Shorter birth length and decreased T-cell production and function predict severe infections in children with non-severe combined immunodeficiency cartilage-hair hypoplasia.

Background: Cartilage-hair hypoplasia (CHH) is a syndromic inborn error of immunity caused by variants in the RMRP gene. Disease manifestations vary, and their ability to predict outcome is uncertain. The optimal management of infants with CHH who do not fulfill classical severe combined immunodeficiency (SCID) criteria is unknown. Objective: We described longitudinal changes in lymphocyte counts during childhood and explored correlations of early childhood clinical and laboratory features with clinical outcomes on long-term follow-up of CHH patients. Methods: Immunologic laboratory parameters, birth length, the presence of Hirschsprung disease, and severe anemia correlated to the primary end points of respiratory and severe infections. We implemented traditional statistical methods and machine learning techniques. Results: Thirty-two children with CHH were followed up for 2.7 to 22.1 years (median, 8.2 years, in total 331.3 patient-years). None of the patients had classical SCID. Median lymphocyte subclass counts, apart from CD16+/56+ cells, were subnormal throughout childhood, but did not show age-related decline seen in healthy children. Low immunoglobulin levels were uncommon and often transient. Respiratory and/or severe infections developed in 14 children, 8 of whom had low naive T-cell counts, absent T-cell receptor excision circles, and/or partial "leaky" SCID-level lymphopenia. Shorter birth length correlated with lower lymphocyte counts and the occurrence of infections. Of the laboratory parameters, decreased naive T-cell counts and abnormal lymphocyte proliferation responses contributed most to the development of severe infections. In addition, all participants with absent T-cell receptor excision circles developed severe infections. Opportunistic infections occurred only in children with leaky SCID-level lymphopenia. Conclusions: Shorter birth length and a combination of laboratory abnormalities can predict the development of severe infections in children with CHH.

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.

The Safety and Efficacy of Live Viral Vaccines in Patients With Cartilage-Hair Hypoplasia.

Background: Live viral vaccines are generally contraindicated in patients with combined immunodeficiency including cartilage-hair hypoplasia (CHH); however, they may be tolerated in milder syndromes. We evaluated the safety and efficacy of live viral vaccines in patients with CHH. Methods: We analyzed hospital and immunization records of 104 patients with CHH and measured serum antibodies to measles, mumps, rubella, and varicella zoster virus (VZV) in all patients who agreed to blood sampling (n = 50). We conducted a clinical trial (ClinicalTrials.gov identifier: NCT02383797) of live VZV vaccine on five subjects with CHH who lacked varicella history, had no clinical symptoms of immunodeficiency, and were seronegative for VZV; humoral and cellular immunologic responses were assessed post-immunization. Results: A large proportion of patients have been immunized with live viral vaccines, including measles-mumps-rubella (MMR) (n = 40, 38%) and VZV (n = 10, 10%) vaccines, with no serious adverse events. Of the 50 patients tested for antibodies, previous immunization has been documented with MMR (n = 22), rubella (n = 2) and measles (n = 1) vaccines. Patients with CHH demonstrated seropositivity rates of 96%/75%/91% to measles, mumps and rubella, respectively, measured at a medium of 24 years post-immunization. Clinical trial participants developed humoral and cellular responses to VZV vaccine. One trial participant developed post-immunization rash and knee swelling, both resolved without treatment. Conclusion: No serious adverse events have been recorded after immunization with live viral vaccines in Finnish patients with CHH. Patients generate humoral and cellular immune response to live viral vaccines. Immunization with live vaccines may be considered in selected CHH patients with no or clinically mild immunodeficiency.

A 30-Year Prospective Follow-Up Study Reveals Risk Factors for Early Death in Cartilage-Hair Hypoplasia.

