Timo Hautala

TLR3 deficiency in herpes simplex encephalitis: High allelic heterogeneity and recurrence risk

Objective: To determine the proportion of children with herpes simplex encephalitis (HSE) displaying TLR3 deficiency, the extent of TLR3 allelic heterogeneity, and the specific clinical features of TLR3 deficiency. Methods: We determined the sequence of all exons of TLR3 in 110 of the 120 patients with HSE enrolled in our study who do not carry any of the previously described HSE-predisposing mutations of TLR3 pathway genes (TLR3, UNC93B1, TRIF, TRAF3, and TBK1). All the new mutant TLR3 alleles detected were characterized experimentally in-depth to establish the causal relationship between the genotype and phenotype. Results: In addition to the 3 previously reported TLR3-deficient patients from the same cohort, 6 other children or young adults with HSE carry 1 of 5 unique or extremely rare (minor allele frequency <0.001) missense TLR3 alleles. Two alleles (M374T, D592N) heterozygous in 3 patients are not deleterious in vitro. The other 3 are deleterious via different mechanisms: G743D+R811I and L360P heterozygous in 2 patients are loss-of-function due to low levels of expression and lack of cleavage, respectively, and R867Q homozygous in 1 patient is hypomorphic. The 3 patients fibroblasts display impaired TLR3 responses and enhanced herpes simplex virus 1 susceptibility. Overall, TLR3 deficiency is therefore found in 6 (5%) of the 120 patients studied. There is high allelic heterogeneity, with 3 forms of autosomal dominant partial defect by negative dominance or haploinsufficiency, and 2 forms of autosomal recessive defect with complete or partial deficiency. Finally, 4 (66%) of the 6 TLR3-deficient patients had at least 1 late relapse of HSE, whereas relapse occurred in only 12 (10%) of the total cohort of 120 patients. Conclusions: Childhood-onset HSE is due to TLR3 deficiency in a traceable fraction of patients, in particular the ones with HSE recurrence. Mutations in TLR3 and TLR3 pathway genes should be searched and experimentally studied in children with HSE, and patients with proven TLR3 deficiency should be followed carefully.

Central nervous system-related symptoms and findings are common in acute Puumala hantavirus infection.

Abstract Background. Puumala hantavirus (PUUV) causes a hemorrhagic fever with renal syndrome (HFRS) also called nephropathia epidemica (NE). Recent case reports and retrospective studies suggest that NE may damage the pituitary gland. Based on these observations, our goal was to explore the nature of this complication prospectively. Methods. A total of 58 hospitalized patients with acute NE volunteered to participate. Central nervous system (CNS) symptoms were recorded, cerebrospinal fluid (CSF) samples were collected, human leukocyte antigen (HLA) haplotype was analyzed, brain magnetic resonance imaging (MRI) was acquired, and electroencephalography (EEG) was recorded. Patients with abnormal pituitary MRI finding were examined by an endocrinologist. Results. Most patients experienced CNS symptoms, and half of the CSF samples were positive for PUUV IgM, elevated protein level, or leukocyte count. CSF of patients negative for DR15(2)-DQ6 haplotype was less frequently affected. MRI revealed pituitary hemorrhage in two patients; these two patients suffered sudden loss of vision associated with headache, and they both developed hypopituitarism. Only one patient required long-term hormonal replacement therapy. Conclusion. CNS-related symptoms and inflammation in the CSF are common in acute NE. Genetic properties of the host may predispose to CNS involvement. It does seem that pituitary injury and subsequent hormonal insufficiency may complicate the recovery.

Prospective study on ocular findings in acute Puumala hantavirus infection in hospitalised patients

Aims To appraise the ocular manifestations of Puumala hantavirus evoked haemorrhagic fever with renal syndrome nephropathia epidemica (NE) and to clarify the mechanisms of ocular changes in the largest series of patients examined to date. Methods 92 eyes of 46 patients with serologically proven NE were examined during the acute phase and after clinical recovery. Ocular symptoms were recorded, and visual acuity, refraction, intraocular pressure and ocular dimensions were evaluated. Results 88% of the patients experienced decreased intraocular pressure (IOP) (p<0.001), 87% reduced visual acuity, 87% conjunctival chemosis, 82% thickening of the lens (p<0.05), 78% myopic shift (p<0.001), 64% shallowing of the anterior chamber (p<0.05) and 52% shallowing of vitreous length (p<0.05) during the acute phase compared with that measured after clinical recovery. In all, 70% of the patients reported ocular symptoms. Conclusion Ocular symptoms and disturbances are common in acute NE, and the symmetry of the ocular changes reflects the systemic nature of the disease. A decrease in IOP and myopic shift mainly due to thickening of the lens are evident in acute disease. The myopic shift only partially explains the visual disturbances supporting the possible multifactorial origin of the ocular findings in patients with NE.

