Circulating hematopoietic stem/progenitor cell subsets contribute to human hematopoietic homeostasis.

ABSTRACT: In physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPCs) are present in the peripheral blood, but their contribution to human hematopoiesis remain unsolved. By integrating advanced immunophenotyping, single-cell transcriptional and functional profiling, and integration site (IS) clonal tracking, we unveiled the biological properties and the transcriptional features of human cHSPC subpopulations in relationship to their bone marrow (BM) counterpart. We found that cHSPCs reduced in cell count over aging and are enriched for primitive, lymphoid, and erythroid subpopulations, showing preactivated transcriptional and functional state. Moreover, cHSPCs have low expression of multiple BM-retention molecules but maintain their homing potential after xenotransplantation. By generating a comprehensive human organ-resident HSPC data set based on single-cell RNA sequencing data, we detected organ-specific seeding properties of the distinct trafficking HSPC subpopulations. Notably, circulating multi-lymphoid progenitors are primed for seeding the thymus and actively contribute to T-cell production. Human clonal tracking data from patients receiving gene therapy (GT) also showed that cHSPCs connect distant BM niches and participate in steady-state hematopoietic production, with primitive cHSPCs having the highest recirculation capability to travel in and out of the BM. Finally, in case of hematopoietic impairment, cHSPCs composition reflects the BM-HSPC content and might represent a biomarker of the BM state for clinical and research purposes. Overall, our comprehensive work unveiled fundamental insights into the in vivo dynamics of human HSPC trafficking and its role in sustaining hematopoietic homeostasis. GT patients' clinical trials were registered at ClinicalTrials.gov (NCT01515462 and NCT03837483) and EudraCT (2009-017346-32 and 2018-003842-18).

Expanding the spectrum of neonatal-onset AIFM1-associated disorders.

OBJECTIVES: Pathogenic variants in AIFM1 have been associated with a wide spectrum of disorders, spanning from CMT4X to mitochondrial encephalopathy. Here we present a novel phenotype and review the existing literature on AIFM1-related disorders. METHODS: We performed EEG recordings, brain MRI and MR Spectroscopy, metabolic screening, echocardiogram, clinical exome sequencing (CES) and family study. Effects of the variant were established on cultured fibroblasts from skin punch biopsy. RESULTS: The patient presented with drug-resistant, electro-clinical, multifocal seizures 6 h after birth. Brain MRI revealed prominent brain swelling of both hemispheres and widespread signal alteration in large part of the cortex and of the thalami, with sparing of the basal nuclei. CES analysis revealed the likely pathogenic variant c.5T>C; p.(Phe2Ser) in the AIFM1 gene. The affected amino acid residue is located in the mitochondrial targeting sequence. Functional studies on cultured fibroblast showed a clear reduction in AIFM1 protein amount and defective activities of respiratory chain complexes I, III and IV. No evidence of protein mislocalization or accumulation of precursor protein was observed. Riboflavin, Coenzyme Q10 and thiamine supplementation was therefore given. At 6 months of age, the patient exhibited microcephaly but did not experience any further deterioration. He is still fed orally and there is no evidence of muscle weakness or atrophy. INTERPRETATION: This is the first AIFM1 case associated with neonatal seizures and diffuse white matter involvement with relative sparing of basal ganglia, in the absence of clinical signs suggestive of myopathy or motor neuron disease.

Case Report - Multinodular goiter in a patient with Congenital Hypothyroidism and Bannayan-Riley-Ruvalcaba syndrome: the possible synergic role of TPO and PTEN mutation.

We report the case of a paediatric female patient affected by Bannayan-Riley-Ruvalcaba syndrome (BRRS) and congenital hypothyroidism (CH) with homozygous mutation of the TPO gene. She underwent total thyroidectomy at the age of seven years because of the development of a multinodular goiter. BRRS patients present an increased risk of benign and malignant thyroid disease since childhood because of inactivating mutation of PTEN, an onco-suppressor gene. Instead, homozygous mutations in the TPO gene can be associated with severe forms of hypothyroidism with goiter; previous studies have described cases of follicular and papillary thyroid cancer in CH patients with TPO mutation despite a perfectly controlled thyroid function with Levothyroxine therapy. To our knowledge, this is the first case that describes the possible synergic role of coexisting mutation of both TPO and PTEN in the development of multinodular goiter underlining the importance of a tailored surveillance program in these patients, especially during childhood.

