Common bone marrow signature in COVID-19-associated multisystem inflammatory syndrome in children: A first-wave small case series experience.

The hyper-inflammatory response, also known as multisystem inflammatory syndrome in children (MIS-C), represents a major concern in children with SARS-CoV-2 infection. We report bone marrow features of three patients with MIS-C who were diagnosed during the first wave of the SARS-CoV-2 pandemic. A bone marrow evaluation was performed at onset of the inflammatory condition in order to exclude secondary hemophagocytic lymphohistiocytosis (sHLH). The bone marrows of the patients presented common features: the erythroid and megakaryocytic lineages were prominently affected and hemophagocytosis was moderately increased, differently than observed in sHLH. Megakaryocytopoiesis was increased, representing a peculiar feature of MIS-C differing from sHLH. SARS-CoV-2 RT-PCR and viral panel were studied in bone marrow aspiration samples. MIS-C is a rare complication of SARS-CoV-2 infections in children. An immuno-dysregulation considering both innate and adaptive immunity together with vascular inflammation and endothelial dysfunction play a major role. Our observations, although limited due to the small sample size, suggest that there are unique features in the bone marrow of patients with MIS-C that are likely secondary to immuno-dysregulation, and there are notable differences in bone marrow features compared to those reported in sHLH.

Increased proportions of γδ T lymphocytes in atypical SCID associate with disease manifestations.

Severe combined immunodeficiencies (SCID) comprise a group of genetic diseases characterized by abrogated development of T lymphocytes. In some case reports of atypical SCID patients elevated proportions of γδ T lymphocytes have been reported. However, it is unknown whether these γδ T cells modulate or reflect the patient's clinical phenotype. We investigated the frequency of elevated γδ T cell proportions and associations with clinical disease manifestations in a cohort of 76 atypical SCID patients. Increased proportions of γδ T lymphocytes were present in approximately 60% of these patients. Furthermore, we identified positive correlations between elevated proportions of γδ T cells and the occurrence of CMV infections and autoimmune cytopenias. We discuss that CMV infections might trigger an expansion of γδ T lymphocytes, which could drive the development of autoimmune cytopenias. We advocate that atypical SCID patients should be screened for elevated proportions of γδ T lymphocytes, CMV infection and autoimmune cytopenias.

Efficacy and Adverse Events During Janus Kinase Inhibitor Treatment of SAVI Syndrome.

OBJECTIVES: Mutations affecting the TMEM173 gene cause STING-associated vasculopathy with onset in infancy (SAVI). No standard immunosuppressive treatment approach is able to control disease progression in patients with SAVI. We studied the efficacy and safety of targeting type I IFN signaling with the Janus kinase inhibitor, ruxolitinib. METHODS: We used DNA sequencing to identify mutations in TMEM173 in patients with peripheral blood type I IFN signature. The JAK1/2 inhibitor ruxolitinib was administered on an off-label basis. RESULTS: We identified three patients with SAVI presenting with skin involvement and progressive severe interstitial lung disease. Indirect echocardiographic signs of pulmonary hypertension were present in one case. Following treatment with ruxolitinib, we observed improvements of respiratory function including increased forced vital capacity in two patients, with discontinuation of oxygen therapy and resolution of echocardiographic abnormalities in one case. Efficacy was persistent in one patient and only transitory in the other two patients. Clinical control of skin complications was obtained, and one patient discontinued steroid treatment. One patient, who presented with kidney involvement, showed resolution of hematuria. One patient experienced increased recurrence of severe viral respiratory infections. Monitoring of peripheral blood type I interferon signature during ruxolitinib treatment did not show a stable decrease. CONCLUSIONS: We conclude that targeting type I IFN receptor signaling may represent a promising therapeutic option for a subset of patients with SAVI syndrome and severe lung involvement. However, the occurrence of viral respiratory infection might represent an important cautionary note for the application of such form of treatment.

Mechanisms of genotype-phenotype correlation in autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency.