Damaging heterozygous mutations in NFKB1 lead to diverse immunologic phenotypes

Background The nuclear factor &kgr; light‐chain enhancer of activated B cells (NF‐&kgr;B) signaling pathway is a key regulator of immune responses. Accordingly, mutations in several NF‐&kgr;B 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‐&kgr;B pathway. Graphical abstract Figure. No Caption available.

Rubella persistence in epidermal keratinocytes and granuloma M2 macrophages in patients with primary immunodeficiencies

To the Editor: Cutaneous granulomas are a well-recognized pathologic feature in patients with various primary immunodeficiency diseases (PIDs) and may be self-limited or can progress to a persisting granulomatous disorder. Rubella virus (RV) vaccine strain RA27/3 has been recently detected in disseminated cutaneous granulomas of 2 patients with ataxia telangiectasia (AT) and a patient with Simpson-Golabi-Behmel syndrome (who had combined immunodeficiency [CID]). However, a more detailed study of a larger series of granuloma cases in patients with different PIDs was required to confirm and extend this observation. Patients with cutaneous granulomas and with diverse PIDs were selected from the United States Immune Deficiency Network registry. Additional cases (cases 1 and 11) were recruited from the Clinical Immunology Society immune deficiency Listserv. Presence of RV in granuloma-containing tissue samples (Fig 1, A-C) was examined by immunofluorescence staining with 2 different RV capsid-specific antibodies (see this article’s Methods section in the Online Repository at www.jacionline.org) by a reader blinded to the diagnosis. Seven out of 14 patients (50%) exhibited positive RV antigen staining (Table I), whereas the tissue samples of 5 non-PID granuloma patients were negative. Staining intensity varied substantially between patients and did not correlate with the severity of granulomatous disease. RV immunostaining was typically observed in both epidermis and granulomas in dermis (Fig 1, D-F); however, staining only in granulomas (case 3) was seen (Table I). Multiple granulomas within a sample contained RV antigen with typically a few positive cells in the middle except cases 2 and 6, in which virtually all cells in the granulomas were positive. RV was found exclusively in patients with CIDs: CID cause unknown (n 5 2), AT (n5 4), and cartilage hair hypoplasia (n5 1). The immune deficiencies in which granulomas were not found to be positive for rubella were common variable immune deficiency (n 5 2), AT (n 5 2), X-linked agammaglobulinemia (n 5 1), MardenWalker syndrome (n 5 1), and nuclear factor kappa B essential modulator (n 5 1). None of the nonimmune deficient samples was positive. RV-positive cells in granulomas were positive for CD14 and CD68, markers of monocyte/macrophage cell lineage, and CD206 and CD163, activation markers for M2 macrophages, but negative for iNOS, an M1 macrophage marker (see this article’s Methods section; see Table E1 in this article’s Online Repository at www.jacionline.org; Fig 1, G-H). Endothelial cells (vWF), T cells (CD3), B cells (CD20), dermal Langerhans (CD1a), and dendritic (CD11c) cells were negative for RV antigen (see this article’s Results section in the Online Repository at www.jacionline.org; Table E1). These results demonstrate that M2 macrophages were the cell type harboring RV antigen in granulomas. There was a high production of cytokeratin in many RV-positive keratinocytes, suggesting that RV replication in keratinocytes can lead to dysregulation of keratin synthesis (Fig 1, I). Overexpression of keratin is known to alter the architecture of the epidermis and impact healing of ulcers. The RV-immunostaining patterns were unchanged in the 3 skin samples obtained within a 7-year period (case 1), indicating longterm persistence of RVantigen. Antigen persistence is a hallmark of granulomas. Biopsy specimens collected from different body sites contained both RV-positive and RV-negative specimens (case 3), indicating focal distribution of RVantigen-positive cells in patient tissues. In addition to the skin samples from case 6, a bone periosteum tissue (collected 5 years later) contained RVpositive M2 macrophages in the granulomas. PCR fragments covering the entire genome were amplified and sequenced from 1 patient. Phylogenetic analysis revealed that rubella virus of genotype 1a was present in the patient skin (RVs/ Oulu.FIN/22.15/PID; see Fig E1 in this article’s Online Repository at www.jacionline.org). The sequence was similar (97.4% identity) to that of the RA27/3 vaccine virus. In RVs/Oulu.FIN/ 22.15/PID, 2 out of 7 RA27/3-specific amino acid residues had reverted to the wild type (see this article’s Results section; see Table E2 in this article’s Online Repository at www.jacionline.org). There were 69 amino acid substitutions in RVs/Oulu.FIN/22.15/ PID compared with RA27/3; 52 of them were not found in wild-type RV genomes (see Table E3 in this article’s Online Repository at www.jacionline.org). Most neutralizing epitopes, which are located in the E1 protein, were conserved including the immunodominant epitope E1214-233, whereas each of 3 known CD8 T-cell epitopes (largely predicted to be A2 binding), all located in the capsid, contained single mutations (see this article’s Results section; see Table E4 in this article’s Online Repository at www.jacionline.org), suggesting a role for CD8 T-cell–selective pressure in viral evolution during chronic RA27/3 infection. Similar to the previous report, ATwas the most common single diagnosis among RV-positive patients. We have also identified additional PIDs (cartilage hair hypoplasia and CID) in which RV was found in granulomas. Thus, current data have clearly shown an association between defects in T-cell immunity, granulomas, and RV. One of the important findings of this study is the identification of RV-positive cells in granulomas as M2 macrophages. ProinflammatoryM1macrophages play an essential role in eliminating pathogens, whereas anti-inflammatory M2 macrophages are crucial for maintaining tissue homeostasis. RV can infect most cell types and can persistently infect a macrophage-like cell line, but it is currently unknown whether this occurs in vivo. Another novel finding is persisting RVantigen in epidermal keratinocytes, in all epidermal layers except the basal layer. In contrast, in acute postnatal rubella cases, rubella antigen was found only in the deep dermis (cell types not defined) in skin biopsies from rubella rashes, whereas the epidermis was negative. Wild-type RV can establish persistent infections and cause disease in immunologically normal individuals if infection occurs in immune-privileged sites, for example, Fuchs’ uveitis or fetal development. A role for vaccine virus in Fuchs’ uveitis is also suspected. Granulomas have been reported only once in CRS. We hypothesize that the immune deficiency allows persistence of the attenuated virus, polarization of macrophages to M2 occurs,

