Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis.

In monogenic autoinflammatory diseases, mutations in genes regulating innate immune responses often lead to uncontrolled activation of inflammasome pathways or the type I interferon (IFN-I) response. We describe a mechanism of autoinflammation potentially predisposing patients to life-threatening necrotizing soft tissue inflammation. Six unrelated families are identified in which affected members present with necrotizing fasciitis or severe soft tissue inflammations. Exome sequencing reveals truncating monoallelic loss-of-function variants of nuclear factor κ light-chain enhancer of activated B cells (NFKB1) in affected patients. In patients' macrophages and in NFKB1-variant-bearing THP-1 cells, activation increases both interleukin (IL)-1ß secretion and IFN-I signaling. Truncation of NF-κB1 impairs autophagy, accompanied by the accumulation of reactive oxygen species and reduced degradation of inflammasome receptor nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein 3 (NLRP3), and Toll/IL-1 receptor domain-containing adaptor protein inducing IFN-ß (TRIF), thus leading to combined excessive inflammasome and IFN-I activity. Many of the patients respond to anti-inflammatory treatment, and targeting IL-1ß and/or IFN-I signaling could represent a therapeutic approach for these patients.

Hybrid Immunity Improves the Immune Response after the Fourth COVID-19 Vaccine Dose in Individuals with Medical Conditions Predisposing to Severe COVID-19.

Data on immune responses following COVID-19 booster vaccinations and subsequent infections in the immunocompromised are limited. We studied antibody responses after the fourth dose and subsequent infections to define patient groups benefiting most from boosters. Fourth vaccine (booster) doses were, in Finland, first recommended for severely immunocompromised individuals, whom we invited to participate in our study in 2022. We assessed spike protein-specific IgG and neutralizing antibodies (NAb) against the ancestral and Omicron BA.1 strains one month after the fourth dose from 488 adult participants and compared them to the levels of 35 healthy controls after three doses. We used Bayesian generalized linear modeling to assess factors explaining antibody levels and assessed vaccine-induced and hybrid immunity six months after the last vaccine dose. Chronic kidney disease (CKD) and immunosuppressive therapy (IT) were identified as factors explaining sub-optimal antibody responses. The proportion of participants with a normal antibody response and NAbs was significantly lower regarding CKD patients compared to the controls. By the 6-month sampling point, one-third of the participants became infected (documented by serology and/or molecular tests), which notably enhanced antibody levels in most immunocompromised participants. Impaired antibody responses, especially NAbs against the Omicron lineage, suggest limited protection in individuals with CKD and highlight the need for alternative pharmaceutical preventive strategies. Vaccination strategies should take into account the development of robust hybrid immunity responses also among the immunocompromised.

Clinical and functional spectrum of RAC2-related immunodeficiency.

ABSTRACT: Mutations in the small Rho-family guanosine triphosphate hydrolase RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 patients (23 previously reported) from 37 families yielding 15 novel RAC2 missense mutations, including one present only in homozygosity. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n = 5), infantile LAD-like disease (n = 5), or CID (n = 44). Disease correlated to RAC2 activity: constitutively active RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, whereas dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration, and visible neutrophil macropinosomes. Among 42 patients with CID with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, p21-activated kinase 1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction, ranging from early onset SCID to later-onset combined immunodeficiencies depending on RAC2 activity. This trial was registered at www.clinicaltrials.gov as #NCT00001355 and #NCT00001467.

Long-term follow up of families with pathogenic NFKB1 variants reveals incomplete penetrance and frequent inflammatory sequelae.

Nuclear factor κ light-chain enhancer of activated B cells (NF-κB) family of evolutionarily conserved transcription factors are involved in key cellular signaling pathways. Previously, hypogammaglobulinemia and common variable immunodeficiency (CVID)-like phenotypes have been associated with NFKB1 variants and loss-of-function NFKB1 variants have been reported as the most common monogenic cause for CVID among Europeans. Here, we describe a Finnish cohort of NFKB1 carriers consisting of 31 living subjects in six different families carrying five distinct heterozygous variants. In contrast to previous reports, the clinical penetrance was not complete even with advancing age and the prevalence of CVID/hypogammaglobulinemia was significantly lower, whereas (auto)inflammatory manifestations were more common (42% of the total cohort). At current stage of knowledge, routine genetic screening of asymptomatic individuals is not recommended, but counseling of potential adult carriers seems necessary.

Pulmonary Follow-Up Imaging in Cartilage-Hair Hypoplasia: a Prospective Cohort Study.

