Type 1 interferonopathy presenting as juvenile idiopathic arthritis with interstitial lung disease: report of a new phenotype.

BACKGROUND: STING-associated vasculopathy with onset in infancy (SAVI) is a type 1 interferonopathy manifesting as a pulmonary and vascular syndrome resulting from gain-of-function mutations in TMEM173, the gene encoding STING. Familial reports in the literature are sparse. CASE PRESENTATION: We report a case series of SAVI in a three generation kindred, with a phenotype of interstitial lung disease (ILD) and rheumatoid factor positive polyarticular juvenile idiopathic arthritis (JIA). Current and historical medical records were reviewed for clinical and laboratory information. Whole blood from cases 1 and 2, plus stored appendicectomy tissue from case 3, underwent DNA sequencing of the TMEM173 gene. Peripheral blood RNA was obtained from cases 1 and 2 for functional assessment of the TMEM173 mutation. DNA sequencing identified the same heterozygous TMEM173 mutation (c.463G > A; p.Val155Met) in all three cases, consistent with a diagnosis of the autosomal dominant condition SAVI. Functional assessment of this mutation identified a prominent interferon signature which was confirmed on repeat testing. CONCLUSIONS: SAVI presented in this family as ILD with early onset juvenile rheumatoid arthritis. This condition should be considered in all rheumatoid arthritis patients with early-onset ILD and in all JIA patients with ILD.

Impaired glucose tolerance attenuates bone accrual by promoting the maturation of osteoblasts: Role of Beclin1-mediated autophagy.

Patients with type 2 diabetes mellitus (T2DM) experience a 1.5-3.5 fold increase in fracture risk, but the mechanisms responsible for these alterations in bone biomechanical properties remain elusive. Macroautophagy, often referred to as autophagy, is regulated by signaling downstream of the insulin receptor. Metabolic changes associated with the progression of glucose intolerance have been shown to alter autophagy in various tissues, but limited information is available in relation to bone cells. The aim of this study was to (a) investigate whether autophagy is altered in bone tissue during impaired glucose tolerance, and (b) determine how autophagy impacts osteoblast differentiation, activity, and maturation. Four-week-old, male C57BL/6 mice were fed a control (Con) or high fat (HF) diet for 2, 8, or 16 wks. Mice on the HF diet demonstrated elevated fasting blood glucose and impaired glucose tolerance. Reduced trabecular bone in the femoral neck was evident in the mice on the HF diet by 8 wks compared to Con mice. Histological evaluation of the tibia suggested that the high fat diet promoted terminal differentiation of the osteoblast to an osteocyte. This shift of the osteoblasts towards a non-mineralizing, osteocyte phenotype appears to be coordinated by Beclin1-mediated autophagy. Consistent with these changes in the osteoblast in vivo, the induction of autophagy was able to direct MC3T3-E1 cells towards a more mature osteoblast phenotype. Although these data are somewhat observational, further investigation is warranted to determine if Beclin1-mediated autophagy is essential for the terminal differentiation of the osteoblasts and whether autophagy is having a protective or deleterious effect on bone in T2DM.

The child with joint pain in primary care.

Joint pains are a common reason for children to present to primary care. The differential diagnosis is large including some diseases that do not primarily affect the musculoskeletal system. Although the cause for many patients will be benign and self-resolving, in rare cases the diagnosis is associated with long-term morbidity and mortality if not detected early and appropriately treated. These include primary and secondary malignancies, septic arthritis, osteomyelitis, inflammatory arthritis, slipped upper femoral epiphysis (SUFE) and non-accidental injury. We highlight the importance of a thorough history and directed yet comprehensive examination. A diagnostic algorithm is provided to direct primary care physicians' clinical assessment and investigation with the evidence base where available. In many cases, tests are not required, but if there is suspicion of malignancy, infection or inflammatory conditions, laboratory tests including full blood count, blood film, erythrocyte sedimentation rate, C-reactive protein and lactate dehydrogenase help to support or exclude the diagnosis. Autoimmune tests, such as antinuclear antibodies and rheumatoid factor, have no diagnostic role in juvenile idiopathic arthritis; therefore, we advise against any form of 'rheumatological/autoimmune disease screen' in primary care. Imaging does have a place in the diagnosis of joint pains in children, with plain radiographs being most appropriate for suspected fractures and SUFE, whilst ultrasound is better for the detection of inflammatory or infective effusions. The appropriate referral of children to paediatric rheumatologists, oncologists, orthopaedic surgeons and the emergency department are discussed.