Young male patients are at elevated risk of developing serious central nervous system complications during acute Puumala hantavirus infection

BackgroundOur aim was to characterize clinical properties and laboratory parameters in patients with or without cerebrospinal fluid (CSF) findings suggestive of central nervous system (CNS) involvement, and especially those who developed serious CNS complications during acute nephropathia epidemica (NE) caused by Puumala hantavirus (PUUV) infection.MethodsA prospective cohort of 40 patients with acute NE and no signs of major CNS complications was analyzed. In addition, 8 patients with major CNS complications associated with NE were characterized. We collected data of CNS symptoms, CSF analysis, brain magnetic resonance imaging (MRI) results, electroencephalography (EEG) recordings, kidney function, and a number of laboratory parameters. Selected patients were evaluated by an ophthalmologist.ResultsPatients with a positive CSF PUUV IgM finding or major CNS complications were more often males (p < 0.05) and they had higher plasma creatinine values (p < 0.001) compared to those with negative CSF PUUV IgM. The degree of tissue edema did not explain the CSF findings. Patients with major CNS complications were younger than those with negative CSF PUUV IgM finding (52.9 vs. 38.5 years, p < 0.05). Some patients developed permanent neurological and ophthalmological impairments.ConclusionsCNS and ocular involvement during and after acute NE can cause permanent damage and these symptoms seem to be attributable to true infection of the CNS rather than increased tissue permeability. The possibility of this condition should be borne in mind especially in young male patients.

