P2RX7 gene variants associate with altered inflammasome assembly and reduced pyroptosis in chronic nonbacterial osteomyelitis (CNO).

Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1ß and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care.

Chronic Recurrent Multifocal Osteomyelitis (CRMO): Presentation, Pathogenesis, and Treatment.

PURPOSE OF REVIEW: Chronic non-bacterial osteomyelitis (CNO) with its most severe form chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder. We summarize the clinical presentation, diagnostic approaches, most recent advances in understanding the pathophysiology, and available treatment options and outcomes in CNO/CRMO. RECENT FINDINGS: Though the exact molecular pathophysiology of CNO/CRMO remains somewhat elusive, it appears likely that variable defects in the TLR4/MAPK/inflammasome signaling cascade result in an imbalance between pro- and anti-inflammatory cytokine expressions in monocytes from CNO/CRMO patients. In this context, we present previously unpublished data on cytokine and chemokine expression in monocytes and tissues. CNO/CRMO is an autoinflammatory bone disorder resulting from imbalanced cytokine expression from innate immune cells. Though the exact molecular pathophysiology remains unclear, variable molecular defects appear to result in inflammasome activation and pro-inflammatory cytokine expression in monocytes from CNO/CRMO patients. Recent advances suggest signaling pathways and single molecules as biomarkers for CNO/CRMO as well as future treatment targets.

A clinical and pathomechanistic profile of chronic nonbacterial osteomyelitis/chronic recurrent multifocal osteomyelitis and challenges facing the field.

Sporadic chronic nonbacterial osteomyelitis (CNO) is the most common auto-inflammatory bone disorder. The clinical picture is highly variable ranging from nonsymptomatic monofocal lesions to chronic recurrent multifocal disease. Symptoms include pain, local swelling and warmth in the absence or presence of fever. A subset of CNO patients exhibits additional symptoms of psoriatic skin involvement, palmoplantar pustulosis and inflammatory bowel disease. Novel insights into the pathogenesis of CNO link the failure to produce IL-10 and the resulting imbalance toward pro-inflammatory IL-6 and TNF-α with disease expression. Treatment is empiric and includes NSAIDs, corticosteroids, sulfasalazine, methotrexate, TNF inhibitors and bisphosphonates. Laboratory studies and clinical trials are warranted to: establish biomarkers that predict flares and clinical outcomes, identify patients that require additional treatment on top of NSAIDs, establish evidence-based treatment regimens, allow a risk-benefit assessment for the available therapeutic strategies, and identify novel therapeutic targets.

Autoinflammatory bone disorders.

Autoinflammatory bone disorders are characterized by chronic non-infectious osteomyelitis and inflammation-induced bone resorption and result from aberrant activation of the innate immune system. Sporadic chronic non-bacterial osteomyelitis (CNO) is the most common disease subtype. The clinical picture is highly variable and the exact underlying pathophysiology remains to be determined. Recently, novel insights in the pathophysiology of sterile bone inflammation have been gathered by analyzing patients with rare, monogenic inflammatory diseases. In this overview CNO and Majeed syndrome, cherubism, hypophosphatasia and primary hypertrophic osteoarthropathy will be discussed. For the latter four disorders, a genetic cause affecting bone metabolism and leading to chronic bone inflammation has been described. The exact pathophysiology of CNO remains to be determined. Insights from monogenic autoinflammatory bone diseases and the identification of distinct inflammatory pathways may help to understand the pathogenesis of bone inflammation and inflammation-induced bone resorption in more common diseases.

Attenuated TLR4/MAPK signaling in monocytes from patients with CRMO results in impaired IL-10 expression.

Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder of unknown origin. We previously demonstrated that monocytes from CRMO patients fail to express the immune-modulatory cytokine interleukin-10 (IL-10) in a chromatin dependent manner. Here, we demonstrate that attenuated extracellular-signal regulated kinase (ERK)1 and 2 signaling in response to TLR4 activation results in failure to induce IL-10 expression in monocytes from CRMO patients. Attenuated ERK1/2 activation results in 1) reduced levels of Sp-1, a transcription factor that induces IL-10 expression in monocytes, and 2) impaired H3S10 phosphorylation of the IL10 promoter, an activating epigenetic mark. The pro-inflammatory cytokines tumor necrosis factor (TNF)α and IL-6 are not negatively affected, resulting in an imbalance towards pro-inflammatory cytokines. Thus, impaired ERK1/2 signaling with subsequently reduced Sp-1 expression and H3S10 phosphorylation of the IL10 promoter may centrally contribute to the pathophysiology of CRMO.