Guided Growth in Leg Length Discrepancy in Beckwith-Wiedemann Syndrome: A Consecutive Case Series.

Beckwith-Wiedemann Syndrome (BWS) is a rare genetic disorder characterized by overgrowth, macroglossia, abdominal wall defects, neonatal hypoglycemia, predisposition to embryonal tumor, lateralized overgrowth, and leg length discrepancy (LLD), which can affect normal posture and gait. Aim of this study was to evaluate the effects of guided growth (temporary epiphysiodesis technique) as LLD management in BWS patients. Between 2007 and 2021, 22 BWS patients (15 F, 7 M) with a mean age of 7.9 years (2.9-14.4) and a mean LLD at first surgery of 3.65 cm (2-10), underwent temporary proximal tibial (PTE) and distal femur epiphysiodesis (DFE). In 18 patients the first surgical procedure was PTE, in one, DFE, and in 3 cases, PTE and DFE at the same time, respectively. Eleven patients reached equality of leg length after a mean follow-up of 7.7 years (3.7-13.0) and mean age of 13.3 years (12.7-27.5); 10 patients underwent 3 surgical procedures, one 7 procedures. Fifteen patients had no complications. No severe complications, infection, articular stiffness, or neuro-vascular lesions occurred in remaining patients; complications included secondary varus or valgus axial deviation in a total of 6 patients, and two screw breakages in two patients. Guided growth as a minimally invasive procedure seems efficient for LLD treatment with low complication rate in BWS patients.

Expression of glucose transporters in duodenal mucosa of patients with type 1 diabetes.

AIMS: A higher SGLT1 and GLUT2 gene expression was shown in the intestine of subjects with type 2 diabetes, while no data have been reported in type 1 diabetes (T1D). The purpose of our study was to evaluate the expression of glucose transporters in duodenal mucosa of subjects with T1D, compared to healthy controls (CTRL) and to patients with celiac disease (CD), as gut inflammatory disease control group. MATERIALS AND METHODS: Gene expression of GLUT1, GLUT2, SGLT1 and SGLT2 was quantified on duodenal mucosa biopsies of subjects with T1D (n = 19), CD (n = 16), T1D and CD (n = 6) and CTRL (n = 12), recruited at San Raffaele Hospital (Milan, Italy), between 2009 and 2018. SGLT2 expression was further evaluated by immunohistochemical and immunofluorescence staining. RESULTS: The expression of all four glucose transporters was detected in duodenal mucosa of all groups. A reduced GLUT2, SGLT1 and SGLT2 expression was observed in CD in comparison with T1D and CTRL, as expected; GLUT1 was significantly more expressed in T1D compared to CTRL. SGLT2 expression was quantified at much lower levels than other transporters, with no differences between groups. SGLT2 expression was confirmed by immunohistochemistry in a restricted number of enterocytes lining in the mucosa of intestinal villi, also shown on immunofluorescence. CONCLUSIONS: Our results show that glucose transporters expression in duodenal mucosa of subjects with T1D, except an increased GLUT1, is not different from that observed in healthy controls. The expression of SGLT2 in human duodenal mucosa, although at low intensity, represents a novel finding.

Reduced PD-1 expression on circulating follicular and conventional FOXP3+ Treg cells in children with new onset type 1 diabetes and autoantibody-positive at-risk children.

