High incidence of multisystem inflammatory syndrome and other autoimmune diseases after SARS-CoV-2 infection compared to COVID-19 vaccination in children and adolescents in south central Europe.

OBJECTIVES: To estimate the incidence and describe the spectrum of inflammatory and autoimmune diseases linked to SARS-CoV-2 infection and COVID-19 vaccination in children from two neighbouring south central European countries. METHODS: We performed a multi-centre prospective cohort study of children under 18 years diagnosed with inflammatory/autoimmune diseases linked to SARS-CoV-2 infection or COVID-19 vaccination, who were admitted to the paediatric tertiary care hospitals in Slovenia and Friuli Venezia Giulia, Italy, from January 1, 2020, to December 31, 2021. Disease incidence was calculated based on laboratory-confirmed cases only. RESULTS: Inflammatory and autoimmune diseases linked to SARS-CoV-2 were diagnosed in 192 children (127 laboratory-confirmed), of whom 112 had multisystem inflammatory syndrome (MIS-C), followed by vasculitis, neurological and cardiac diseases. Calculated risk of MIS-C was 1 in 860 children after SARS-CoV-2 infection and cumulative incidence of MIS-C was 18.3/100,000 of all children. Fifteen children had severe COVID-19. Two patients with MIS-C and a patient with myositis presented after COVID-19 vaccination. All 3 had at presentation also a serologically proven recent SARS-CoV-2 infection. After MIS-C, nine patients were vaccinated against COVID-19 and 25 patients had a SARS-CoV-2 reinfection, without recurrence of MIS-C. CONCLUSIONS: Autoimmune diseases following SARS-CoV-2 infection in children were 8.5 times as common as severe COVID-19. MIS-C was the most common manifestation and its incidence in this predominantly white population was higher than previously reported. MIS-C does not seem to recur after SARS-CoV-2 reinfection or COVID-19 vaccination. Autoimmune diseases were much more common after SARS-CoV-2 infection than after COVID-19 vaccination.

International retrospective study of allogeneic hematopoietic cell transplantation for activated PI3K-delta syndrome.

BACKGROUND: Activated phosphoinositide 3-kinase delta syndrome (APDS) is a combined immunodeficiency with a heterogeneous phenotype considered reversible by allogeneic hematopoietic cell transplantation (HCT). OBJECTIVES: This study sought to characterize HCT outcomes in APDS. METHODS: Retrospective data were collected on 57 patients with APDS1/2 (median age, 13 years; range, 2-66 years) who underwent HCT. RESULTS: Pre-HCT comorbidities such as lung, gastrointestinal, and liver pathology were common, with hematologic malignancy in 26%. With median follow-up of 2.3 years, 2-year overall and graft failure-free survival probabilities were 86% and 68%, respectively, and did not differ significantly by APDS1 versus APDS2, donor type, or conditioning intensity. The 2-year cumulative incidence of graft failure following first HCT was 17% overall but 42% if mammalian target of rapamycin inhibitor(s) (mTORi) were used in the first year post-HCT, compared with 9% without mTORi. Similarly, 2-year cumulative incidence of unplanned donor cell infusion was overall 28%, but 65% in the context of mTORi receipt and 23% without. Phenotype reversal occurred in 96% of evaluable patients, of whom 17% had mixed chimerism. Vulnerability to renal complications continued post-HCT, adding new insights into potential nonimmunologic roles of phosphoinositide 3-kinase not correctable through HCT. CONCLUSIONS: Graft failure, graft instability, and poor graft function requiring unplanned donor cell infusion were major barriers to successful HCT. Post-HCT mTORi use may confer an advantage to residual host cells, promoting graft instability. Longer-term post-HCT follow-up of more patients is needed to elucidate the kinetics of immune reconstitution and donor chimerism, establish approaches that reduce graft instability, and assess the completeness of phenotype reversal over time.

Vaccination coverage in children with rheumatic diseases.