Activated memory B cells may function as antigen-presenting cells in the joints of children with juvenile idiopathic arthritis.

OBJECTIVE: B cells impact the perpetuation of chronic inflammatory or autoimmune diseases in multiple ways. A role of B cells as antigen-presenting cells (APCs) in the pathogenesis of chronic arthritis in humans has been suggested; however, as of yet the presence of such B cells at the site of inflammation has not been demonstrated. This study was undertaken to investigate whether synovial B cells in patients with juvenile idiopathic arthritis (JIA) might display features of APCs. METHODS: The frequency, phenotype, and immunoglobulin repertoire of synovial B cells were studied by flow cytometry and single-cell polymerase chain reaction (PCR). Cytokine expression by B cells was analyzed by real-time PCR, and interaction between B cells and T cells was investigated in a mixed lymphocyte culture. RESULTS: CD27+IgD- and CD27-IgD- B cells accumulated in the joints of JIA patients and displayed an activated phenotype. Both B cell subsets expressed hypermutated and class-switched immunoglobulins, indicators of memory B cells. The accumulating memory B cells expressed the costimulatory molecules CD80/CD86 and showed a higher capacity to activate allogeneic T cells and prime a Th1 phenotype than their peripheral blood counterparts. CONCLUSION: Activated immunoglobulin class-switched CD27- and CD27+ memory B cells, indicating a phenotype of APCs with expression of costimulatory molecules, accumulate in the joints of patients with JIA and might be involved in the amplification of pathogenic T cell activation. These findings provide evidence that B cells play an antibody-independent immunopathologic role in human chronic inflammatory arthritis of childhood.

Varicella-zoster virus infections in immunocompromised patients - a single centre 6-years analysis.

BACKGROUND: Infection with varicella-zoster virus (VZV) contemporaneously with malignant disease or immunosuppression represents a particular challenge and requires individualized decisions and treatment. Although the increasing use of varicella-vaccines in the general population and rapid initiation of VZV-immunoglobulins and acyclovir in case of exposure has been beneficial for some patients, immunocompromised individuals are still at risk for unfavourable courses. METHODS: In this single center, 6-year analysis we review incidence, hospitalization and complication rates of VZV-infections in our center and compare them to published data. Furthermore, we report three instructive cases. RESULTS: Hospitalization rate of referred children with VZV-infections was 45%, among these 17% with malignancies and 9% under immunosuppressive therapy. Rate of complications was not elevated in these two high-risk cohorts, but one ALL-patient died due to VZV-related complications. We report one 4-year old boy with initial diagnosis of acute lymphoblastic leukemia who showed a rapidly fatal outcome of his simultaneous varicella-infection, one 1.8-year old boy with an identical situation but a mild course of his disease, and an 8.5-year old boy with a steroid-dependent nephrotic syndrome. This boy developed severe hepatic involvement during his varicella-infection but responded to immediate withdrawl of steroids and administration of acyclovir plus single-dose cidofovir after nonresponse to acyclovir after 48 h. CONCLUSION: Our data show that patients with malignant diseases or immunosuppressive therapy should be hospitalized and treated immediately with antiviral agents. Despite these measures the course of VZV-infections can be highly variable in these patients. We discuss aids to individual decision-making for these difficult situations.

Gastroenteritis in childhood: a retrospective study of 650 hospitalized pediatric patients.

BACKGROUND: Acute diarrhea continues to be an important cause of hospitalization in young children, and deaths still occur as a result. We reviewed a large cohort of hospitalized children affected by gastroenteritis. The hypothesis of our study was that clinical characteristics and a limited set of laboratory data can differentiate between the different causative pathogens of diarrhea. METHODS: A chart review was performed of 650 patients with pathogen-proven diarrhea treated between April 2005 and May 2008 in the children's hospital of the University of Würzburg. Clinical presentation at the time of admission and during hospital stay, laboratory findings, stool pathogen results, and epidemiological data were collected and compared. A severity score was generated. RESULTS: Rotavirus was the most common gastroenteritis pathogen identified, followed by norovirus, adenovirus and Salmonella spp. Nosocomial infections were caused most commonly by norovirus. Rotavirus was the most common agent when there was simultaneous detection of two or more viruses. Rotavirus infections were significantly more severe, with a higher frequency of diarrhea and elevated liver enzymes. Infections due to Salmonella spp showed significantly higher values for C-reactive protein, erythrocyte sedimentation rate, and body temperature. A seasonal distribution was noted, with the peak for rotaviruses/noroviruses in winter/spring, the peak for adenoviruses in November/December, and the peak for Salmonella spp in the summer months. Younger children and toddlers had significantly higher gastroenteritis and airway inflammation scores. Of note, respiratory symptoms and parameters of systemic inflammation differed between the different pathogens. CONCLUSIONS: Gastroenteritis is a common reason for hospital admission in previously healthy children during the first years of life. Rotaviruses were found to be the most common pathogens in our cohort. On the basis of clinical and laboratory parameters it appears possible to distinguish between the different causative agents. This may have implications for hospital hygiene management and for the identification of predictive markers of a severe course.