Cartilage-hair hypoplasia (CHH) is a skeletal dysplasia with combined immunodeficiency, variable clinical course and increased risk of malignancy. Management of CHH is complicated by a paucity of long-term follow-up data, as well as knowledge on prognostic factors. We assessed clinical course and risk factors for mortality in a prospective cohort study of 80 patients with CHH recruited in 1985-1991 and followed up until 2016. For all patients we collected additional health information from health records and from the national Medical Databases and Cause-of-death Registry. The primary outcome was immunodeficiency-related death, including death from infections, lung disease and malignancy. Standardized mortality ratios (SMRs) were calculated using national mortality rates as reference. Half of the patients (57%, n = 46) manifested no symptoms of immunodeficiency during follow-up while 19% (n = 15) and 24% (n = 19) demonstrated symptoms of humoral or combined immunodeficiency, including six cases of adult-onset immunodeficiency. In a significant proportion of patients (17/79, 22%), clinical features of immunodeficiency progressed over time. Of the 15 patients with non-skin cancer, eight had no preceding clinical symptoms of immunodeficiency. Altogether 20 patients had deceased (SMR = 7.0, 95%CI = 4.3-11); most commonly from malignancy (n = 7, SMR = 10, 95%CI = 4.1-21) and lung disease (n = 4, SMR = 46, 95%CI = 9.5-130). Mortality associated with birth length below -4 standard deviation (compared to normal, SMR/SMR ratio = 5.4, 95%CI = 1.5-20), symptoms of combined immunodeficiency (compared to asymptomatic, SMR/SMR ratio = 3.9, 95%CI = 1.3-11), Hirschsprung disease (odds ratio (OR) 7.2, 95%CI = 1.04-55), pneumonia in the first year of life or recurrently in adulthood (OR = 7.6/19, 95%CI = 1.3-43/2.6-140) and autoimmunity in adulthood (OR = 39, 95%CI = 3.5-430). In conclusion, patients with CHH may develop adult-onset immunodeficiency or malignancy without preceding clinical symptoms of immune defect, warranting careful follow-up. Variable disease course and risk factors for mortality should be acknowledged.

Novel DSP Spectrin 6 Region Variant Causes Neonatal Erythroderma, Failure to Thrive, Severe Herpes Simplex Infections and Brain Lesions.

Desmoplakin (DSP) and Desmoglein 1 (DSG1) variants result in skin barrier defects leading to erythroderma, palmoplantar keratoderma and variable [AQ4] other features. Some DSG1 variant carriers present with SAM syndrome (Severe dermatitis, multiple Allergies, Metabolic wasting) and a SAM-like phenotype has been reported in 4 subjects with different heterozygous DSP variants. We report here a patient with a novel DSP spectrin region (SR) 6 variant c.1756C>T, p.(His586Tyr), novel features of brain lesions and severe recurrent mucocutaneous herpes simplex virus infections, with a favourable response to ustekinumab. Through a review of reported cases of heterozygous variants in DSP SR6 (n = 15) and homozygous or compound heterozygous variants in DSG1 (n = 12) and SAM-like phenotype, we highlight phenotypic variability. Woolly hair, nail abnormalities and cardiomyopathy characterize patients with DSP variants, while elevated immunoglobulin E and food allergies are frequent in patients with DSG1 variants. Clinicians should be aware of the diverse manifestations of desmosomopathies.

Immune cell phenotype and functional defects in Netherton syndrome.