OBJECTIVES: To assess vaccination status in a cohort of children with rheumatic diseases followed at the University Children's Hospital Ljubljana and to evaluate the most common reasons for vaccination dropout. METHODS: Patients with rheumatic diseases who were evaluated at the rheumatology outpatient clinic between January 2015 and January 2017 received a questionnaire about their vaccination status and reasons for potential vaccination dropout. Vaccination coverage for individual vaccines was determined at 5, 10, 18 years and at the time of their last clinic visit. RESULTS: Data were received from 187 out of 424 enrolled patients (44.1%). Majority of included patients had juvenile idiopathic arthritis (n=165), followed by childhood-onset systemic lupus erythematosus (n=6), juvenile dermatomyositis (n=5), mixed connective tissue disease (n=3), chronic recurrent multifocal osteomyelitis, juvenile systemic sclerosis, Takayasu's arteritis (n=2 each), granulomatous polyangiitis and fibromyalgia (n=1 each). Vaccination coverage was complete in 91.9%, 70.3%, 66.7% and 64.7% of patients at 5, 10, 18 years and at their last clinic visit, respectively. Most commonly omitted vaccines were hepatitis B and second dose of measles, mumps and rubella vaccine. Most common additional vaccine was against rotavirus. Most common reason for vaccination dropout was suggestion of the treating rheumatologist. CONCLUSIONS: Thirty-five percent of our patients remain incompletely vaccinated and thus susceptible to vaccine-preventable diseases. Physicians play a crucial role in the decision to vaccinate.

Rezidivierende fulminante Myokarditis mit wiederholtem Einsatz der ECMO bei einem Kind.

Recurrent myocarditis is rare with only few reports having been published for paediatric cases. Repeated use of extracorporeal membrane oxygenation is also uncommon. In this paper we will present a very rare case of a 7-year old girl with recurrent fulminant myocarditis with heart failure requiring cardiopulmonary resuscitation and mechanical circulatory support with extracorporeal membrane oxygenation. Both episodes were precipitated by a viral upper respiratory tract infection, and in both cases the cardiac function eventually completely recovered. The second episode of fulminant myocarditis was particularly complex with markedly elevated markers of myocardiocytolysis, multiorgan dysfunction and the need for prolonged mechanical circulatory support. Nevertheless, the patient made a remarkable recovery. A comprehensive diagnostic workup pointed towards an aberrant immune response as the likely cause of the girl's susceptibility for fulminant myocarditis.

Clinical spectrum and features of activated phosphoinositide 3-kinase δ syndrome: A large patient cohort study.

BACKGROUND: Activated phosphoinositide 3-kinase δ syndrome (APDS) is a recently described combined immunodeficiency resulting from gain-of-function mutations in PIK3CD, the gene encoding the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ). OBJECTIVE: We sought to review the clinical, immunologic, histopathologic, and radiologic features of APDS in a large genetically defined international cohort. METHODS: We applied a clinical questionnaire and performed review of medical notes, radiology, histopathology, and laboratory investigations of 53 patients with APDS. RESULTS: Recurrent sinopulmonary infections (98%) and nonneoplastic lymphoproliferation (75%) were common, often from childhood. Other significant complications included herpesvirus infections (49%), autoinflammatory disease (34%), and lymphoma (13%). Unexpectedly, neurodevelopmental delay occurred in 19% of the cohort, suggesting a role for PI3Kδ in the central nervous system; consistent with this, PI3Kδ is broadly expressed in the developing murine central nervous system. Thoracic imaging revealed high rates of mosaic attenuation (90%) and bronchiectasis (60%). Increased IgM levels (78%), IgG deficiency (43%), and CD4 lymphopenia (84%) were significant immunologic features. No immunologic marker reliably predicted clinical severity, which ranged from asymptomatic to death in early childhood. The majority of patients received immunoglobulin replacement and antibiotic prophylaxis, and 5 patients underwent hematopoietic stem cell transplantation. Five patients died from complications of APDS. CONCLUSION: APDS is a combined immunodeficiency with multiple clinical manifestations, many with incomplete penetrance and others with variable expressivity. The severity of complications in some patients supports consideration of hematopoietic stem cell transplantation for severe childhood disease. Clinical trials of selective PI3Kδ inhibitors offer new prospects for APDS treatment.

Deficiency in Mucosa-associated Lymphoid Tissue Lymphoma Translocation 1: A Novel Cause of IPEX-Like Syndrome.