Cartilage-hair hypoplasia is a syndromic immunodeficiency with short stature, chondrodysplasia, and variable degree of immune dysfunction. Patients with cartilage-hair hypoplasia are prone to recurrent respiratory tract infections, and the prevalence of bronchiectasis ranges from 29 to 52%. Pulmonary complications contribute significantly to the mortality; therefore, regular lung imaging is essential. However, the optimal schedule for repeated lung imaging remains unestablished. We determined the rate and correlates of progression of structural lung changes in a prospectively followed cohort of 16 patients with cartilage-hair hypoplasia. We analyzed clinical, laboratory, and pulmonary functional testing data and performed lung magnetic resonance imaging at a median interval of 6.8 years since previous imaging. Imaging findings remained identical or improved due to disappearance of inflammatory changes in all evaluated patients. Patients with subtle signs of bronchiectasis on imaging tended to have low immunoglobulin M levels, as well as suffered from pneumonia during the follow-up. In conclusion, our results suggest slow if any development of bronchiectasis in selected subjects with cartilage-hair hypoplasia.

Somatic mutations and T-cell clonality in patients with immunodeficiency.

Common variable immunodeficiency and other late-onset immunodeficiencies often co-manifest with autoimmunity and lymphoproliferation. The pathogenesis of most cases is elusive, as only a minor subset harbors known monogenic germline causes. The involvement of both B and T cells is however implicated. To study whether somatic mutations in CD4+ and CD8+ T cells associate with immunodeficiency, we recruited 17 patients and 21 healthy controls. Eight patients had late-onset common variable immunodeficiency and nine patients other immunodeficiency and/or severe autoimmunity. In total, autoimmunity occurred in 94% and lymphoproliferation in 65%. We performed deep sequencing of 2533 immune-associated genes from CD4+ and CD8+ cells. Deep T-cell receptor beta sequencing was used to characterize CD4+ and CD8+ T-cell receptor repertoires. The prevalence of somatic mutations was 65% in all immunodeficiency patients, 75% in common variable immunodeficiency and 48% in controls. Clonal hematopoiesis-associated variants in both CD4+ and CD8+ cells occurred in 24% of immunodeficiency patients. Results demonstrated mutations in known tumor suppressors, oncogenes, and genes that are critical for immune- and proliferative functions, such as STAT5B (two patients), C5AR1 (two patients), KRAS (one patient), and NOD2 (one patient). Additionally, as a marker of T-cell receptor repertoire perturbation, common variable immunodeficiency patients harbored increased frequencies of clones with identical complementarity determining region 3 sequences despite unique nucleotide sequences when compared to controls. In conclusion, somatic mutations in genes implicated for autoimmunity and lymphoproliferation are common in CD4+ and CD8+ cells of patients with immunodeficiency. They may contribute to immune dysregulation in a subset of immunodeficiency patients.

Three Separate Clinical Entities of Infective Endocarditis-A Population-Based Study From Southern Finland 2013-2017.

BACKGROUND: Health care-associated infective endocarditis (HAIE) and intravenous drug use-related IE (IDUIE) have emerged as major groups in infective endocarditis (IE). We studied their role and clinical picture in a population-based survey. METHODS: A population-based retrospective study including all adult patients diagnosed with definite or possible IE in Southern Finland in 2013-2017. IE episodes were classified according to the mode of acquisition into 3 groups: community-acquired IE (CAIE), HAIE, and IDUIE. RESULTS: Total of 313 episodes arising from 291 patients were included. Incidence of IE was 6.48/100 000 person-years. CAIE accounted for 38%, HAIE 31%, and IDUIE 31% of IE episodes. Patients in the IDUIE group were younger, and they more frequently had right-sided IE (56.7% vs 5.0%; P < .001) and S. aureus as etiology (74.2% vs 17.6%; P < .001) compared with the CAIE group. In-hospital (15.1% vs 9.3%; P = .200) and cumulative 1-year case fatality rates (18.5% vs 17.5%; P = .855) were similar in CAIE and IDUIE. Patients with HAIE had more comorbidities, prosthetic valve involvement (29.9% vs 10.9%; P = .001), enterococcal etiology (20.6% vs 5.9%; P = .002), and higher in-hospital (27.8% vs 15.1%; P = .024) and cumulative 1-year case fatality rates (43.3% vs 18.5%; P < .001) than patients with CAIE. Staphylococcus aureus caused one-fifth of IE episodes in both groups. CONCLUSIONS: Our study indicates that in areas where injection drug use is common IDUIE should be regarded as a major risk group for IE, along with HAIE, and not seen as part of CAIE. Three different risk groups, CAIE, HAIE, and IDUIE, with variable characteristics and outcome should be recognized in IE.