Corticosteroid-induced spinal epidural lipomatosis in the pediatric age group: report of a new case and updated analysis of the literature.

Spinal epidural lipomatosis is a rare complication of chronic corticosteroid treatment. We report a new pediatric case and an analysis of this and 19 pediatric cases identified in the international literature. The youngest of these combined 20 patients was 5 years old when lipomatosis was diagnosed. Lipomatosis manifested after a mean of 1.3 (+/- 1.5) years (SD) (median, 0.8 years; range, 3 weeks - 6.5 years) of corticosteroid treatment. The corticosteroid dose at the time of presentation of the lipomatosis ranged widely, between 5 and 80 mg of prednisone/day. Back pain was the most common presenting symptom. Imaging revealed that lipomatosis almost always involved the thoracic spine, extending into the lumbosacral region in a subset of patients. Predominantly lumbosacral involvement was documented in only two cases. Although a neurological deficit at presentation was documented in about half of the cases, surgical decompression was not performed in the cases reported after 1996. Instead, reducing the corticosteroid dose (sometimes combined with dietary restriction to mobilize fat) sufficed to induce remission. In summary, pediatric spinal epidural lipomatosis remains a potentially serious untoward effect of corticosteroid treatment, which, if recognized in a timely manner, can have a good outcome with conservative treatment.

Pitfalls in diagnostics of hip pain: osteoid osteoma and osteoblastoma.

Osteoid osteoma and osteoblastoma are benign bone tumors that occur most often in adolescents and predominantly in males. Typical clinical symptoms, such as reduced range of motion of adjacent joints, nocturnal bone pain and relief of pain using non-steroidal anti-inflammatory drug therapy especially in osteoid osteoma may lead to the correct diagnosis. However, these symptoms are not always apparent and specific. In radiographic examinations, the initial changes are often uncharacteristic causing further delay in diagnosis. Magnetic resonance imaging is increasingly used for screening, but early findings in the course of disease might not lead to the definite diagnosis. Both entities (especially osteoid osteoma) occur frequently in the area of the hip. To demonstrate pitfalls in the diagnostic pathway of hip pain caused by benign bone tumors, we present two cases with osteoid osteoma and one with osteoblastoma.

Tumor necrosis factor receptor 1-associated periodic syndrome without fever: cytokine profile before and during etanercept treatment.

The objectives of this study are autoinflammatory syndromes which are usually characterized by repeated attacks of fever, especially in children. The presentation of these diseases, however, varies between entities and between patients of a particular syndrome. We report a 16-year-old female patient, who suffered from periodic erythema and myositis/fasciitis. She experienced at least nine attacks of dermatitis and myositis, while no fever episodes were noted over a 3-year period. A delay of puberty with amenorrhea and a short stature were also present. Laboratory investigations consistently showed markedly increased inflammatory parameters (especially a high serum amyloid A) and dysproteinemia. Because the patient's mother complained about chronic and periodic abdominal pain with also persistently elevated inflammatory parameters, the differential diagnosis included hereditary disorders resulting in chronic inflammation. The diagnosis of an inherited tumor necrosis factor receptor (TNFR) 1-associated periodic syndrome (TRAPS) was confirmed by genetic analyses. Long-term anti-inflammatory treatment with etanercept resulted in a significant clinical improvement and reduction of the inflammatory parameters ESR, CRP, interleukin-6, TNF-α, and soluble TNF-α receptor 1, but not of interleukin-12. Monitoring of the cytokine profile suggested partial effectiveness of etanercept in the treatment of TRAPS. Hereditary fever syndromes have to be considered in case of chronic unexplained inflammation even if fever is no presenting symptom.

Treatment of Lyme borreliosis.

Borrelia burgdorferi sensu lato is the causative agent of Lyme borreliosis in humans. This inflammatory disease can affect the skin, the peripheral and central nervous system, the musculoskeletal and cardiovascular system and rarely the eyes. Early stages are directly associated with viable bacteria at the site of inflammation. The pathogen-host interaction is complex and has been elucidated only in part. B. burgdorferi is highly susceptible to antibiotic treatment and the majority of patients profit from this treatment. Some patients develop chronic persistent disease despite repeated antibiotics. Whether this is a sequel of pathogen persistence or a status of chronic auto-inflammation, auto-immunity or a form of fibromyalgia is highly debated. Since vaccination is not available, prevention of a tick bite or chemoprophylaxis is important. If the infection is manifest, then treatment strategies should target not only the pathogen by using antibiotics but also the chronic inflammation by using anti-inflammatory drugs.

