Trends in Serum Cytokine Expression in Pediatric Skeletal Dysplasia.

The skeletal dysplasias are a heterogeneous group of genetic conditions caused by abnormalities of growth, development, and maintenance of bone and cartilage. Little is known about the roles that cytokines play in the inflammatory and non-inflammatory pathophysiology of skeletal dysplasia. We sought to test our hypothesis that cytokines would be differentially expressed in children with skeletal dysplasia as compared to typically growing controls. Cytokine levels were analyzed using the Cytokine Human Magnetic 25-Plex Panel (Invitrogen, Waltham, MA, USA); 136 growing individuals with skeletal dysplasia and compared to a cohort of 275 healthy pediatric control subjects. We focused on the expression of 12 cytokines across nine dysplasia cohorts. The most common skeletal dysplasia diagnoses were: achondroplasia (58), osteogenesis imperfecta (19), type II collagenopathies (11), multiple epiphyseal dysplasia (MED: 9), diastrophic dysplasia (8), metatropic dysplasia (8), and microcephalic osteodysplastic primordial dwarfism type II (MOPDII: 8). Of the 108 specific observations made, 45 (41.7%) demonstrated statistically significant differences of expression between controls and individuals with skeletal dysplasia. Four of the 12 analyzed cytokines demonstrated elevated expression above control levels in all of the dysplasia cohorts (interleukin 12 [IL-12], IL-13, interferon γ-induced protein 10 kDa [IP-10], regulated on activation, normal T cell expressed and secreted [RANTES]) and two demonstrated expression below control levels across all dysplasia cohorts (monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein-1ß [MIP-1ß]). The highest levels of overexpression were seen in MOPDII, with expression levels of IP-10 being increased 3.8-fold (p < 0.0001). The lowest statistically significant levels of expressions were in type II collagenopathies, with expression levels of MCP-1 being expressed 0.43-fold lower (p < 0.005). With this data, we hope to lay the groundwork for future directions in dysplasia research that will enhance our understanding of these complex signaling pathways. Looking forward, validating these early trends in cytokine expression, and associating the observed variations with trends in the progression of dysplasia may offer new candidates for clinical biomarkers or even new therapeutics. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

The role of transforming growth factor ß signaling in fibroblast-like synoviocytes from patients with oligoarticular juvenile idiopathic arthritis: dysregulation of transforming growth factor ß signaling, including overexpression of bone morphogenetic protein 4, may lead to a chondrocyte phenotype and may contribute to bony hypertrophy.

OBJECTIVE: This study was designed to investigate the pathogenic contributions of fibroblast-like synoviocytes (FLS) to juvenile idiopathic arthritis (JIA) by identifying pathways with dysregulated gene expression in FLS from patients with oligoarticular JIA. METHODS: FLS were derived from synovial fluid obtained by arthrocentesis from patients with JIA undergoing intraarticular steroid injections and from orthopedic control patients. Gene expression profiles of the JIA and control FLS were obtained using the Affymetrix platform, with application of Ingenuity Pathway Analysis and Gene Set Enrichment Analysis software to define gene sets in dysregulated pathways and networks of potential pathologic relevance in this disease. Biologically relevant differentially expressed genes were confirmed by RNA and protein analysis. RESULTS: Exploration of global gene expression profiles of the JIA FLS revealed important dysregulated pathways, including the transforming growth factor ß (TGFß) signaling, as well as endochondral bone formation, cartilage formation, and ß-catenin networks. Importantly, bone morphogenetic protein 4 (BMP-4) was significantly overexpressed in the JIA FLS. FLS from patients with oligoarticular JIA exhibit a chondrocyte phenotype, as evidenced by expression of type II collagen and aggrecan. CONCLUSION: Dysregulation of the pathways involved in the pathogenesis of oligoarticular JIA were revealed through gene expression profiling. JIA FLS displayed dysregulated TGFß signaling and exhibited a hypertrophic chondrocyte phenotype. These characteristics, along with contributions from the ß-catenin network may have implications for endochondral bone formation and local growth disturbances in oligoarticular JIA. Overexpression of BMP-4 in FLS from patients with oligoarticular JIA in particular may play an important role in disease pathogenesis, with a direct effect on functional outcome and with implications for future treatment.