BACKGROUND: Netherton syndrome (NS) is a rare life-threatening syndrome caused by SPINK5 mutations leading to a skin barrier defect and a severe atopic diathesis. NS patients are prone to bacterial infections, but the understanding of the underlying immune deficiency is incomplete. RESULTS: We analyzed blood lymphocyte phenotypes and function in relation to clinical infections in 11 Finnish NS patients, aged 3 to 17 years, and healthy age-matched controls. The proportion of B cells (CD19+) and naïve B cells (CD27-, IgD+) were high while memory B cells (CD27+) and switched memory B cells (CD27+IgM-IgD-), crucial for the secondary response to pathogens, was below or in the lowest quartile of the reference values in 8/11 (73%) and 9/11 (82%) patients, respectively. The proportion of activated non-differentiated B cells (CD21low, CD38low) was below or in the lowest quartile of the reference values in 10/11 (91%) patients. Despite normal T cell counts, the proportion of naïve CD4+ T cells was reduced significantly and the proportion of CD8+ T central memory significantly elevated. An increased proportion of CD57+ CD8+ T cells indicated increased differentiation potential of the T cells. The proportion of cytotoxic NK cells was elevated in NS patients in phenotypic analysis based on CD56DIM, CD16+ and CD27- NK cells but in functional analysis, decreased expression of CD107a/b indicated impaired cytotoxicity. The T and NK cell phenotype seen in NS patients also significantly differed from that of age-matched atopic dermatitis (AD) patients, indicating a distinctive profile in NS. The frequency of skin infections correlated with the proportion of CD62L+ T cells, naïve CD4+ and CD27+ CD8+ T cells and with activated B cells. Clinically beneficial intravenous immunoglobulin therapy (IVIG) increased naïve T cells and terminal differentiated effector memory CD8+ cells and decreased the proportion of activated B cells and plasmablasts in three patients studied. CONCLUSIONS: This study shows novel quantitative and functional aberrations in several lymphocyte subpopulations, which correlate with the frequency of infections in patients with Netherton syndrome. IVIG therapy normalized some dysbalancies and was clinically beneficial.

A Wide Spectrum of Autoimmune Manifestations and Other Symptoms Suggesting Immune Dysregulation in Patients With Cartilage-Hair Hypoplasia.

Background: Mutations in RMRP, encoding a non-coding RNA molecule, underlie cartilage-hair hypoplasia (CHH), a syndromic immunodeficiency with multiple pathogenetic mechanisms and variable phenotype. Allergy and asthma have been reported in the CHH population and some patients suffer from autoimmune (AI) diseases. Objective: We explored AI and allergic manifestations in a large cohort of Finnish patients with CHH and correlated clinical features with laboratory parameters and autoantibodies. Methods: We collected clinical and laboratory data from patient interviews and hospital records. Serum samples were tested for a range of autoantibodies including celiac, anti-cytokine, and anti-21-hydroxylase antibodies. Nasal cytology samples were analyzed with microscopy. Results: The study cohort included 104 patients with genetically confirmed CHH; their median age was 39.2 years (range 0.6-73.6). Clinical autoimmunity was common (11/104, 10.6%) and included conditions previously undescribed in subjects with CHH (narcolepsy, psoriasis, idiopathic thrombocytopenic purpura, and multifocal motor axonal neuropathy). Patients with autoimmunity more often had recurrent pneumonia, sepsis, high immunoglobulin (Ig) E and/or undetectable IgA levels. The mortality rates were higher in subjects with AI diseases ( χ(2)2 = 14.056, p = 0.0002). Several patients demonstrated serum autoantibody positivity without compatible symptoms. We confirmed the high prevalence of asthma (23%) and allergic rhinoconjunctivitis (39%). Gastrointestinal complaints, mostly persistent diarrhea, were also frequently reported (32/104, 31%). Despite the history of allergic rhinitis, no eosinophils were observed in nasal cytology in five tested patients. Conclusions: AI diseases are common in Finnish patients with CHH and are associated with higher mortality, recurrent pneumonia, sepsis, high IgE and/or undetectable IgA levels. Serum positivity for some autoantibodies was not associated with clinical autoimmunity. The high prevalence of persistent diarrhea, asthma, and symptoms of inflammation of nasal mucosa may indicate common pathways of immune dysregulation.

Fecal Calprotectin Test Performed at Home: A Prospective Study of Pediatric Patients With Inflammatory Bowel Disease.