OBJECTIVE: Early-onset inflammatory bowel diseases can result from a wide spectrum of rare mendelian disorders. Early molecular diagnosis is crucial in defining treatment and in improving life expectancy. Herein we aimed at defining the mechanism of an immunodeficiency-polyendrocrinopathy and enteropathy-X-linked (IPEX)-like disease combined with a severe immunodeficiency in 2 siblings born from distantly related parents. METHODS: Whole exome sequencing was performed on blood-extracted genomic DNA from the 2 affected children and their parents on the genomic platform of Institut IMAGINE. Candidate gene mutation was identified using the in-house software PolyWeb and confirmed by Sanger sequencing. Protein expression was determined by western blot. Flow cytometry was used to assess consequences of the mutation on lymphocyte phenotype and nuclear factor-kappa B (NF-κB) activation at diagnosis and after treatment by hematopoietic stem cell transplantation. RESULTS: We identified a homozygous missense mutation in mucosa-associated lymphoid tissue lymphoma translocation 1 gene (MALT1), which precluded protein expression. In keeping with the known function of MALT1, NF-κB-dependent lymphocyte activation was severely impaired. Moreover, there was a drastic reduction in Forkhead box P3 (FOXP3) regulatory T cells accounting for the IPEX-like phenotype. Following identification of the mutation, both children received hematopoietic stem cell transplantation, which permitted full clinical recovery. Immunological workup at 6 and 12 months after transplantation showed normal NF-κB activation and correction of regulatory T cells frequency. CONCLUSIONS: Along with FOXP3, interleukin 2 receptor alpha chain (IL2RA), and cytotoxic T-lymphocyte protein 4 precursor (CTLA-4) mutations, MALT1 deficiency should now be considered as a possible cause of IPEX-like syndrome associated with immunodeficiency that can be cured by hematopoietic stem cell transplantation.

Autoimmune and Inflammatory Manifestations in 247 Patients with Primary Immunodeficiency-a Report from the Slovenian National Registry.

An abnormal regulation of immune responses leads to autoimmune and inflammatory manifestations in patients with primary immunodeficiencies (PIDs). The objective of our study was to evaluate the frequency of non-infectious and non-malignant manifestations in a large cohort of patients included in the Slovenian national PID registry and to assess the time of manifestation onset with respect to the time of PID diagnosis. Medical records of registered patients were reviewed. Data on autoimmunity, lymphoproliferation, autoinflammation, allergies, PID diagnosis, and underlying genetic defects were collected and analyzed. The time of each manifestation onset was determined and compared with the time of PID diagnosis. As of May 2015, 247 patients with 50 different PIDs were registered in the Slovenian national PID registry (147 males, 100 females; mean age 20 years). Mean disease duration was 14 years; 78 % of patients were younger than 18 years; and 22 % of patients were adults. Diagnosis of PID was genetically confirmed in 51 % of patients. Non-infectious and non-malignant manifestations were present in 69/235 (29 %) patients, including autoimmune manifestations in 52/235 (22 %), lymphoproliferative/granulomatous in 28/235 (12 %), autoinflammatory in 12/247 (5 %), and allergic manifestations in 10/235 (4 %) of all registered patients. Autoimmune manifestations were present in all patients whose PIDs were classified as diseases of immune dysregulation, 47 % of patients with chronic granulomatous disease, and 38 % of patients with predominantly antibody immune deficiencies. A high prevalence of non-infectious and non-malignant manifestations among patients in the Slovenian national PID registry suggests common genetic factors of autoimmunity, inflammation, and immunodeficiency. Patients with PID should be routinely screened for autoimmune and inflammatory manifestations at the time of PID diagnosis and during the long-term follow up.

Long-term follow-up of 109 children with juvenile idiopathic oligoarthritis after first intra-articular corticosteroid injection.