Curcumin-induced HDAC inhibition and attenuation of medulloblastoma growth in vitro and in vivo.

BACKGROUND: Medulloblastoma is the most common brain tumor in children, and its prognosis is worse than for many other common pediatric cancers. Survivors undergoing treatment suffer from serious therapy-related side effects. Thus, it is imperative to identify safer, effective treatments for medulloblastoma. In this study we evaluated the anti-cancer potential of curcumin in medulloblastoma by testing its ability to induce apoptosis and inhibit tumor growth in vitro and in vivo using established medulloblastoma models. METHODS: Using cultured medulloblastoma cells, tumor xenografts, and the Smo/Smo transgenic medulloblastoma mouse model, the antitumor effects of curcumin were tested in vitro and in vivo. RESULTS: Curcumin induced apoptosis and cell cycle arrest at the G2/M phase in medulloblastoma cells. These effects were accompanied by reduced histone deacetylase (HDAC) 4 expression and activity and increased tubulin acetylation, ultimately leading to mitotic catastrophe. In in vivo medulloblastoma xenografts, curcumin reduced tumor growth and significantly increased survival in the Smo/Smo transgenic medulloblastoma mouse model. CONCLUSIONS: The in vitro and in vivo data suggest that curcumin has the potential to be developed as a therapeutic agent for medulloblastoma.

Prolonged synovitis in pediatric Lyme arthritis cannot be predicted by clinical or laboratory parameters.

Ten percent of Lyme arthritis (LA) patients have continued synovitis despite antimicrobial therapy. The current study was designed to (1) investigate predictors of prolonged disease and (2) further define natural history of pediatric LA. Medical records of 94 children fulfilling Centers for Disease Control criteria for Lyme disease were reviewed, classified into groups according to duration of synovitis, and SPSS statistical software was used for analysis. Thirty-nine percent required >6 months and 13% required >12 months to resolve LA. Pearson correlation between duration of symptoms of LA pretreatment and duration of synovitis was not significant. When patients were stratified by group, no differences were found for age, antinuclear antibodies positivity, enzyme-linked immunosorbent assay titer, or reactivity of Western blot using parametric and nonparametric tests. Linear and logistic regression showed no predictors of disease duration. One third of pediatric LA patients require >6 months to resolve synovitis. Duration is not associated with delay in treatment, age, or seroreactivity.

Long-term effects of immunization with recombinant lipoprotein outer surface protein a on serologic test for lyme disease.

Immunization with recombinant lipoprotein outer surface protein A vaccine is known to interfere with some serologic tests for Lyme disease. We tested sera from 152 vaccine recipients by using in-house and commercial Western blot assays and found that vaccination caused interference in up to 25% of recipients and can persist for over 6 years.

Comparison of PCR and clinical laboratory tests for diagnosing H. pylori infection in pediatric patients.