OBJECTIVES: Measuring fecal calprotectin (FC) in a laboratory is time-consuming and that is why home tests have been developed. We studied the use of an FC home test in pediatric patients with inflammatory bowel disease (PIBD) in real-life settings. METHODS: The patients were asked to perform the IBDoc FC home test monthly for 6 months and to report their clinical disease activity at testing. Clinical decision-making, however, was guided by routine FC enzyme-linked immunosorbent assay (ELISA) for patients with raised IBDoc values. Spare frozen samples were analyzed using ELISA and IBDoc in the laboratory. The participants completed a questionnaire about FC testing at the start and end of the study. RESULTS: Of the 52 patients, 35 (67%) ages 5 to 18 years completed the study, and 197 home tests were performed. Of these, 15% failed, mainly because of technical reasons. Just under half of the patients (47%) considered home testing comparable or superior to routine testing. In contrast, the parents were unsatisfied (61%), mostly because the IBDoc results were significantly different from ELISA and they found the phone application difficult to handle but whenever the IBDoc was performed by a laboratory professional it was comparable with ELISA, suggesting that practical issues hampered home testing. Despite their reservations, more than 80% of parents felt that home testing would improve disease management. CONCLUSIONS: PIBD patients and their families were interested in FC home monitoring and willing to adopt testing as a part of their disease management, but this approach requires thorough guidance.

Decreased telomere length in children with cartilage-hair hypoplasia.

BACKGROUND: Cartilage-hair hypoplasia (CHH) is an autosomal recessive chondrodysplasia caused by RMRP (RNA component of mitochondrial RNA processing endoribonuclease) gene mutations. Manifestations include short stature, variable immunodeficiency, anaemia and increased risk of malignancies, all of which have been described also in telomere biology disorders. RMRP interacts with the telomerase RT (TERT) subunit, but the influence of RMRP mutations on telomere length is unknown. We measured relative telomere length (RTL) in patients with CHH, their first-degree relatives and healthy controls and correlated RTL with clinical and laboratory features. METHODS: The study cohort included 48 patients with CHH with homozygous (n=36) or compound heterozygous RMRP mutations (median age 38.2 years, range 6.0-70.8 years), 86 relatives (74 with a heterozygous RMRP mutation) and 94 unrelated healthy controls. We extracted DNA from peripheral blood, sequenced the RMRP gene and measured RTL by qPCR. RESULTS: Compared with age-matched and sex-matched healthy controls, median RTL was significantly shorter in patients with CHH (n=40 pairs, 1.05 vs 1.21, p=0.017), but not in mutation carriers (n=48 pairs, 1.16 vs 1.10, p=0.224). RTL correlated significantly with age in RMRP mutation carriers (r=-0.482, p<0.001) and non-carriers (r=-0.498, p<0.001), but not in patients (r=-0.236, p=0.107). In particular children (<18 years) with CHH had shorter telomeres than controls (median RTL 1.12 vs 1.26, p=0.008). In patients with CHH, RTL showed no correlation with genotype, clinical or laboratory characteristics. CONCLUSIONS: Telomere length was decreased in children with CHH. We found no correlation between RTL and clinical or laboratory parameters.

Altered Fecal Microbiota in Paediatric Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Several factors support the view of inflammatory bowel disease [IBD] origin in the host responsiveness to intestinal bacteria, although no single bacterial species has been shown as a causative agent in the pathogenesis. Our aim was to analyse the fecal microbiota of paediatric IBD patients at different stages of the disease. In addition, the characteristics of immune response to the bacterial isolates showing very low abundance in IBD were studied. METHODS: Fecal samples [1-3 samples/child] were collected from 10 paediatric patients with crohn's disease [CD], and 12 with ulcerative colitis [UC] and from 8 healthy children, for polyphasic microbiological analysis (culture, real-time polymerase chain reaction [PCR], and denaturing gradient gel electrophoresis). In addition, in vitro cytokine responses of peripheral blood mononuclear cells to the bacterial isolates, which showed very low abundance in IBD, were studied. RESULTS: Although predominant bacterial diversity was higher in IBD, the numbers of Lachnospiraceae and Coriobacteriaceae bacteria were lower in IBD patients as compared with control children [p < 0.05]. In addition, Ruminococcaceae population diversity was lower in IBD [p < 0.05] and correlated negatively with fecal calprotectin levels. Both abundance and diversity of bifidobacterial populations were lower in children with IBD [p < 0.05], and particularly low numbers of certain bifidobacterial isolates were detected. In CD, we found enhanced up-regulation of interleukin-6 transcripts and impaired RAR-related orphan receptor C response to bifidobacteria, whereas decreased interferon-gamma response was observed in both CD and UC. CONCLUSION: We demonstrate altered fecal microbiota in paediatric IBD, particularly low numbers and diversity of bifidobacterial populations. Interestingly, immunological response to bifidobacteria differed between paediatric CD patients and control children.