MicroRNAs--micro in size but macro in function.

MicroRNAs (miRNAs) are endogenous small RNAs that can regulate target mRNAs by binding to their 3'-UTRs. A single miRNA can regulate many mRNA targets, and several miRNAs can regulate a single mRNA. These have been reported to be involved in a variety of functions, including developmental transitions, neuronal patterning, apoptosis, adipogenesis metabolism and hematopoiesis in different organisms. Many oncogenes and tumor suppressor genes are regulated by miRNAs. Studies conducted in the past few years have demonstrated the possible association between miRNAs and several human malignancies and infectious diseases. In this article, we have focused on the mechanism of miRNA biogenesis and the role of miRNAs in human health and disease.

Abscess-forming lymphadenopathy and osteomyelitis in children with Bartonella henselae infection.

Bartonella henselae is the agent of cat-scratch disease (CSD), a chronic lymphadenopathy among children and adolescents. A systemic infection is very rare and most of these cases are found in patients with immunodeficiency. Here, cases involving four children of 6-12 years of age are reported. Three of the children had an abscess-forming lymphadenopathy and surrounding myositis in the clavicular region of the upper arm. The diagnosis was made serologically and, in one case, using eubacterial universal PCR. One child was treated with erythromycin for 10 days, the second received cefotaxime and flucloxacillin for 14 days and the third child was not treated with antibiotics. The fourth child had a different course: a significantly elevated signal intensity affecting the complete humerus was found in magnetic resonance imaging, consistent with osteomyelitis. A lymph node abscess was also found in the axilla. Diagnosis was established by indirect fluorescence assay and lymph node biopsy. Antibiotic therapy using clarithromycin, clindamycin and rifampicin was gradually successful. Immunodeficiency was excluded. All described lesions healed without residues. In immunocompetent patients, infection affects skin and draining lymph nodes; however, prolonged fever of unknown origin as in the fourth patient indicated a systemic complication of CSD.

Chronic recurrent multifocal osteomyelitis: what is it and how should it be treated?

BACKGROUND: Chronic recurrent multifocal osteomyelitis (CRMO) is the most severe form of chronic nonbacterial osteomyelitis. In children and adolescents, chronic nonbacterial osteomyelitis predominantly affects the metaphyses of the long bones, but lesions can occur at any site in the skeleton. Other organs (the skin, eyes, gastrointestinal tract and lungs) can also be affected. Clinical diagnosis is often difficult because the symptoms and course of disease vary significantly. We present a 10-year-old girl diagnosed with CRMO involving several vertebrae, the femur and the metatarsus. INVESTIGATIONS: Physical examination, abdominal ultra sonography, conventional X-ray, MRI, technetium bone scan, esophagogastroduodenoscopy, colonoscopy, tests for HLA-B27 and thiopurine methyltransferase, polymerase chain reaction and thoracic vertebral bone biopsies. DIAGNOSIS: CRMO and Crohn's disease. MANAGEMENT: The patient's condition improved whilst being treated with NSAIDs for 3 months; however, the patient had an allergic skin reaction to this therapy. Treatment was switched to sulfasalazine, accompanied by 3 weeks of therapy using oral prednisone, but sulfasalazine was discontinued 2 months later because the patient exhibited a minor elevation in the levels of liver enzymes. The patient was free of musculoskeletal symptoms for 6 months, at which time she started to complain again about pain in her back and bowel. Multimodal therapy, consisting of mesasalazine, corticosteroids (budesonide) and azathioprine, induced clinical remission of Crohn's disease.

Magnetic resonance imaging in child abuse.

Magnetic resonance imaging is a particularly important diagnostic method when bone and soft tissue lesions of inflammatory or malignant origin need to be analyzed. Traumatic lesions often are evaluated using standard radiographs or computed tomography. Both of these methods evaluate fractures appropriately; however, bone bruise, bone bending, and soft tissue lesions might be underestimated. Especially in the evaluation of suspected child abuse, magnetic resonance imaging can contribute significantly to making the diagnosis, especially when the reported history is not conclusive.

Chronic multifocal non-bacterial osteomyelitis in hypophosphatasia mimicking malignancy.