A High Prevalence of Gastrointestinal Manifestations in Common Variable Immunodeficiency.

OBJECTIVES: Common variable immunodeficiency (CVID) is associated with a spectrum of autoimmune complications. We studied the prevalence of gastrointestinal (GI) manifestations and infections in patients with CVID. METHODS: Complete clinical data of 132 Finnish patients with CVID (106 probable and 26 possible CVID) followed up between 2007 and 2016 were collected to a structured database. Data on endoscopies, histology, and laboratory studies were retrieved from patient files. RESULTS: Most common referral indications were diarrhea and/or weight loss (47%-67%). Patients with probable CVID had higher fecal calprotectin and α1-antitrypsin and lower blood vitamin B12 than patients with possible CVID. Gastroscopy and colonoscopy were done to 71 (67%) and 63 (59%) patients with probable CVID, respectively. Endoscopies showed that 15% of them had chronic active gastritis and 17% atrophic gastritis and 3% had gastric adenocarcinoma. A celiac sprue-like condition was found in 7 patients (10%), of whom 3 responded to a gluten-free diet. Colonoscopies demonstrated unspecific colitis (14%), ulcerative colitis (8%), microscopic colitis (10%), and Crohn's disease (2%). Colonic polyps were noted in 30% of patients, and 3% had lower GI malignancies. Thirty-five patients with CVID had bacterial or parasitic gastroenteritis; chronic norovirus was detected in 4 patients with probable CVID. Patients with GI inflammation had higher levels of fecal calprotectin and blood CD8 T lymphocytes but lower counts of CD19CD27 memory B cells and/or CD19 B cells. Immunophenotype with low B-cell counts was associated with higher fecal calprotectin levels. DISCUSSION: Patients with CVID had a high prevalence of GI manifestations and infections of the GI tract. GI inflammation was associated with a distinct immunophenotype and elevated fecal calprotectin.

Impact of pre-operative antimicrobial treatment on microbiological findings from endocardial specimens in infective endocarditis.

Treatment of infective endocarditis (IE) should be initiated promptly. This might hamper the chances to identify the causative organism in blood cultures. Microbiological sampling of infected valve in patients undergoing surgery might identify the causative organism. The impact of pre-operative antimicrobial treatment on the yield of valve samples is not known. This study evaluated the impact of the duration of the pre-operative antibiotic treatment on valve culture and 16S rRNA PCR findings from resected endocardial samples. Patients meeting the modified Duke criteria of definite or possible IE and undergoing valve surgery due to IE during 2011-2016 were included from Southern Finland. Eighty-seven patients were included. In patients with shorter than 2 weeks of pre-operative antimicrobial treatment, PCR was positive in 91% (n = 42/46) and valve culture in 41% (n = 19/46) of cases. However, in patients who had 2 weeks or longer therapy before operation, PCR was positive in 53% (n = 18/34) and all valve cultures were negative. In 14% of patients, PCR had a diagnostic impact. In blood-culture negative cases (n = 13), PCR could detect the causative organism in ten patients (77%). These included five cases of Bartonella quintana, one Tropheryma whipplei, and one Coxiella burnetii. Long pre-operative antimicrobial treatment was shown to have a negative impact on microbiological tests done on resected endocardial material. After 2 weeks of therapy, all valve cultures were negative, but PCR was positive in half of the cases. PCR aided in diagnostic work-up, especially in blood culture negative cases.

Impact of short-incubation MALDI-TOF MS on empiric antibiotic therapy in bloodstream infections caused by Pseudomonas aeruginosa, Enterococcus spp. and AmpC-producing Enterobacteriaceae.

The impact of the short-incubation matrix-assisted laser desorption ionization time-of-flight (si-MALDI-TOF) mass spectrometry technique was evaluated in the treatment of bloodstream infections (BSIs) caused by Pseudomonas aeruginosa, Enterococcus spp., and Amp-C producing Enterobacteriaceae. A total of 124 bacteremia episodes were divided into 2 groups: i) si-MALDI-TOF group (n = 69) and ii) control group (n = 55). Identification by si-MALDI-TOF resulted in 12.8% increase in cases receiving appropriate antibiotic treatment within 48 h from blood culture draw. The importance of the rapid identification was emphasized in BSIs caused by enterococci (n = 62), where si-MALDI-TOF led to appropriate antibiotic treatment in 87.9% of cases (versus control group 65.5%, P = 0.036). Implementation of si-MALDI-TOF technology for microbial identification was associated with increased proportion of patients receiving effective antibiotic treatment within 48 h from blood culture draw. The effect was most significant in BSIs caused by enterococcal species and in a subgroup of immunosuppressed patients.

