investigate the causes of clinical variability in an extended family with 14 affected subjects, four of whom showed particular severe manifestations of cardiomyopathy requiring heart transplantation in early adulthood. This analysis, followed by confirmative conventional sequencing, identified the mutation p.K219T in the lamin A/C gene in all 14 affected patients. An additional variant in the gene for titin, p.L4855F, was identified in the severely affected patients. The age for heart transplantation was substantially less for LMNA:p.K219T/TTN:p.L4855F double heterozygotes than that for LMNA:p.K219T single heterozygotes. Myocardial specimens of doubly heterozygote individuals showed increased nuclear length, sarcomeric disorganization, and myonuclear clustering compared with samples from single heterozygotes. In conclusion, our results show that WES can be used for the identification of causal and modifier variants in families with variable manifestations of DCM. In addition, they not only indicate that LMNA and TTN mutational status may be useful in this family for risk stratification in individuals at risk for DCM but also suggest titin as a modifier for DCM.     

Analysis of LMNB1 Duplications in Autosomal Dominant Leukodystrophy Provides Insights into Duplication Mechanisms and Allele‐Specific Expression

Autosomal dominant leukodystrophy (ADLD) is an adult onset demyelinating disorder that is caused by duplications of the lamin B1 (LMNB1) gene. However, as only a few cases have been analyzed in detail, the mechanisms underlying LMNB1 duplications are unclear. We report the detailed molecular analysis of the largest collection of ADLD families studied, to date. We have identified the minimal duplicated region necessary for the disease, defined all the duplication junctions at the nucleotide level and identified the first inverted LMNB1 duplication. We have demonstrated that the duplications are not recurrent; patients with identical duplications share the same haplotype, likely inherited from a common founder and that the duplications originated from intrachromosomal events. The duplication junction sequences indicated that nonhomologous end joining or replication‐based mechanisms such fork stalling and template switching or microhomology‐mediated break induced repair are likely to be involved. LMNB1 expression was increased in patients’ fibroblasts both at mRNA and protein levels and the three LMNB1 alleles in ADLD patients show equal expression, suggesting that regulatory regions are maintained within the rearranged segment. These results have allowed us to elucidate duplication mechanisms and provide insights into allele‐specific LMNB1 expression levels.     

Cytoskeletal behaviour in spectrin and in band 3 deficient spherocytic red cells: evidence for a differentiated splenic conditioning role

Based on quantitative analysis of red cell membrane proteins, hereditary spherocytosis (HS) can be divided into two main groups including isolated or ankyrin combined spectrin deficiency and band 3 reduction. Protein methyl esterification catalysed by protein carboxyl methyltransferase (PCMT type II; EC 2.1.1.77) is a post-biosynthetic modification which is involved in the metabolism of damaged membrane proteins. We utilized the evaluation of erythrocyte membrane protein methyl esterification as a marker of cytoskeletal disarray in seven HS subjects with spectrin reduction and in seven patients with HS due to band 3 deficiency. Our results support the notion that band 3 deficient erythrocytes are not affected by an extensive cytoskeletal derangement. On the contrary, we found a remarkable increase of membrane methylation in the unsplenectomized, spectrin-deficient, HS patients, suggesting a striking membrane skeleton disarray. This phenomenon was not observed in the spectrin-deficient red cells of splenectomized patients. Therefore in spectrin deficient erythrocytes the induction of cytoskeletal damage, specifically recognized by PCMT type II, could be one of the splenic steps producing conditioned spherocytes.     

Placental genomic risk scores and early neurodevelopmental outcomes

Tracing the early paths leading to developmental disorders is critical for prevention. In previous work, we detected an interaction between genomic risk scores for schizophrenia (GRSs) and early-life complications (ELCs), so that the liability of the disorder explained by genomic risk was higher in the presence of a history of ELCs, compared with its absence. This interaction was specifically driven by loci harboring genes highly expressed in placentae from normal and complicated pregnancies [G. Ursini et al., Nat. Med. 24, 792–801 (2018)]. Here, we analyze whether fractionated genomic risk scores for schizophrenia and other developmental disorders and traits, based on placental gene-expression loci (PlacGRSs), are linked with early neurodevelopmental outcomes in individuals with a history of ELCs. We found that schizophrenia’s PlacGRSs are negatively associated with neonatal brain volume in singletons and offspring of multiple pregnancies and, in singletons, with cognitive development at 1 y and, less strongly, at 2 y, when cognitive scores become more sensitive to other factors. These negative associations are stronger in males, found only with GRSs fractionated by placental gene expression, and not found in PlacGRSs for other developmental disorders and traits. The relationship of PlacGRSs with brain volume persists as an anlage of placenta biology in adults with schizophrenia, again selectively in males. Higher placental genomic risk for schizophrenia, in the presence of ELCs and particularly in males, alters early brain growth and function, defining a potentially reversible neurodevelopmental path of risk that may be unique to schizophrenia.