BACKGROUND: Autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD EDA-ID) is caused by heterozygous point mutations at or close to serine 32 and serine 36 or N-terminal truncations in IκBα that impair its phosphorylation and degradation and thus activation of the canonical nuclear factor κ light chain enhancer of activated B cells (NF-κB) pathway. The outcome of hematopoietic stem cell transplantation is poor in patients with AD EDA-ID despite achievement of chimerism. Mice heterozygous for the serine 32I mutation in IκBα have impaired noncanonical NF-κB activity and defective lymphorganogenesis. OBJECTIVE: We sought to establish genotype-phenotype correlation in patients with AD EDA-ID. METHODS: A disease severity scoring system was devised. Stability of IκBα mutants was examined in transfected cells. Immunologic, biochemical, and gene expression analyses were performed to evaluate canonical and noncanonical NF-κB signaling in skin-derived fibroblasts. RESULTS: Disease severity was greater in patients with IκBα point mutations than in those with truncation mutations. IκBα point mutants were expressed at significantly higher levels in transfectants compared with truncation mutants. Canonical NF-κB-dependent IL-6 secretion and upregulation of the NF-κB subunit 2/p100 and RELB proto-oncogene, NF-κB subunit (RelB) components of the noncanonical NF-κB pathway were diminished significantly more in patients with point mutations compared with those with truncations. Noncanonical NF-κB-driven generation of the transcriptionally active p100 cleavage product p52 and upregulation of CCL20, intercellular adhesion molecule 1 (ICAM1), and vascular cell adhesion molecule 1 (VCAM1), which are important for lymphorganogenesis, were diminished significantly more in LPS plus α-lymphotoxin ß receptor-stimulated fibroblasts from patients with point mutations compared with those with truncations. CONCLUSIONS: IκBα point mutants accumulate at higher levels compared with truncation mutants and are associated with more severe disease and greater impairment of canonical and noncanonical NF-κB activity in patients with AD EDA-ID.

B-cell activation with CD40L or CpG measures the function of B-cell subsets and identifies specific defects in immunodeficient patients.

Around 65% of primary immunodeficiencies are antibody deficiencies. Functional tests are useful tools to study B-cell functions in vitro. However, no accepted guidelines for performing and evaluating functional tests have been issued yet. Here, we report our experience on the study of B-cell functions in infancy and throughout childhood. We show that T-independent stimulation with CpG measures proliferation and differentiation potential of memory B cells. Switched memory B cells respond better than IgM memory B cells. On the other hand, CD40L, a T-dependent stimulus, does not induce plasma cell differentiation, but causes proliferation of naïve and memory B cells. During childhood, the production of plasmablasts in response to CpG increases with age mirroring the development of memory B cells. The response to CD40L does not change with age. In patients with selective IgA deficiency (SIgAD), we observed that switched memory B cells are reduced due to the absence of IgA memory B cells. In agreement, IgA plasma cells are not generated in response to CpG. Unexpectedly, B cells from SIgAD patients show a reduced proliferative response to CD40L. Our results demonstrate that functional tests are an important tool to assess the functions of the humoral immune system.

A prospective study on the natural history of patients with profound combined immunodeficiency: An interim analysis.

BACKGROUND: Absent T-cell immunity resulting in life-threatening infections provides a clear rationale for hematopoetic stem cell transplantation (HSCT) in patients with severe combined immunodeficiency (SCID). Combined immunodeficiencies (CIDs) and "atypical" SCID show reduced, not absent T-cell immunity. If associated with infections or autoimmunity, they represent profound combined immunodeficiency (P-CID), for which outcome data are insufficient for unambiguous early transplant decisions. OBJECTIVES: We sought to compare natural histories of severity-matched patients with/without subsequent transplantation and to determine whether immunologic and/or clinical parameters may be predictive for outcome. METHODS: In this prospective and retrospective observational study, we recruited nontransplanted patients with P-CID aged 1 to 16 years to compare natural histories of severity-matched patients with/without subsequent transplantation and to determine whether immunologic and/or clinical parameters may be predictive for outcome. RESULTS: A total of 51 patients were recruited (median age, 9.6 years). Thirteen of 51 had a genetic diagnosis of "atypical" SCID and 14 of 51 of CID. About half of the patients had less than 10% naive T cells, reduced/absent T-cell proliferation, and at least 1 significant clinical event/year, demonstrating their profound immunodeficiency. Nineteen patients (37%) underwent transplantation within 1 year of enrolment, and 5 of 51 patients died. Analysis of the HSCT decisions revealed the anticipated heterogeneity, favoring an ongoing prospective matched-pair analysis of patients with similar disease severity with or without transplantation. Importantly, so far neither the genetic diagnosis nor basic measurements of T-cell immunity were good predictors of disease evolution. CONCLUSIONS: The P-CID study for the first time characterizes a group of patients with nontypical SCID T-cell deficiencies from a therapeutic perspective. Because genetic and basic T-cell parameters provide limited guidance, prospective data from this study will be a helpful resource for guiding the difficult HSCT decisions in patients with P-CID.