BACKGROUND: To evaluate long-term outcomes and prognostic factors in patients with juvenile idiopathic arthritis (JIA), presenting as oligoarthritis, who received IAC as the first treatment for their disease. METHODS: We conducted retrospective study at the University Children's Hospital Ljubljana, Slovenia, from January 2015 to May 2023 in children with JIA, clinically presenting as oligoarthritis receiving intra-articular corticosteroid injection (IAC) as the initial treatment. Patient and treatment data were collected, and the outcomes were categorized into three groups based on the later need for therapy: no therapy needed, only additional IAC needed and systemic therapy needed. The last group was further divided based on the requirement of bDMARD. Log-rank (Mantel-Cox) survival analyses compared different outcome groups. RESULTS: We included 109 patients with JIA, presenting as oligoarthritis (63% female), who were first treated with IAC. The mean age at IAC was 8.0 years, with a 4.3-year follow-up. Notably, 38.5% of patients did not require additional therapy post-IAC, whereas 15.5% required only additional IAC. Systemic therapy, mainly methotrexate (MTX), was necessary for 45.9% of patients, initiated in average 7.8 months post-IAC. Biologic therapy was initiated in 22% in average 2.2 years post-IAC. Number of injected joints correlated with the need for biologics. At the last follow-up, 88.9% had inactive disease. ANA positivity (P = 0.049, chi square 3.89) and HLA B27 antigen presence (P = 0.050, chi square 3.85) were associated with the need for systemic therapy. A subgroup of children older than 8 years, ANA and HLA B27 negative required significantly less systemic (25.8%) and biologic therapy (9.6%) compared to other patients (p = 0.050, chi square 3.77). CONCLUSION: Almost 40% of children with oligoarticular JIA requiring IAC did not progress to chronic disease. Younger age, ANA positivity, and HLA B27 presence were predictive factors for systemic therapy, while the number of injected joints predicted the future need for biologic therapy.

Systemic auto-inflammatory manifestations in patients with spondyloarthritis.

OBJECTIVES: (1) characterizing a group of spondyloarthritis (SpA) patients with systemic auto-inflammatory symptoms (S-SpA); (2) comparing SpA features with and without auto-inflammatory symptoms; (3) comparing the auto-inflammatory features of S-SpA and Still's disease (SD). METHODS: Retrospective observational study. Clinical data of adult and pediatric patients with S-SpA, SD or SpA were collected retrospectively and analyzed. RESULTS: Forty-one subjects with S-SpA, 39 with SD and 42 with SpA were enrolled. The median latency between systemic and articular manifestations in S-SpA was 4.4 (IQR: 7.2) years. S-SpA and SpA had similar frequency of peripheral arthritis and enthesitis (N.S.), while tenosynovitis was more frequent (P=0.01) and uveitis less frequent (P<0.01) in S-SpA. MRI showed signs of sacroiliac inflammation and damage in both S-SpA and SpA equally (N.S.). S-SpA patients had less corner inflammatory lesions (P<0.05) and inflammation at the facet joints (P<0.01), more interspinous enthesitis (P=0.01) and inter-apophyseal capsulitis (P<0.01). Compared to SD, S-SpA patients had lower-grade fever (P<0.01), less rash (P<0.01) and weight loss (P<0.05), but more pharyngitis (P<0.01), gastrointestinal symptoms (P<0.01) and chest pain (P<0.05). ESR, CRP, WBC, ANC, LDH tested higher in SD (P<0.01). Resolution of systemic symptoms was less frequent in S-SpA than SD on corticosteroid (P<0.01) and methotrexate (P<0.05) treatment. When considering all SD patients, a complete response to corticosteroids in the systemic phase significantly reduced the likelihood of developing SpA (OR=0.06, coefficient -2.87 [CI: -5.0 to -0.8]). CONCLUSIONS: SpA should be actively investigated in patients with auto-inflammatory manifestations, including undifferentiated auto-inflammatory disease and SD.

Case report: The success of empagliflozin therapy for glycogen storage disease type 1b.

Introduction: Glycogen storage disease type 1b (GSD-1b) is characterized by neutropenia and neutrophil dysfunction generated by the accumulation of 1,5-anhydroglucitol-6-phosphate in neutrophils. Sodium-glucose co-transporter 2 inhibitors, such as empagliflozin, facilitate the removal of this toxic metabolite and ameliorate neutropenia-related symptoms, including severe infections and inflammatory bowel disease (IBD). Our case series presents the treatment of three pediatric GSD-1b patients with empagliflozin over a follow-up of three years; the most extended reported follow-up period to date. Cases description: A retrospective analysis of empagliflozin treatment of three pediatric GSD-1b patients (two male and one female; ages at treatment initiation: 4.5, 2.5 and 6 years) was performed. Clinical and laboratory data from a symmetrical period of up to three years before and after the therapy introduction was reported. Data on the clinical course of the treatment, IBD activity, the need for antibiotic treatment and hospitalizations, neutrophil count and function, and markers of inflammation were assessed. Prior the introduction of empagliflozin, patients had recurrent oral mucosa lesions and infections, abdominal pain, and anemia. During empagliflozin treatment, the resolution of aphthous stomatitis, termination of abdominal pain, reduced frequency and severity of infections, anemia resolution, increased appetite, and improved wound healing was observed in all patients, as well as an increased body mass index in two of them. In a patient with IBD, long-term deep remission was confirmed. An increased and stabilized neutrophil count and an improved neutrophil function enabled the discontinuation of G-CSF treatment in all patients. A trend of decreasing inflammation markers was detected. Conclusions: During the three-year follow-up period, empagliflozin treatment significantly improved clinical symptoms and increased the neutrophil count and function, suggesting that targeted metabolic treatment could improve the immune function in GSD-1b patients.