Autoantibodies (AAbs) are a hallmark of Type 1 diabetes (T1D). Alterations in the frequency and phenotype of follicular helper (Tfh) T cells have been previously documented in patients with type 1 diabetes (T1D), but the contribution of follicular regulatory T (Treg) cells, which are responsible for suppressing AAb development, is less clear. Here, we investigated the frequency and activation status of follicular (CXCR5+) and conventional (CXCR5-) Treg cells in the blood of children with new-onset T1D, and children with risk for developing T1D (AAb-positive) and compared them to AAb-negative controls. Blood follicular and conventional Treg cells were higher in frequency in children with new onset T1D, but expressed reduced amounts of PD-1 as compared to AAb-negative children. Interestingly, the proportion of circulating FOXP3+ Tregs expressing PD-1 was also reduced in AAb-positive at-risk children as compared to AAb-negative controls, suggesting its potential use as a biomarker of disease progression. Follicular Treg cells were reduced in frequency in the spleens of prediabetic NOD mice as they became older and turned diabetic. Interestingly, PD-1 expression declined also on circulating follicular and conventional Treg cells in prediabetic NOD mice as they aged. Together, these findings show that the frequency of circulating follicular and conventional Treg cells and their levels of PD-1 change with disease progression in children at-risk for developing T1D and in NOD mice.

Treatment with rapamycin can restore regulatory T-cell function in IPEX patients.

BACKGROUND: Immune-dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a lethal disease caused by mutations in a transcription factor critical for the function of thymus-derived regulatory T (Treg) cells (ie, FOXP3), resulting in impaired Treg function and autoimmunity. At present, hematopoietic stem cell transplantation is the therapy of choice for patients with IPEX syndrome. If not available, multiple immunosuppressive regimens have been used with poor disease-free survival at long-term follow-up. Rapamycin has been shown to suppress peripheral T cells while sparing Treg cells expressing wild-type FOXP3, thereby proving beneficial in the clinical setting of immune dysregulation. However, the mechanisms of immunosuppression selective to Treg cells in patients with IPEX syndrome are unclear. OBJECTIVE: We sought to determine the cellular and molecular basis of the clinical benefit observed under rapamycin treatment in 6 patients with IPEX syndrome with different FOXP3 mutations. METHODS: Phenotype and function of FOXP3-mutated Treg cells from rapamycin-treated patients with IPEX syndrome were tested by flow cytometry and in vitro suppression assays, and the gene expression profile of rapamycin-conditioned Treg cells by droplet-digital PCR. RESULTS: Clinical and histologic improvements in patients correlated with partially restored Treg function, independent of FOXP3 expression or Treg frequency. Expression of TNF-receptor-superfamily-member 18 (TNFRSF18, glucocorticoid-induced TNF-receptor-related) and EBV-induced-3 (EBI3, an IL-35 subunit) in patients' Treg cells increased during treatment as compared with that of Treg cells from untreated healthy subjects. Furthermore inhibition of glucocorticoid-induced TNF-receptor-related and Ebi3 partially reverted in vitro suppression by in vivo rapamycin-conditioned Treg cells. CONCLUSIONS: Rapamycin is able to affect Treg suppressive function via a FOXP3-independent mechanism, thus sustaining the clinical improvement observed in patients with IPEX syndrome under rapamycin treatment.

Duodenal Mucosa of Patients With Type 1 Diabetes Shows Distinctive Inflammatory Profile and Microbiota.

Context: Increasing evidences suggest a correlation between gut and type 1 diabetes (T1D). Objective: The objective of this study is to evaluate the gut inflammatory profile and microbiota in patients with T1D compared with healthy control (CTRL) subjects and patients with celiac disease (CD) as gut inflammatory disease controls. Design/Setting/Participants: The inflammatory status and microbiome composition were evaluated in biopsies of the duodenal mucosa of patients with T1D (n = 19), in patients with CD (n = 19), and CTRL subjects (n = 16) recruited at San Raffaele Scientific Institute, in Milan, Italy, between 2009 and 2015. Main Outcome Measures: Inflammation was evaluated by gene expression study and immunohistochemistry. Microbiome composition was analyzed by 16S ribosomal RNA gene sequencing. Results: An increased expression of CCL13, CCL19, CCL22, CCR2, COX2, IL4R, CD68, PTX3, TNFα, and VEGFA was observed in patients with T1D compared with CTRL subjects and patients with CD. Immunohistochemical analysis confirmed T1D-specific inflammatory status compared with healthy and CD control tissues, mainly characterized by the increase of the monocyte/macrophage lineage infiltration. The T1D duodenal mucosal microbiome results were different from the other groups, with an increase in Firmicutes and Firmicutes/Bacteroidetes ratio and a reduction in Proteobacteria and Bacteroidetes. The expression of genes specific for T1D inflammation was associated with the abundance of specific bacteria in the duodenum. Conclusions: This study shows that duodenal mucosa in T1D presents disease-specific abnormalities in the inflammatory profile and microbiota. Understanding the mechanisms underlying these features is critical to disentangle the complex pathogenesis of T1D and to gain new perspectives for future therapies targeting the intestine.