Interleukin-17 immunity in pediatric Crohn disease and ulcerative colitis.

OBJECTIVE: The present understanding of inflammatory bowel disease pathogenesis mainly relies on studies of adult patients. Therefore, we studied the balance between T-effector and regulatory cells in pediatric inflammatory bowel disease. METHODS: Quantitative polymerase chain reaction and immunohistochemistry served to quantify the expression of immunological markers in mucosal biopsies and flow cytometry analysis was used in peripheral blood mononuclear cells. RESULTS: Colonic interleukin (IL)-17+, IL-22, and IL-6 mRNA upregulation and increase in the number of colonic IL-17 cells were demonstrated in both Crohn disease (CD) and ulcerative colitis (UC). Likewise, colonic forkhead box P3 (FOXP3+) mRNA expression and the number of colonic FOXP3 cells were increased both in CD and in UC and were accompanied in CD also with increased numbers of FOXP3+CD25 High CD4 cells in peripheral blood. Ileal relation of IL-17/CD4 cells was increased only in CD. CONCLUSIONS: We showed activation of colonic IL-17/IL-22 axis and upregulation of FOXP3 to occur both in pediatric CD and in UC, indicating shared immunological characteristics. Upregulation of IL-17 was restricted to colon in UC, but existed in the ileum and in the colon in active CD.

Dendritic cells from Crohn's disease patients show aberrant STAT1 and STAT3 signaling.

Abnormalities of dendritic cells (DCs) and STAT proteins have been reported in Crohn's disease (CD). Studies on JAK/STAT signaling in DCs are, however, lacking in CD. We applied a flowcytometric single-cell-based phosphoepitope assay to evaluate STAT1 and STAT3 pathways in DC subsets from CD patients. In addition, circulating DC counts were determined, together with the activation-related immunophenotype. We found that IL-6- and IFN-α-induced STAT3 phosphorylation and IFN-α-induced STAT1 phosphorylation were impaired in plasmacytoid DCs (pDCs) from CD patients (P = 0.005, P = 0.013, and P = 0.006, respectively). In myeloid DCs (mDCs), IFN-α-induced STAT1 and STAT3 phosphorylation were attenuated (P<0.001 and P = 0.048, respectively), but IL-10-induced STAT3 phosphorylation was enhanced (P = 0.026). IFN-γ-induced STAT1 signaling was intact in both DC subtypes. Elevated plasma IL-6 levels were detected in CD (P = 0.004), which strongly correlated with disease activity (ρ = 0.690, P<0.001) but not with IL-6-induced STAT3 phosphorylation. The numbers of pDCs and BDCA3+ mDCs were decreased, and CD40 expression on CD1c+ mDCs was increased in CD. When elucidating the effect of IL-6 signaling on pDC function, we observed that IL-6 treatment of healthy donor pDCs affected the maturation of and modified the T-cell priming by pDCs, favoring Th2 over Th1 type of response and the expression of IL-10 in T cells. Our results implicate DC signaling in human CD. Reduced IL-6 responsiveness in pDCs, together with the attenuated IFN-α-induced signaling in both DC subtypes, may contribute to the immunological dysregulation in CD patients.

In Crohn's Disease, Anti-TNF-α Treatment Changes the Balance between Mucosal IL-17, FOXP3, and CD4 Cells.