The case of an APDS patient: Defects in maturation and function and decreased in vitro anti-mycobacterial activity in the myeloid compartment.

Activated PI3-kinase delta syndrome (APDS) was recently reported as a novel primary immunodeficiency caused by heterozygous gain-of-function mutations in PIK3CD gene. Here we describe immunological studies in a 19year old APDS patient for whom genetic diagnosis was discovered by Whole Exome Sequencing (WES) analysis. In addition to the progressive lymphopenia and defective antibody production we showed that the ability of the patient's B cells to differentiate in vitro is severely reduced. An in depth analysis of the myeloid compartment showed an increased expression of CD83 activation marker on monocytes and mono-derived DC cells. Moreover, monocytes-derived macrophages (MDMs) failed to solve the Mycobacterium bovis bacillus Calmette Guèrin (BCG) infection in vitro. Selective p110δ inhibitor IC87114 restored the MDM capacity to kill BCG in vitro. Our data show that the constitutive activation of Akt-mTOR pathway induces important alterations also in the myeloid compartment providing new insights in order to improve the therapeutic approach in these patients.

Agammaglobulinemia associated to nasal polyposis due to a hypomorphic RAG1 mutation in a 12 years old boy.

Recombination-activating gene (RAG) 1 and 2 mutations in humans cause T- B- NK+ SCID and Omenn syndrome, but milder phenotypes associated with residual protein activity have been recently described. We report a male patient with a diagnosis of common variable immunodeficiency (CVID) born from non-consanguineous parents, whose immunological phenotype was characterized by severe reduction of B cells and agammaglobulinemia for which several candidate genes were excluded by targeted Sanger sequencing. Next Generation Sequencing revealed two compound heterozygous mutations in the RAG1 gene: the previously described p.R624H, and the novel p.Y728H mutation, as well as the known polymorphism p.H249R. This case reinforces the notion of large phenotypic spectrum in RAG deficiency and opens questions on the management and follow-up of these patients.

Longitudinal Evaluation of Immune Reconstitution and B-cell Function After Hematopoietic Cell Transplantation for Primary Immunodeficiency.

PURPOSE: Hematopoietic cell transplantation (HCT) provides a curative therapy for severe forms of primary immunodeficiencies (PID). While the timing and extent of T-cell reconstitution following transplant for PID has been studied in depth, less is known about the kinetics of B-cell development and long-term restoration of humoral functions, which been often reported to be suboptimal after HCT. METHODS: We studied longitudinally B-cell development and function in a cohort of 13 PID patients transplanted between 1997 and 2010, with a follow-up ranging from 0.7 to 15 years. Flow cytometric analysis of naïve and antigen-experienced B-cell subsets and in vitro functional responses to CpG were compared with data from healthy children and correlated with the degree of B-cell chimerism and in vivo antibody production. RESULTS: We found that total memory B-cells count remained below normal levels for the first 2 years of follow up and progressively normalized. Switched memory B-cells (CD19+CD27+IgD-IgM-) were restored early and better than IgM memory B-cells (CD19+CD27+IgD+IgM+), which remained significantly reduced long-term. The recovery of memory B-cells correlated with good in vivo humoral function and normalization of CpG-response. A complete B-cell reconstitution was usually associated with donor B-cells chimerism and pre-transplant conditioning. Donor source and the underlying genetic defect represented also important variables. CONCLUSION: Monitoring of phenotypic and functional changes on B-cells following HCT may prove clinically relevant to tailor patients' care. In particular the analysis of IgM memory and switched memory B-cells in addition to in vitro B-cells stimulation are recommended before Ig replacement therapy (IgRT) discontinuation.

The burden of central line-associated bloodstream infections in children with medical complexity.