Comprehensive Care and Education Can Improve Nutritional and Growth Outcomes in Children with Persistent Cow's Milk, Egg, or Peanut Allergies: A Five-Year Follow-Up Study of Nutritional Status.

BACKGROUND: Data suggest that food allergies greatly impact a child's health and growth due to inadequate nutrient intake. Our study aimed to establish the long-term outcome of children with food allergies compared to a control group. METHODS: This study was a retrospective cohort study with longitudinal follow-up with a mean period of 4.85 years from the diagnosis to the last study visit. The patients' nutritional intake was assessed using a three-day food diary and analysed by a dietitian. Patients (61 boys and 33 girls, mean age 6.9 years) had a single food allergy including 21 patients with cow's milk, 34 with egg, and 39 with peanut allergies. The control group included 36 children (19 boys and 17 girls, mean age 8.03 years). Blood analysis was performed on all participants. RESULTS: Data from our study showed that patients with cow's milk, egg or peanut allergies had normal growth and achieved catch-up growth from the diagnosis until the last study visit. In the cow's milk allergy group, the allergy was shown to affect calcium intake (p < 0.05), while egg and peanut allergies did not impact the dietary intake of nutrients. None of the investigated food allergies affected blood results (p < 0.05). CONCLUSIONS: In the present study, we showed that single food allergies do not compromise growth in children if they are provided with appropriate support and that the affected children reach catch-up growth from the diagnosis.

Clinical and genetic characteristics of a patient with phosphoribosyl pyrophosphate synthetase 1 deficiency and a systematic literature review.

Phosphoribosylpyrophosphate synthetase 1 (PRS-I) is an enzyme involved in nucleotide metabolism. Pathogenic variants in the PRPS1 are rare and PRS-I deficiency can manifest as three clinical syndromes: X-linked non-syndromic sensorineural deafness (DFN2), X-linked Charcot-Marie-Tooth neuropathy type 5 (CMTX5) and Arts syndrome. We present a Slovenian patient with PRS-I enzyme deficiency due to a novel pathogenic variant - c.424G > A (p.Val142Ile) in the PRPS1 gene, who presented with gross motor impairment, severe sensorineural deafness, balance issues, ataxia, and frequent respiratory infections. In addition, we report the findings of a systemic literature review of all described male cases of Arts syndrome and CMTX5 as well as intermediate phenotypes. As already proposed by other authors, our results confirm PRS-I deficiency should be viewed as a phenotypic continuum rather than three separate syndromes because there are multiple reports of patients with an intermediary clinical presentation.

Autoimmune and autoinflammatory manifestations in inborn errors of immunity.

PURPOSE OF REVIEW: Autoimmune and inflammatory complications have been shown to arise in all age groups and across the spectrum of inborn errors of immunity (IEI). This review aims to highlight recent ground-breaking research and its impact on our understanding of IEI. RECENT FINDINGS: Three registry-based studies of unprecedented size revealed the high prevalence of autoimmune, inflammatory and malignant complications in IEI. Two novel IEI were discovered: an autoinflammatory relopathy, cleavage-resistant RIPK1-induced autoinflammatory syndrome, as well as an inheritable phenocopy of PD-1 blockade-associated complication (as seen in cancer therapy) manifesting with multiorgan autoimmunity and Mycobacterium tuberculosis infection. A study examining patients with partial RAG deficiency pinpointed the specific defects leading to the failure of central and peripheral tolerance resulting in wide-ranging autoimmunity. A novel variant of Immunodeficiency Polyendocrinopathy Enteropathy X-linked syndrome was described, associated with preferential expression of a FOXP3 isoform lacking exon 2, linking exon-specific functions and the phenotypes corresponding to their absence. Lastly, we touch on recent findings pertaining actinopathies, the prototypical IEI with autoimmune, inflammatory and atopic complications. SUMMARY: Dysregulated immunity has been associated with IEI since their discovery. Recently, large concerted efforts have shown how common these complications actually are while providing insight into normal and dysregulated molecular mechanisms, as well as describing novel diseases.