Diagnostics of rare disorders: whole-exome sequencing deciphering locus heterogeneity in telomere biology disorders.

BACKGROUND: The telomere biology disorders (TBDs) include a range of multisystem diseases characterized by mucocutaneous symptoms and bone marrow failure. In dyskeratosis congenita (DKC), the clinical features of TBDs stem from the depletion of crucial stem cell populations in highly proliferative tissues, resulting from abnormal telomerase function. Due to the wide spectrum of clinical presentations and lack of a conclusive laboratory test it may be challenging to reach a clinical diagnosis, especially if patients lack the pathognomonic clinical features of TBDs. METHODS: Clinical sequencing was performed on a cohort of patients presenting with variable immune phenotypes lacking molecular diagnoses. Hypothesis-free whole-exome sequencing (WES) was selected in the absence of compelling diagnostic hints in patients with variable immunological and haematological conditions. RESULTS: In four patients belonging to three families, we have detected five novel variants in known TBD-causing genes (DKC1, TERT and RTEL1). In addition to the molecular findings, they all presented shortened blood cell telomeres. These findings are consistent with the displayed TBD phenotypes, addressing towards the molecular diagnosis and subsequent clinical follow-up of the patients. CONCLUSIONS: Our results strongly support the utility of WES-based approaches for routine genetic diagnostics of TBD patients with heterogeneous or atypical clinical presentation who otherwise might remain undiagnosed.

Unexpectedly High Prevalence of Common Variable Immunodeficiency in Finland.

BACKGROUND: Common variable immunodeficiency (CVID) is the most common primary immunodeficiency. Prevalence varies greatly between countries and studies. Most diagnostic criteria include hypogammaglobulinemia and impaired vaccine response. AIM: To evaluate the minimum prevalence as well as the clinical and immunological phenotypes of CVID in Southern Finland. METHODS: We performed a cross-sectional study to assess all adult CVID patients followed up in three hospital districts in Southern and South-Eastern Finland between April 2007 and August 2015. CVID diagnosis was based, with a minor modification, on the ESID/PAGID criteria for primary CVID. Antipolysaccharide responses to Pneumovax® were defined as impaired only if 50% or more of the serotypes did not reach a level of 0.35 µg/mL after vaccination. We further characterized the patients' B cell phenotypes and complications associated with CVID. RESULTS: In total, 9 patients were excluded due to potential secondary causes before diagnosis. ESID/PAGID criteria were met by 132 patients (males 52%), of whom, 106 had "probable" and 26 "possible CVID." Based on the population statistics in the three hospital districts, the minimum adult prevalence per 100,000 inhabitants in Finland for all CVID ("probable CVID," respectively) patients was 6.9 (5.5). In the highest prevalence district (Helsinki and Uusimaa), the prevalence was 7.7 (6.1). CVID patients suffer from frequent complications. Ten patients died during follow-up. Of probable CVID patients, 73% had more than one clinical phenotype. Intriguingly, gradual B cell loss from peripheral blood during follow-up was seen in as many as 16% of "probable CVID" patients. Patients with possible CVID displayed somewhat milder clinical and laboratory phenotypes than probable CVID patients. We also confirm that large granular lymphocyte lymphoproliferation is a CVID-associated complication. CONCLUSION: The prevalence of CVID in Finland appears the highest recorded, likely reflecting the genetic isolation and potential founder effects in the Finnish population. Studies to discover potential gene variants responsible for the high prevalence in Finland thus seem warranted. Increased awareness of CVID among physicians would likely lead to earlier diagnosis and improved quality of care.

Damaging heterozygous mutations in NFKB1 lead to diverse immunologic phenotypes.