Bulb biopsies for the diagnosis of celiac disease in pediatric patients.

BACKGROUND: Celiac disease (CD) is a gluten-dependent enteropathy. The current standard for diagnosing CD involves obtaining 4 biopsy samples from the descending duodenum. It has been suggested that duodenal bulb biopsies may also be useful. OBJECTIVE: To assess the utility of bulbar biopsies for the diagnosis of CD in pediatric patients. DESIGN: Prospective study. SETTING: Single center. PATIENTS: Forty-seven consecutively enrolled pediatric patients with celiac serologies and a clinical suspicion of CD. INTERVENTIONS: All patients underwent EGD, and 4 biopsy samples were obtained from the duodenal bulb and 4 from the descending duodenum of each child. MAIN OUTCOME MEASUREMENTS: The pathologist blindly reported the Marsh histological grade for the diagnosis of CD of the bulb and descending duodenum. RESULTS: The diagnosis of CD was histologically confirmed in 89.4% (42/47) of the cases of biopsy samples obtained from the descending duodenum and in all 47 obtained from the bulb. In 35 patients (74.5%), histology was the same in the bulb and duodenum; in 11 (23.4%) cases, the grade of atrophy was higher in the bulb than in the descending duodenum, and 5 (10.6%) had bulb histology positive for CD but negative duodenal findings. One child (2.1%) had a higher histological grade in the duodenum than in the bulb. The diagnostic gain with bulbar biopsies was 10.6%. LIMITATIONS: Small sample and absence of a comparison group (asymptomatic children with normal CD antibodies). CONCLUSIONS: We suggest examining 4 biopsy samples from the duodenal bulb and 4 from the descending duodenum to improve diagnostic accuracy of CD.

Role of reduced intensity conditioning in T-cell and B-cell immune reconstitution after HLA-identical bone marrow transplantation in ADA-SCID.

The treatment of choice for severe combined immunodeficiency is bone marrow transplantation from an HLA-identical donor sibling without conditioning. However, this may result in low donor stem cell chimerism, leading to reduced long-term immune reconstitution. We compared engraftment, metabolic, and T-cell and B-cell immune reconstitution of HLA-identical sibling bone marrow transplantation performed in 2 severe combined immunodeficiency infants with adenosine deaminase deficiency from the same family treated with or without a reduced intensity conditioning regimen (busulfan/fludarabine). Only the patient who received conditioning showed a stable mixed chimerism in all lineages, including bone marrow myeloid and B cells. The use of conditioning resulted in higher thymus-derived naïve T cells and T-cell receptor excision circles, normalization of the T-cell repertoire, and faster and complete B-cell and metabolic reconstitution. These results suggest the utility of exploring the use of reduced intensity conditioning in bone marrow transplantation from HLA-identical donor in severe combined immunodeficiency to improve long-term immune reconstitution.

IgA anti-actin antibodies in children with celiac disease: comparison of immunofluorescence with Elisa assay in predicting severe intestinal damage.