Care of patients with inborn errors of immunity in thirty J Project countries between 2004 and 2021.

Introduction: The J Project (JP) physician education and clinical research collaboration program was started in 2004 and includes by now 32 countries mostly in Eastern and Central Europe (ECE). Until the end of 2021, 344 inborn errors of immunity (IEI)-focused meetings were organized by the JP to raise awareness and facilitate the diagnosis and treatment of patients with IEI. Results: In this study, meeting profiles and major diagnostic and treatment parameters were studied. JP center leaders reported patients' data from 30 countries representing a total population of 506 567 565. Two countries reported patients from JP centers (Konya, Turkey and Cairo University, Egypt). Diagnostic criteria were based on the 2020 update of classification by the IUIS Expert Committee on IEI. The number of JP meetings increased from 6 per year in 2004 and 2005 to 44 and 63 in 2020 and 2021, respectively. The cumulative number of meetings per country varied from 1 to 59 in various countries reflecting partly but not entirely the population of the respective countries. Altogether, 24,879 patients were reported giving an average prevalence of 4.9. Most of the patients had predominantly antibody deficiency (46,32%) followed by patients with combined immunodeficiencies (14.3%). The percentages of patients with bone marrow failure and phenocopies of IEI were less than 1 each. The number of patients was remarkably higher that those reported to the ESID Registry in 13 countries. Immunoglobulin (IgG) substitution was provided to 7,572 patients (5,693 intravenously) and 1,480 patients received hematopoietic stem cell therapy (HSCT). Searching for basic diagnostic parameters revealed the availability of immunochemistry and flow cytometry in 27 and 28 countries, respectively, and targeted gene sequencing and new generation sequencing was available in 21 and 18 countries. The number of IEI centers and experts in the field were 260 and 690, respectively. We found high correlation between the number of IEI centers and patients treated with intravenous IgG (IVIG) (correlation coefficient, cc, 0,916) and with those who were treated with HSCT (cc, 0,905). Similar correlation was found when the number of experts was compared with those treated with HSCT. However, the number of patients treated with subcutaneous Ig (SCIG) only slightly correlated with the number of experts (cc, 0,489) and no correlation was found between the number of centers and patients on SCIG (cc, 0,174). Conclusions: 1) this is the first study describing major diagnostic and treatment parameters of IEI care in countries of the JP; 2) the data suggest that the JP had tremendous impact on the development of IEI care in ECE; 3) our data help to define major future targets of JP activity in various countries; 4) we suggest that the number of IEI centers and IEI experts closely correlate to the most important treatment parameters; 5) we propose that specialist education among medical professionals plays pivotal role in increasing levels of diagnostics and adequate care of this vulnerable and still highly neglected patient population; 6) this study also provides the basis for further analysis of more specific aspects of IEI care including genetic diagnostics, disease specific prevalence, newborn screening and professional collaboration in JP countries.

Clinical and immunological manifestations of patients with atypical severe combined immunodeficiency.

Hypomorphic mutations in genes associated with severe combined immunodeficiency (SCID) or Omenn syndrome can also cause milder immunodeficiencies. We report 10 new patients with such "atypical" SCID and summarize 63 patients from the literature. The patient groups with T(low)B(low) (n=28), T(low)B(+) (n=16) and ADA (n=29) SCID variants had similar infection profiles but differed in the frequency of immune dysregulation, which was observed predominantly in patients with recombination defects. Most immunological parameters were remarkably similar in the three groups. Of note, 19/68 patients with "atypical" SCID had normal T cell counts, 48/68 had normal IgG and 23/46 had at least one normal specific antibody titer. Elevated IgE was a characteristic feature of ADA deficiency. This overview characterizes "atypical" SCID as a distinct disease with immune dysregulation in addition to infection susceptibility. Lymphopenia, reduced naïve T cells and elevated IgE are suggestive, but not consistent features of the disease.

The expansion of human T-bethighCD21low B cells is T cell dependent.