BACKGROUND: The nuclear factor κ light-chain enhancer of activated B cells (NF-κB) signaling pathway is a key regulator of immune responses. Accordingly, mutations in several NF-κB pathway genes cause immunodeficiency. OBJECTIVE: We sought to identify the cause of disease in 3 unrelated Finnish kindreds with variable symptoms of immunodeficiency and autoinflammation. METHODS: We applied genetic linkage analysis and next-generation sequencing and functional analyses of NFKB1 and its mutated alleles. RESULTS: In all affected subjects we detected novel heterozygous variants in NFKB1, encoding for p50/p105. Symptoms in variant carriers differed depending on the mutation. Patients harboring a p.I553M variant presented with antibody deficiency, infection susceptibility, and multiorgan autoimmunity. Patients with a p.H67R substitution had antibody deficiency and experienced autoinflammatory episodes, including aphthae, gastrointestinal disease, febrile attacks, and small-vessel vasculitis characteristic of Behçet disease. Patients with a p.R157X stop-gain experienced hyperinflammatory responses to surgery and showed enhanced inflammasome activation. In functional analyses the p.R157X variant caused proteasome-dependent degradation of both the truncated and wild-type proteins, leading to a dramatic loss of p50/p105. The p.H67R variant reduced nuclear entry of p50 and showed decreased transcriptional activity in luciferase reporter assays. The p.I553M mutation in turn showed no change in p50 function but exhibited reduced p105 phosphorylation and stability. Affinity purification mass spectrometry also demonstrated that both missense variants led to altered protein-protein interactions. CONCLUSION: Our findings broaden the scope of phenotypes caused by mutations in NFKB1 and suggest that a subset of autoinflammatory diseases, such as Behçet disease, can be caused by rare monogenic variants in genes of the NF-κB pathway.

Prognosis of Good syndrome: mortality and morbidity of thymoma associated immunodeficiency in perspective.

Good syndrome (GS) or thymoma-associated immunodeficiency, is a rare condition that has only been studied in retrospective case series. General consensus was that GS has a worse prognosis than other humoral immunodeficiencies. In this study, physicians of GS patients completed two questionnaires with a two year interval with data on 47 patients, 499 patient years in total. Results on epidemiology, disease characteristics, and outcome are presented. Mean age at diagnosis was 60years and median follow-up from onset of symptoms was 9years. There was a high frequency of respiratory tract infections due to encapsulated bacteria. Median survival was 14years. Survival was reduced compared to age-matched population controls (5-year survival: 82% versus 95%, p=0.008). In this cohort survival was not associated with gender (HR 0.9, 95% CI 0.3-3.0), autoimmune diseases (HR 2.9, 95% CI 0.8-10.1) or immunosuppressive use (HR 0.3, 95% CI: 0.1-1.2).

Nosocomial bloodstream infections caused by Escherichia coli and Klebsiella pneumoniae resistant to third-generation cephalosporins, Finland, 1999-2013: Trends, patient characteristics and mortality.

BACKGROUND: Few systematically collected multi-centre surveillance data on nosocomial bloodstream infections (BSI) caused by extended-spectrum ß-lactamase (ESBL) producing Escherichia coli or Klebsiella pneumoniae have been published. AIM: To evaluate trends, patient characteristics and mortality of such infections, nosocomial BSI data reported by the 4-17 hospitals participating in the prospective laboratory-based surveillance during 1999-2013 were analysed. METHODS: Data were collected by local infection control nurses, patient-days were obtained from the hospital's administrative database, and dates of deaths from the population registry. Resistance to third-generation cephalosporins was further examined in the national reference laboratory. FINDINGS: A total of 16 028 nosocomial BSIs were identified; 2217 (14%) were caused by E. coli and 661 (4%) by K. pneumoniae; 207 (7%) were non-susceptible to third-generation cephalosporins, with an increasing trend from 0% in 1999 to 17% in 2013. Patient characteristics did not differ significantly between BSIs caused by third-generation susceptible and resistant E. coli and K. pneumonia, but the case fatality tended to be higher. Most (88%) of the isolates reported as non-susceptible to third-generation cephalosporins had ESBL phenotype, CTX-M (79%) being the most common enzyme. CONCLUSION: A sharp increase in nosocomial BSIs caused by ESBL producing bacteria was observed. Identification of patients for screening pose a challenge, emphasising the role of infection control guidelines and antibiotic policy in prevention.

[Whipple's disease--a rare and severe systemic infection]. / Whipplen tauti--harvinainen ja vakava systeemi-infektio.

Whipple's disease is a very rare systemic infection caused by the bacterium Tropheryma whipplei. If untreated it can be fatal. Approximately one thousand infections caused by this microorganism have been reported globally. Our two patients with Whipple's disease suffered from weight loss, diarrhea and abdominal pain and distention, and were diagnosed with microcytic anemia and significant hypoalbuminemia. In the third patient the manifestation was blood culture negative endocarditis causing aortic insufficiency, atrial fibrillation and coronary embolisation. Antimicrobial drug therapy was effective for all three patients.