BACKGROUND: Previous studies have demonstrated that the presence of serum IgA antibodies against actin filaments (AAA) in patients with celiac disease (CD) is strongly associated with mucosal damage and severe degrees of villous atrophy.The aims of the present study were (1) to verify the effectiveness of IgA-AAA in newly diagnosed CD patients in a clinical setting (2) to compare the immunofluorescence assay with ELISA assay; (3) to compare the correlation of our IgA anti-tissue transglutaminase antibodies (tTG-Ab) class with mucosal intestinal lesions. METHODS: 90 patients underwent endoscopy and multiple biopsies for suspected CD on the basis of symptoms, in presence of positive tTG-Ab tests. Twenty biopsied and 25 not-biopsied subjects with negative tTG-Ab were tested as control groups. IgA-AAA assays were performed by indirect immunofluorescence using rat epithelial intestinal cells, and by ELISA with a commercial kit. tTG-Ab assay was a radio-binding assay. Intestinal specimens were collected by upper endoscopy and the histological study was done according to the Marsh's classification modified by Oberhuber (M/O). Auto-antibodies assays and histological evaluation have been performed blindly by skilled operators. RESULTS: CD diagnosis was confirmed in 82 patients (type I M/O in 2 patients, IIIA in 18 patients, IIIB in 29 patients and IIIC in 33 patients). Two patients with type 1 lesion in presence of positive tTG-Ab and abdominal complaints, started a gluten free diet. The rate of IgA-AAA positivity (sensitivity) by IFI and ELISA in histologically proven celiac disease patients, were 5.5% and 25% patients in IIIA, 27.5% and 34.4% patients in IIIB, 78.8% and 75% in IIIC patients, respectively.Patients with normal or nearly normal mucosa, regardless of tTG-Ab status, presented negative IgA-AAA IFI assay. On the other hand, 1 patient with normal mucosa but positive tTG-Ab, also presented positive IgA-AAA ELISA. All healthy non biopsied controls had negative IgA-AAA. tTG-Ab serum concentration was significantly correlated with more severe intestinal lesion (IIIB, IIIC M/O). CONCLUSIONS: IgA-AAA may be undetectable in presence of severe mucosal damage. Histology is still necessary to diagnose celiac disease and IgA-AAA cannot be included in usual screening tests, because it has little to offer if compared to the well-established tTG-Ab.IgA-AAA could be an adjunctive, very useful tool to support the diagnosis of CD in case of suboptimal histology, when the biopsy is to be avoided for clinical reasons, or in case of negative parents' consensus.

Occurrence of celiac disease after onset of type 1 diabetes: a 6-year prospective longitudinal study.

OBJECTIVE: To investigate the prevalence of celiac disease in a large cohort of children and adolescents at the onset of type 1 diabetes and the occurrence of new cases during a 6-year follow-up. METHODS: We prospectively studied, by repeated serologic screening, 274 consecutive patients at the onset of type 1 diabetes (age [mean +/- standard deviation]: 8.28 +/- 4.65 years) for 6 subsequent years. One patient had a diagnosis of celiac disease before the onset of diabetes. The immunoglobulin A-antiendomysium antibody test was selected as the screening test; patients with positive results (++ or +++) or with 2 consecutive weak positive tests (+) were considered appropriate for the jejunal biopsy. RESULTS: At diabetes onset, 15 (5.5%) of 273 patients tested positive with the antiendomysium test; jejunal biopsy was performed in 10, and celiac disease was diagnosed in 9. The prevalence of biopsy-confirmed celiac disease at the manifestation of diabetes was 3.6% (10 of 274 patients). Twelve more patients with a negative antiendomysium antibody test at diabetes onset tested positive during the follow-up within 4 years; 10 of them had biopsies performed, and 7 had celiac disease. Therefore, the overall prevalence of biopsy-confirmed celiac disease in the entire cohort of patients was 6.2%. The age at diabetes onset in patients with and without celiac disease was not different (7.88 +/- 5.69 vs 8.3 +/- 4.58 years). The majority of cases of celiac disease were asymptomatic in their presentation, and no signs of overt malnutrition were documented. CONCLUSIONS: The prevalence of celiac disease in patients with type 1 diabetes is approximately 20 times higher than in the general population. Sixty percent of cases are already present at diabetes onset, mostly undetected, but an additional 40% of patients develop celiac disease a few years after diabetes onset. Extending screening programs for celiac disease after the onset of type 1 diabetes is recommended, even in the absence of clinical symptoms.