Accumulation of human CD21low B cells in peripheral blood is a hallmark of chronic activation of the adaptive immune system in certain infections and autoimmune disorders. The molecular pathways underpinning the development, function, and fate of these CD21low B cells remain incompletely characterized. Here, combined transcriptomic and chromatin accessibility analyses supported a prominent role for the transcription factor T-bet in the transcriptional regulation of these T-bethighCD21low B cells. Investigating essential signals for generating these cells in vitro established that B cell receptor (BCR)/interferon-γ receptor (IFNγR) costimulation induced the highest levels of T-bet expression and enabled their differentiation during cell cultures with Toll-like receptor (TLR) ligand or CD40L/interleukin-21 (IL-21) stimulation. Low proportions of CD21low B cells in peripheral blood from patients with defined inborn errors of immunity (IEI), because of mutations affecting canonical NF-κB, CD40, and IL-21 receptor or IL-12/IFNγ/IFNγ receptor/signal transducer and activator of transcription 1 (STAT1) signaling, substantiated the essential roles of BCR- and certain T cell­derived signals in the in vivo expansion of T-bethighCD21low B cells. Disturbed TLR signaling due to MyD88 or IRAK4 deficiency was not associated with reduced CD21low B cell proportions. The expansion of human T-bethighCD21low B cells correlated with an expansion of circulating T follicular helper 1 (cTfh1) and T peripheral helper (Tph) cells, identifying potential sources of CD40L, IL-21, and IFNγ signals. Thus, we identified important pathways to target autoreactive T-bethighCD21low B cells in human autoimmune conditions, where these cells are linked to pathogenesis and disease progression.

Wiskott Aldrich syndrome protein regulates non-selective autophagy and mitochondrial homeostasis in human myeloid cells.

The actin cytoskeletal regulator Wiskott Aldrich syndrome protein (WASp) has been implicated in maintenance of the autophagy-inflammasome axis in innate murine immune cells. Here, we show that WASp deficiency is associated with impaired rapamycin-induced autophagosome formation and trafficking to lysosomes in primary human monocyte-derived macrophages (MDMs). WASp reconstitution in vitro and in WAS patients following clinical gene therapy restores autophagic flux and is dependent on the actin-related protein complex ARP2/3. Induction of mitochondrial damage with CCCP, as a model of selective autophagy, also reveals a novel ARP2/3-dependent role for WASp in formation of sequestrating actin cages and maintenance of mitochondrial network integrity. Furthermore, mitochondrial respiration is suppressed in WAS patient MDMs and unable to achieve normal maximal activity when stressed, indicating profound intrinsic metabolic dysfunction. Taken together, we provide evidence of new and important roles of human WASp in autophagic processes and immunometabolic regulation, which may mechanistically contribute to the complex WAS immunophenotype.

The Clinical and Genetic Spectrum of 82 Patients With RAG Deficiency Including a c.256_257delAA Founder Variant in Slavic Countries.

Background: Variants in recombination-activating genes (RAG) are common genetic causes of autosomal recessive forms of combined immunodeficiencies (CID) ranging from severe combined immunodeficiency (SCID), Omenn syndrome (OS), leaky SCID, and CID with granulomas and/or autoimmunity (CID-G/AI), and even milder presentation with antibody deficiency. Objective: We aim to estimate the incidence, clinical presentation, genetic variability, and treatment outcome with geographic distribution of patients with the RAG defects in populations inhabiting South, West, and East Slavic countries. Methods: Demographic, clinical, and laboratory data were collected from RAG-deficient patients of Slavic origin via chart review, retrospectively. Recombinase activity was determined in vitro by flow cytometry-based assay. Results: Based on the clinical and immunologic phenotype, our cohort of 82 patients from 68 families represented a wide spectrum of RAG deficiencies, including SCID (n = 20), OS (n = 37), and LS/CID (n = 25) phenotypes. Sixty-seven (81.7%) patients carried RAG1 and 15 patients (18.3%) carried RAG2 biallelic variants. We estimate that the minimal annual incidence of RAG deficiency in Slavic countries varies between 1 in 180,000 and 1 in 300,000 live births, and it may vary secondary to health care disparities in these regions. In our cohort, 70% (n = 47) of patients with RAG1 variants carried p.K86Vfs*33 (c.256_257delAA) allele, either in homozygous (n = 18, 27%) or in compound heterozygous (n = 29, 43%) form. The majority (77%) of patients with homozygous RAG1 p.K86Vfs*33 variant originated from Vistula watershed area in Central and Eastern Poland, and compound heterozygote cases were distributed among all Slavic countries except Bulgaria. Clinical and immunological presentation of homozygous RAG1 p.K86Vfs*33 cases was highly diverse (SCID, OS, and AS/CID) suggestive of strong influence of additional genetic and/or epigenetic factors in shaping the final phenotype. Conclusion: We propose that RAG1 p.K86Vfs*33 is a founder variant originating from the Vistula watershed region in Poland, which may explain a high proportion of homozygous cases from Central and Eastern Poland and the presence of the variant in all Slavs. Our studies in this cohort of RAG1 founder variants confirm that clinical and immunological phenotypes only partially depend on the underlying genetic defect. As access to HSCT is improving among RAG-deficient patients in Eastern Europe, we anticipate improvements in survival.