BACKGROUND: Hypophosphatasia (HP) is characterized by a genetic defect in the tissue-nonspecific alkaline phosphatase (TNSALP) gene and predominantly an autosomal recessive trait. HP patients suffer from reduced bone mineralization. Biochemically, elevated concentrations of substrates of TNSALP, including pyridoxal-5'-phosphate and inorganic pyrophosphate occur in serum, tissues and urine. The latter has been associated with chronic inflammation and hyperprostaglandinism. CASE PRESENTATION: We report on 2 affected children presenting with multifocal inflammatory bone lesions mimicking malignancy: A 6 years old girl with short stature had been treated with human growth hormone since 6 months. Then she started to complain about a painful swelling of her left cheek. MRI suggested a malignant bone lesion. Bone biopsy, however, revealed chronic inflammation. A bone scan showed a second rib lesion. Since biopsy was sterile, the descriptive diagnosis of chronic non-bacterial osteomyelitis (CNO) was established. The diagnostic tests related to growth failure were repeated and subsequent analyses demonstrated a molecular defect in the TNSALP gene. The second girl (10 years old) complained about back pain after she had fallen from her bike. X rays of her spine revealed compressions of 2 thoracic vertebrae. At first these were considered trauma related, however a bone scan did show an additional lesion in the right 4th rib. A biopsy of this rib revealed a sterile lympho- plasmocytoid osteomyelitis suggesting multifocal CNO. Further analyses did show a decreased TNSALP in leukocytes and elevated pyridoxal phosphate in plasma, suggesting a heterozygous carrier status of HP. CONCLUSION: Chronic bone oedema in adult HP and chronic hyper-prostaglandinism in childhood HP do suggest that in some HP patients bone inflammation is present in conjunction with the metabolic defect. Sterile multifocal osteomyelitis could be demonstrated. Non-steroidal anti-inflammatory treatment achieved complete remission. These cases illustrate chronic inflammation of the bone as a new feature of HP.

Different patterns of bcl-6 and p53 gene mutations in tonsillar B cells indicate separate mutational mechanisms.

Mutations within the 5'-non-coding region of the bcl-6 gene can occur in lymphomas that originate from germinal centers (GCs), as well as in normal memory and GC B cells. Mutations in the p53 gene occur in 50% of human cancers. Since both bcl-6 and p53 can be mutated in certain circumstances, we investigated the accumulation of mutations in these genes in individual tonsillar B and T cells to determine whether the mutations exhibited a pattern anticipated from the B-cell hypermutation machinery. In tonsillar GC B cells, the overall mutational frequencies in the 5'-non-coding region of the bcl-6 gene was 0.85 x 10(-3)/bp. In contrast, there were no mutations in a region 2.8 kb downstream of the promoter. RGYW (purine, guanine, pyrimidine, A/T) targeting and a significantly lower mutational frequency in nai;ve B and GC founder B cells compared with GC B cells suggested that a similar mutator mechanism was active on Ig genes and this non-Ig gene. The mutational frequency in the exon-7-region of p53 was similar in the GC, memory and nai;ve B-cell subsets (1.02 x 10(-3) to 1.25 x 10(-3)/bp). RGYW/WRCY motifs were not targeted preferentially in the p53 gene. Moreover, a comparable mutational frequency of p53 was noted in tonsillar B and T cells. Hence, mutations in p53 do not appear to be the result of the B-cell hypermutational mechanism.

Expression of recombination activating genes 1 and 2 in peripheral B cells of patients with systemic lupus erythematosus.

OBJECTIVE: To analyze immunoregulatory abnormalities in patients with systemic lupus erythematosus (SLE) by assessing the expression of messenger RNA (mRNA) for types 1 and 2 recombination activating genes (RAG) in the peripheral blood of patients with active SLE. METHODS: We examined B cell populations and also individual B cells from patients with SLE for the expression of RAG mRNA. RESULTS: Analysis of bulk mRNA indicated that RAG1 and RAG2 mRNA were found routinely in peripheral B cells of patients with active SLE, but not in healthy subjects. When assessed on a single-cell basis, there was a 3-fold increase in the frequency of RAG1- and RAG2-expressing B cells in SLE patients compared with healthy subjects. Notably, B cells expressing both RAG1 and RAG2 mRNA expressed only IgD mRNA, but not IgG mRNA. Fifty percent of RAG-expressing B cells also expressed VpreB mRNA, whereas all expressed CD154 mRNA. Phenotypic analysis indicated that RAG-expressing B cells were activated, mature B cells. CONCLUSION: These results indicate that RAG expression is up-regulated in peripheral IgD+ and VpreB+ B cells of patients with active SLE. These cells may contribute to the immunoregulatory abnormalities in patients with SLE.