BACKGROUND: Central line-associated bloodstream infections (CLABSI) are significant cause of complications in pediatric intensive care units (PICUs). An emerging challenge are CLABSIs in children with medical complexity (CMC) admitted to PICU. CMC are patients with chronic conditions with or without neurological impairment needing for tracheostomy and/or home mechanical or non-invasive ventilation and/or gastrostomy/jejunostomy. We evaluate CLABSI incidence in a PICU with high prevalence of CMC. METHODS: This was a retrospective study in the PICU of the Bambino Gesù Children Hospital from January 2017 to December 2020. The medical records were reviewed and demographic, clinical and microbiological data were extracted. CLABSI were defined according to the Center for Disease Control and Prevention's National Healthcare Safety Networks (NHSN) surveillance. RESULTS: A total of 101 children with 125 central lines (CLs) were included; 79/101 (78%) patients were CMC and 50/101 (50%) had a thracheostomy. CLABSI incidence was 2.75/1000 CL-days (9 cases/3269 CL-days); incidence was 0 in patients without underling conditions and 3.14/1000 in CMC (p < 0.001). CLABSI were due to gram negative bacteria in five patients, Candida spp in three and Staphylococcus hominis in one. CLs were removed in eight cases while in the later one, with CLABSI due to Pseudomonas aeruginosa, a conservative strategy was adopted cause of unavailable alternative venous access and removed at discharge with negative culture. All patients recovered. CONCLUSIONS: A target 0% CLABSI was possible in critically ill children without underling condition while a high incidence was reported in CMC and sustained by a peculiar CLABSI ecology. This ecology should be considered when a CLABSI was suspected in CMC for prompt antibiotics stewardship.

In vivo T-cell dynamics during immune reconstitution after hematopoietic stem cell gene therapy in adenosine deaminase severe combined immune deficiency.

BACKGROUND: Gene therapy (GT) with hematopoietic stem cells is a promising treatment for inherited immunodeficiencies. OBJECTIVES: Limited information is available on the relative contribution of de novo thymopoiesis and peripheral expansion to T-cell reconstitution after GT as well as on the potential effects of gene transfer on hematopoietic stem cells and lymphocyte replicative lifespan. We studied these issues in patients affected by adenosine deaminase severe combined immune deficiency after low-intensity conditioning and reinfusion of retrovirally transduced autologous CD34(+) cells. METHODS: Immunophenotype, proliferative status, telomere length, and T-cell receptor excision circles were investigated at early and late time points (up to 9 years) after GT treatment. Control groups consisted of pediatric healthy donors and patients undergoing allogeneic bone marrow transplantation (BMT). RESULTS: We observed no telomere shortening in the bone marrow compartment and in granulocytes, whereas peripheral blood naive T cells from both GT and BMT patients showed a significant reduction in telomere length compared with healthy controls. This was in agreement with the presence of a high fraction of actively cycling naive and memory T cells and lower T-cell receptor excision circles. CONCLUSION: These data indicate that T-cell homeostatic expansion contributes substantially to immune reconstitution, like BMT, and is not associated with senescence in the stem cell compartment.

Glycopyrrolate for drooling in children with medical complexity under three years of age.

BACKGROUND: The aim of the study is to determine that Glycopirrolate is safe and effective in decreasing drooling in children with medical complexity under 3 years of age. Medical treatment is based on anticholinergic drugs as transdermal scopolamine, benzotropine and GLY. GLY (Glycopyrronium bromide) is a synthetic quaternary ammonium anticholinergic agent with poor blood-brain barrier penetration and consequently has limited central effects. Actually, the oral GLY formulation was approved by the United States Food and Drug Administration (FDA) to treat drooling in children aged 3-16 years. Five studies reported on GLY use for the treatment of drooling in children with cerebral palsy and other conditions with neurological impairment; four are prospective studies while one a retrospective review. METHODS: this is a case report of eighteen children (sex ratio 11/8, median age 17 months, range 2-36 months) under three years of age, followed by a multidisciplinary team at the Bambino Gesù Children Hospital. The median follow-up was of 31.5 months (range 1-69 months). Response to treatment was assessed according to the Drooling Impact Scale administered at time 0 and after 1 month. All patients have an important neurological impairment: nine patients have a cerebral palsy (Gross Motor Function Classification System class V) and nine a genetic/malformative syndrome. Twelve patients have a tracheostomy and two need mechanical ventilation. Gastrostomy is present in 16 out of 18 patients. All patients received Glycopirrolate. The median starting daily dose was 0.065 mg/kg/die (range 0.02-0.21 mg/kg/die) three times a day. The drooling impact scale was administered at time O and after 1 month. RESULTS: Four out 18 patients stopped treatment for adverse event, lack of efficacy or parental decision. The mean Drooling Impact Scale at time 0 was 89 (range 81-100) and after 1 month 61(range 43-78); the difference was statistically significant (P < 0.001). The overall response to treatment was 94%. CONCLUSIONS: This is the first study to determine the safety and effectiveness of Glycopyrrolate in decreasing drooling in a specific subset of patients. No major side effects were observed. Further comparative studies are needed to confirm our results.