Flow Cytometric Determination of Actin Polymerization in Peripheral Blood Leukocytes Effectively Discriminate Patients With Homozygous Mutation in ARPC1B From Asymptomatic Carriers and Normal Controls.

Actin nucleators initiate formation of actin filaments. Among them, the Arp2/3 complex has the ability to form branched actin networks. This complex is regulated by members of the Wiscott-Aldrich syndrome protein (WASp) family. Polymerization of actin filaments can be evaluated through flow cytometry by fluorescent phalloidin staining before and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). We identified a missense mutation in the gene ARPC1B (Arp2/3 activator subunit) resulting in defective actin polymerization in four patients (three of them were related). All patients (1 male, 3 female) developed microthrombocytopenia, cellular immune deficiency, eczema, various autoimmune manifestations, recurrent skin abscesses and elevated IgE antibodies. Besides four patients with homozygous mutation in ARPC1B, we also identified six heterozygous carriers without clinical disease (3 males, 3 females) within the same family. We developed a functional test to evaluate Arp2/3 complex function, which consists of flow cytometric detection of intracellular polymerized actin after in vitro fMLP stimulation of leukocytes. Median fluorescence intensities of FITC-phalloidin stained actin were measured in monocytes, neutrophils and lymphocytes of patients, carriers, and healthy control subjects. We detected non-efficient actin polymerization in monocytes and neutrophils of homozygous patients compared to carriers or the healthy subjects. In monocytes, the increase in median fluorescence intensities was significantly lower in patients compared to carriers (104 vs. 213%; p < 0.01) and healthy controls (104 vs. 289%; p < 0.01). Similarly, the increase in median fluorescence intensities in neutrophils was significantly increased in the group with carriers (208%; p < 0.01) and healthy controls (238%; p < 0.01) and significantly decreased in the patient's group (94%). Our functional fMLP/phalloidin test can therefore be used as a practical tool to separate symptomatic patients from asymptomatic mutation associated to actin polymerization.

Functional Complement Analysis Can Predict Genetic Testing Results and Long-Term Outcome in Patients With Complement Deficiencies.

Background: Prevalence of complement deficiencies (CDs) is markedly higher in Slovenian primary immunodeficiency (PID) registry in comparison to other national and international PID registries. Objective: The purposes of our study were to confirm CD and define complete and partial CD in registered patients in Slovenia, to evaluate frequency of clinical manifestations, and to assess the risk for characteristic infections separately for subjects with complete and partial CD. Methods: CD was confirmed with genetic analyses in patients with C2 deficiency, C8 deficiency, and hereditary angioedema or with repeated functional complement studies and measurement of complement components in other CD. Results of genetic studies (homozygous subjects vs. heterozygous carriers) and complement functional studies were analyzed to define complete (complement below the level of heterozygous carriers) and partial CD (complement above the level of homozygous patients). Presence of characteristic infections was assessed separately for complete and partial CD. Results: Genetic analyses confirmed markedly higher prevalence of CD in Slovenian PID registry (26% of all PID) than in other national and international PID registries (0.5-6% of all PID). Complement functional studies and complement component concentrations reliably distinguished between homozygous and heterozygous CD carriers. Subjects with partial CD had higher risk for characteristic infections than previously reported. Conclusion: Results of our study imply under-recognition of CD worldwide. Complement functional studies and complement component concentrations reliably predicted risk for characteristic infections in patients with complete or partial CD. Vaccination against encapsulated bacteria should be advocated also for subjects with partial CD and not limited to complete CD.