Understanding the deviations from normal trajectories of brain development may be crucial for predicting illness and for prevention. Epidemiological studies have consistently identified early antecedents, including complications during pregnancy (1–4) and delays in developmental milestones (5–8). The incidence of many developmental disorders tends to be higher in males (9), and risk is typically highly heritable (10). While rare and moderately penetrant genetic variations account for a minority of cases, genome-wide association studies (GWASs) show that most risk is attributable to common variants across the genome (11, 12). Genomic risk scores (GRSs) from GWASs allow a much greater prediction of liability of the disorder than single common variant genotypes, but GRSs per se are not useful in predicting individual risk (13).We previously identified an environmental context in early life in which genomic risk for schizophrenia may enhance disease susceptibility (14). We found that the liability of schizophrenia explained by genomic risk (that is, schizophrenia GRS, also referred to as polygenic risk score; PRS) was more than five times higher in individuals with a history of obstetrical complications (here, early-life complications; ELCs; i.e., during pregnancy, at labor/delivery, and early in neonatal life) compared with its absence (14). Such interaction was exclusive of the GRSs constructed from the loci with the most significant associations with schizophrenia (GRS1: GWAS P < 5 × 10⁻⁸; GRS2: GWAS P < 1 × 10⁻⁶). Genes in the GRS1 and GRS2 loci were more highly expressed in placental tissue compared with genes in GWAS loci not interacting with ELCs (GRS3 to 10); they were up-regulated in placentae from complicated pregnancies and strongly correlated within placenta with expression of immune response genes (14), consistent with previous evidence linking placenta, inflammation, and brain development (15, 16).To investigate the role of placenta biology in the interaction between schizophrenia GRSs and ELCs, we derived sets of GRSs based on single-nucleotide polymorphisms (SNPs) marking schizophrenia-GWAS loci containing genes highly expressed in placenta and differentially expressed in placentae from complicated compared with normal pregnancies (PlacGRSs; placental genomic risk scores) and also from the remaining GWAS loci (NonPlacGRSs; nonplacental genomic risk scores). We found that only PlacGRSs interacted with ELCs on schizophrenia-case control status, while NonPlacGRSs did not, implicating genes involved in placenta stress as driving the interaction between genomic risk and ELCs. These interactions were specifically related to placental gene expression, in that they were not detected when calculating GRSs based on SNPs marking loci highly expressed or epigenetically regulated in other tissues, including various adult and fetal tissues/embryonic cells, and fetal brain. Finally, we detected a much stronger enrichment of expression of the schizophrenia-risk genes in placentae from male compared with female offspring, suggesting a role of placenta in the higher incidence of schizophrenia in males (14).We here investigate whether placental genomic risk for schizophrenia as well as several other developmental disorders and traits is linked with early neurodevelopmental outcomes in individuals with a history of ELCs associated with placenta pathophysiology. Abundant evidence shows that ELCs have implications for early developmental trajectories, including brain size, intellectual development, and neuromotor function as well as for schizophrenia later in life (3, 5, 17–19). Based on these prior observations and our earlier findings (14), we hypothesized that schizophrenia PlacGRSs, in contrast to NonPlacGRSs, have a negative effect on early developmental outcomes, especially in males. Further consistent with our earlier findings, we hypothesized that this negative relationship is characteristic of the PlacGRSs constructed from the placental schizophrenia-GWAS loci with the strongest association with the disorder (PlacGRS1: GWAS P < 5 × 10⁻⁸; PlacGRS2: GWAS P < 1 × 10⁻⁶). We studied the relationship of PlacGRSs and NonPlacGRSs with brain volume in a unique sample of neonates who underwent MRI scanning shortly after birth, and analyzed the relationship with neurocognitive development at 1 and 2 y of age in the same subjects. Finally, we analyzed the relationship of PlacGRSs and NonPlacGRSs with brain volume in a sample of adult controls and patients with schizophrenia.     

Identification of New Natural CphA Metallo-β-Lactamases CphA4 and CphA5 in Aeromonas veronii and Aeromonas hydrophila Isolates from Municipal Sewage in Central Italy

Two new natural CphA metallo-β-lactamases, the CphA4 and CphA5 enzymes, were identified in water samples from municipal sewage in central Italy. Compared to CphA, the CphA4 and CphA5 enzymes showed numerous point mutations. These enzymes have a narrow spectrum of substrates focused on carbapenems only. CphA5 showed k_cat values about 40-, 12-, and 97-fold higher than those observed for CphA4 versus imipenem, ertapenem, and biapenem, respectively.