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.

Dynamic Viral Severe Acute Respiratory Syndrome Coronavirus 2 RNA Shedding in Children: Preliminary Data and Clinical Consideration from a Italian Regional Center.

We evaluated severe acute respiratory syndrome coronavirus 2 RNA clearance in 22 children. The estimation of positivity at day 14 was 52% for nasopharyngeal swab and 31% for stool samples. These data underline the significance of nasopharyngeal and stoolsample for detecting infected children. Additional studies are needed for transmissibility.

A neonatal cluster of novel coronavirus disease 2019: clinical management and considerations.

BACKGROUND: Lately, one of the major clinical and public health issues has been represented by Coronavirus disease of 2019 (COVID-19) during pregnancy and the risk of transmission of the infection from mother to child. Debate on perinatal management and postnatal care is still ongoing, principally questioning the option of the joint management of mother and child after birth and the safety of breastfeeding. According to the available reports, neonatal COVID-19 appears to have a horizontal transmission and seems to be paucisymptomatic or asymptomatic, compared to older age groups. The aim of this work is to describe a cluster of neonatal COVID-19 and discuss our experience, with reference to current evidence on postnatal care and perinatal management. METHODS: This is a retrospective observational case series of five mother-child dyads, who attended the Labor and Delivery Unit of a first-level hospital in Italy, in March 2020. Descriptive statistics for continuous variables consisted of number of observations, mean and the range of the minimum and maximum values. RESULTS: Five women and four neonates tested positive for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In one case, the mother-child dyad was separated and the neonate remained negative on two consecutive tests. Two positive neonates developed symptoms, with a predominant involvement of the gastrointestinal tract. Blood tests were unremarkable, except for a single patient who developed mild neutropenia. No complications occurred. CONCLUSIONS: We agree that the decision on whether or not to separate a positive/suspected mother from her child should be made on an individual basis, taking into account the parent's will, clinical condition, hospital logistics and the local epidemiological situation. In conformity with literature, in our study, affected neonates were asymptomatic or paucisymptomatic. Despite these reassuring findings, a few cases of severe presentation in the neonatal population have been reported. Therefore, we agree on encouraging clinicians to monitor the neonates with a suspected or confirmed infection.

Clinical, Immunological, and Molecular Features of Typical and Atypical Severe Combined Immunodeficiency: Report of the Italian Primary Immunodeficiency Network.

Severe combined immunodeficiencies (SCIDs) are a group of inborn errors of the immune system, usually associated with severe or life-threatening infections. Due to the variability of clinical phenotypes, the diagnostic complexity and the heterogeneity of the genetic basis, they are often difficult to recognize, leading to a significant diagnostic delay (DD). Aim of this study is to define presenting signs and natural history of SCID in a large cohort of patients, prior to hematopoietic stem cell or gene therapies. To this purpose, we conducted a 30-year retro-prospective multicenter study within the Italian Primary Immunodeficiency Network. One hundred eleven patients, diagnosed as typical or atypical SCID according to the European Society for Immune Deficiencies criteria, were included. Patients were subsequently classified based on the genetic alteration, pathogenic mechanism and immunological classification. A positive relationship between the age at onset and the DD was found. SCID patients with later onset were identified only in the last decade of observation. Syndromic SCIDs represented 28% of the cohort. Eight percent of the subjects were diagnosed in Intensive Care Units. Fifty-three percent had an atypical phenotype and most of them exhibited a discordant genotype-immunophenotype. Pre-treatment mortality was higher in atypical and syndromic patients. Our study broadens the knowledge of clinical and laboratory manifestations and genotype/phenotype correlation in patients with SCID and may facilitate the diagnosis of both typical and atypical forms of the disease in countries where newborn screening programs have not yet been implemented.

Corrigendum: Targeted NGS Platforms for Genetic Screening and Gene Discovery in Primary Immunodeficiencies.

[This corrects the article DOI: 10.3389/fimmu.2019.00316.].