Aim. In Crohn's disease (CD), anti-TNF-α treatment is a potent medication. We aimed to characterize the effect of anti-TNF-α treatment on T effector and regulatory cells. Material and Methods. We studied T-effector and regulatory cells on cellular and mRNA levels in intestinal biopsy samples from 13 Crohn's disease patient. Biopsies were obtained at baseline and 3 months after anti-TNF-α treatment, and from 14 inflammation-free control subjects. Results. Patients had higher numbers of ileal IL-17(+) and forkhead box P3 (FOXP3)(+) cells than did control subjects, both before ( P ≤ 0.001 and P ≤ 0.05, resp.) and after the anti-TNF-α treatment (P ≤ 0.01, P ≤ 0.01). Intestinal interferon-γ and IL-17 mRNA expression was higher in Crohn's disease and remained elevated after anti-TNF-α treatment. The ratio of IL-17(+) cells to CD4(+) cells decreased (P ≤ 0.05) and compared to baseline the ratio of IL-17(+) cells to FOXP3(+) was lower after treatment (P ≤ 0.05). Conclusions. TNF-α-blocking agents improved intestinal balance between IL-17(+) T-effector and regulatory T cells, although intestinal IL-17 upregulation remained elevated.

Combined T regulatory cell and Th2 expression profile identifies children with cow's milk allergy.

The role of T regulatory cells in spontaneous recovery from cow's milk allergy (CMA) is unclear. We investigated the mRNA expression of 12 T-cell markers and the protein expression of CD4, CD25, CD127, FoxP3 after in vitro beta-lactoglobulin stimulation of peripheral blood mononuclear cells from children with persisting CMA (n=16), early recovery (n=20) or no atopy (n=21). Artificial neural networks with exhaustive search for all marker combinations revealed that markers FoxP3, Nfat-C2, IL-16 and GATA-3 distinguished patients with persisting CMA most accurately from other study groups. FoxP3 mRNA expression following beta-lactoglobulin stimulation was highest in children with persisting CMA. Also the FoxP3 intensity in CD4(+) CD25(high)CD127(low) cells was higher in children with CMA compared with non-atopic children. The expression profile of both Th2- and T regulatory cell-related genes thus reflects the clinical activity of CMA. Tolerance, in contrast, is not characterized by activation of circulating T regulatory cells.

IL-23/IL-17 immunity as a hallmark of Crohn's disease.

BACKGROUND: We studied the balance between ileal T-effector cells versus T-regulatory cells in active and inactive Crohn's disease (CD). METHODS: We compared effector and regulatory T-cell-related markers such as interleukin (IL)-17, interferon (IFN)-gamma, IL-4, and Foxp3 transforming growth factor (TGF)-beta CTLA-4 and markers for innate immune activation such as IL-6, IL-10, IL-18, IL-23, tumor necrosis factor (TNF)-alpha, and IL-12p70, studied with immunohistochemistry and RT-PCR in ileal biopsies from patients with active or inactive CD and from control subjects. IL-17 in fecal samples was detected by ELISA. The effect of IL-17 on IL-8 and TNF-alpha mRNA expression in epithelial cell line Caco-2 was studied. RESULTS: The numbers of IL-4-, IL-17-, and IL-23(p19)-positive cells in the lamina propria were higher in patients with CD, both active and inactive, than in the controls. mRNA expression of IL-17A, IL-6, and Foxp3 was increased in the biopsies both from patients with active disease and those in remission, whereas mRNA expression of IL-23 was increased only in active disease. Fecal IL-17 concentration was increased in patients with active disease. IL-17 enhanced the IL-8 and TNF-alpha response of the epithelial cell line to lipopolysaccharide (LPS) in vitro. CONCLUSIONS: Our findings suggest that activation of the IL-23/IL-17 axis is fundamentally connected to the etiology of CD and may represent the basis for the relapsing nature of the disease by increasing the sensitivity of epithelium to microbial LPS.