Major Age Group-Specific Differences in Conjunctival Bacteria and Evolution of Antimicrobial Resistance Revealed by Laboratory Data Surveillance

Purpose: We hypothesized that observation and analysis of microbiological laboratory statistics from patients with suspected bacterial conjunctivitis should increase our understanding of microbiological epidemiology of the disease in age categories. We further assumed that the statistical data should expose evolution of antimicrobial resistance that may eventually have an influence on clinical decisions. Materials and Methods: We analyzed statistical data of bacterial isolates (1139 strains) and their resistance to common antibiotics from 2494 patients with suspected bacterial conjunctivitis. Results: Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus displayed their presence in 0- to 5-year-old children. Staphylococcus aureus and Pseudomonas aeruginosa were the most common in the elderly (>age 70) among whom a rapid increase in resistance of Staphylococcus aureus to methicillin (MRSA) was recognized. Conclusions: Our study demonstrates that the spectrum of conjunctival bacteria varies among age groups. In addition, our results confirm that a shift in antimicrobial susceptibility can be rapid and age-group specific, thus emphasizing the need for continuous surveillance of bacterial findings.

Enrichment of rare variants in population isolates: single AICDA mutation responsible for hyper-IgM syndrome type 2 in Finland

Antibody class-switch recombination and somatic hypermutation critically depend on the function of activation-induced cytidine deaminase (AID). Rare variants in its gene AICDA have been reported to cause autosomal recessive AID deficiency (autosomal recessive hyper-IgM syndrome type 2 (HIGM2)). Exome sequencing of a multicase Finnish family with an HIGM2 phenotype identified a rare, homozygous, variant (c.416T>C, p.(Met139Thr)) in the AICDA gene, found to be significantly enriched in the Finnish population compared with other populations of European origin (38.56-fold, P<0.001). The population history of Finland, characterized by a restricted number of founders, isolation and several population bottlenecks, has caused enrichment of certain rare disease-causing variants and losses of others, as part of a phenomenon called the Finnish Disease Heritage. Accordingly, rare founder mutations cause the majority of observed Finnish cases in these mostly autosomal recessive disorders that consequently are more frequent in Finland than elsewhere. Screening of all currently known Finnish patients with an HIGM2 phenotype showed them to be homozygous for p.(Met139Thr). All the Finnish p.(Met139Thr) carriers with available data on their geographic descent originated from the eastern and northeastern parts of Finland. They were observed to share more of their genome identity by descent (IBD) than Finns in general (P<0.001), and they all carried a 207.5-kb ancestral haplotype containing the variant. In conclusion, the identified p.(Met139Thr) variant is significantly enriched in Finns and explains all thus far found AID deficiencies in Finland.

Signs of general inflammation and kidney function are associated with the ocular features of acute Puumala hantavirus infection.

Abstract Background: Puumala hantavirus (PUUV) causes nephropathia epidemica (NE), a type of viral haemorrhagic fever with renal syndrome (HFRS). This febrile infection may affect the kidneys, central nervous system (CNS), and the eye. Acute illness is associated with increased tissue permeability and tissue oedema, and many patients experience reduced vision. The aim of this study was to explore the physiological events associated with the ocular features of acute NE. Methods: This was a prospective study of 46 NE patients who were examined during the acute infection and 1 month after hospitalization. Visual acuity, refraction, intraocular pressure (IOP), and ocular dimensions were evaluated. Cerebrospinal fluid and blood samples were collected, brain magnetic resonance imaging and electroencephalography were recorded, and HLA haplotype was analyzed. The degrees of tissue oedema and fluid imbalance were evaluated. Results: CNS examinations did not reveal the source of the ocular changes in acute NE. The plasma C-reactive protein concentration correlated with the lens thickness and the IOP. The plasma creatinine level was associated with the change in anterior chamber depth. However, oliguric and polyuric patients displayed similar ocular findings. Patients positive for the DR3-DQ2 haplotype experienced the least diminished visual acuity. Conclusions: The level of systemic inflammation rather than CNS involvement appears to account for the ocular changes during acute PUUV infection, and the severity of kidney dysfunction may also have a significant role. In addition, the genetic properties of the host may well explain the ocular features of acute hantavirus infection.

Long-term hormonal follow-up after human Puumala hantavirus infection.

Nephropathia epidemica (NE) is a haemorrhagic fever with renal syndrome (HFRS) caused by Puumala hantavirus (PUUV). Pituitary haemorrhage and hypopituitarism may complicate recovery from acute NE.