BACKGROUND: Histology and/or culture are generally considered the gold standard for the detection of H. pylori infection. Especially in children, these tests may result in a false negative outcome because of patchy distribution of the organism in the stomach mucosa. We have developed a PCR assay utilizing nested primer pairs directed against a subunit of the H. pylori urease gene (ureA). As part of a prospective evaluation of diagnostic tests to aid in detecting H. pylori infection in children, the aim of this study was to compare our PCR and Western blot assays with results obtained from histologic examination of biopsy specimens, rapid urease tests, and an FDA approved serologic assay and published PCR results to determine if we could validate the assays for diagnostic use on our patient population. RESULTS: Gastric biopsy specimens obtained from 101 pediatric patients were evaluated for the presence of H. pylori using histologic techniques, rapid urease (CLOtest) test and the PCR assay. Serum samples from each patient were assayed using both ELISA and Western Blot for antibodies to H. pylori. A total of 32 patients tested were positive by at least one of the methods evaluated. Thirteen patients had positive histology, 13 had a positive CLOtest, and 17 patients had positive H. pylori PCR. Out of the 13 CLO positive patients, 12 were positive by histologic analysis and all 13 were positive by PCR. Results of serologic tests on the same population did not correlate well with other assays. Twenty-eight patients showed serologic evidence of H. pylori infection, of which 9 were both CLO and histology positive and 12 were positive by PCR. Of the seropositive patients, 26 were ELISA positive, 13 were positive by Western blot, and 11 by both serologic methods. CONCLUSIONS: The results obtained suggest that our nested PCR assay has the specificity and sensitivity necessary for clinical application when compared to standard histologic examination and rapid urease test. In addition, we found the current commercially available approved ELISA method appears unable to accurately detect H. pylori in this population. The Western blot assay yielded better concordance with CLOtest and histology, but not as good as the nested PCR assay.

C6 test as an indicator of therapy outcome for patients with localized or disseminated lyme borreliosis.

Management of Lyme disease would benefit from a test to assess therapy outcome. Such a test could be employed to ascertain if treatment of early Lyme disease was successful and would be helpful to clinicians assessing patients with lingering posttreatment symptoms. We reported recently that levels of the antibody to C(6), a Borrelia burgdorferi-derived peptide that is used as an antigen in the C(6)-Lyme diagnostic test, declined after successful antibiotic treatment of Lyme borreliosis patients. We assessed retrospectively the change in anti-C(6) antibody titers in 131 patients with either early localized disease (erythema migrans) or disseminated disease. All of these patients were treated with antibiotics and were free of the clinical signs shown at presentation within 12 weeks after the initiation of treatment. Decreases in reciprocal geometric mean titers (rGMT) of the anti-C(6) antibody were quantified for the subpopulation of 45 patients whose baseline rGMT were >/=80 and whose second serum specimens were obtained at least 6 months after the baseline specimen. Eighty percent of this patient group (36 of 45) experienced a >/=4-fold decrease in their rGMT (P < 0.0003). These results suggest that a change in the anti-C(6) antibody titer may serve as an indicator of therapy outcome for patients with localized or disseminated Lyme borreliosis.

Growth of Helicobacter pylori in a long spiral form does not alter expression of immunodominant proteins.

BACKGROUND: We have previously reported that altered culture conditions (a broth media with shaking) could induce a strain of Helicobacter pylori to assume a long spiral morphology resembling that described for Helicobacter heilmannii. The present study was initiated to determine if other strains of H. pylori could be induced to assume that morphology and if doing so would alter the expression of immunodominant proteins. RESULTS: The six strains used in this study were American Type Culture Collection 43504, 43579, 49503, 51652, and 51653, and Sydney Strain I. Each strain was grown on solid media and in broth culture using conditions previously shown to induce the long spiral morphology in strain 43504. DNA from each was subjected to urease gene fingerprint analysis. Results of the molecular analysis showed identical fingerprint patterns for each strain independent of culture source, indicating that only a single strain was present in each culture. Expression of immunodominant proteins was assessed by SDS polyacrylamide gel electrophoresis and Western blotting with hyperimmune rabbit anti H. pylori sera or serum from an H. pylori infected patient. Analysis of protein profiles revealed some variation between strains but no significant differences associated with morphologic alterations. CONCLUSIONS: These results indicate that growth of H. pylori in a long spiral form does not affect expression of immunodominant proteins, thus in vivo growth in the long spiral form (not documented to date) would not be distinguishable by serology.

In vitro assessment of antiborrelial activity of OspA vaccine sera.

Prevention of Lyme disease by the recombinant OspA-based vaccine reportedly works by preventing transmission of spirochetes from ticks to humans. We report on an in vitro microculture assay, which can be used to provide an indicator of the need for booster doses of vaccine.