Effects of dried plum supplementation on bone metabolism in adult C57BL/6 male mice.

Dietary supplementation of dried plum (DP) prevents bone loss and restores bone mass in osteopenic animal models. This study was designed to determine the effects of DP supplementation on bone metabolic activity over time using adult (6-month-old) male C57BL/6 mice (n = 40) receiving control (CON = AIN93 M) or CON+DP 25 % (w/w) diets for 4 or 12 weeks. After 4 weeks of treatment, animals consuming the DP diet had a higher whole-body bone mineral density, vertebral trabecular bone volume (BV/TV), and femoral cortical thickness compared to the CON animals. In the distal metaphysis of the femur, BV/TV was increased in the DP-treated animals, but only after 12 weeks. Bone histomorphometric analyses revealed that DP decreased osteoblast surface (67 %) and osteoclast surface (62 %) at 4 weeks, but these surfaces normalized to the CON animals by 12 weeks. Coincident with these changes, the mineralizing surface (MS/BS) and cancellous bone formation rate (BFR/BS) were reduced at 4 weeks in the DP group compared to the CON, but by 12 weeks of DP supplementation, BFR/BS (~twofold) and MS/BS (~1.7-fold) tended to be increased (p < 0.10). The relative abundance of RNA for key regulators of osteoblast and osteoclast differentiation and indicators of osteoblast activity were reduced in the DP group at 4 weeks with no difference between groups at 12 weeks. These results indicate that supplementing the diet with DP initially suppressed cancellous bone turnover, but a biphasic response occurs over time, resulting in a positive effect on bone mass and structure.

The role of protease-activated receptors PAR-1 and PAR-2 in the repair of 16HBE 14o(-) epithelial cell monolayers in vitro.

BACKGROUND: We recently reported that repair following mechanical wounding of epithelial cell layers in vitro is dependent on fibrin formation and the activity of locally expressed coagulation cascade proteins. Serine proteases of the coagulation cascade are an important group of protease-activated receptor (PAR) activators and PAR-1 to 4 are expressed by the normal bronchial epithelium. OBJECTIVE: We tested the hypothesis that activation of PAR-1 and PAR-2 by coagulation cascade proteases stimulates epithelial repair via effects on fibrin formation. METHODS: Using mechanically wounded 16HBE 14o(-) epithelial cell layers in culture, we investigated the effect of PAR-1 and PAR-2 agonist peptides, control partially scrambled peptides and PAR-neutralizing antibodies on the rate of repair and fibrin formation. Coagulation factors in culture supernatants were measured by immunoblot. RT-PCR was used to investigate PAR-1, PAR-2 and PGE2 receptor (EP-1 to EP-4) expression in this model and qRT-PCR to quantify responses to wounding. Additionally, we investigated the effect of exogenously added factor Xa (FXa) and neutrophil elastase and the influence of PGE2 and indomethacin on the repair response. RESULTS: PAR-1 and PAR-2 peptide agonists stimulated the rate of repair and enhanced the formation of a fibrin provisional matrix to support the repair process. Conversely, PAR-neutralizing antibodies inhibited repair. Under serum-free culture conditions, 16HBE 14o(-) cells expressed EP-2 and EP-3, but not EP-1 or EP-4, receptors. Wounding induced an increased expression of EP-3 but did not alter EP-2, PAR-1 or PAR-2 expression. In the absence of PAR agonists, there was no evidence for a role for PGE2 in fibrin formation or the repair process. Indomethacin attenuated fibrin formation in wounded cultures only in the presence of the PAR-2 peptide. FXa stimulated epithelial repair while neutrophil elastase reduced the levels of coagulation factors and inhibited repair. CONCLUSION: Locally expressed serine proteases of the coagulation cascade activate PAR-1 and PAR-2 to enhance fibrin formation and bronchial epithelial repair.

Effects of iron supplementation on binding activity of iron regulatory proteins and the subsequent effect on growth performance and indices of hematological and mineral status of young pigs.

Two experiments were conducted to evaluate the effects of supplemental Fe on the binding activity of iron regulatory proteins (IRP) and the subsequent effect on growth performance and indices of hematological and mineral status of young pigs. In Exp. 1, male pigs (n = 10; 1.8 kg; age = 14 +/- 1 h) were allotted by BW to two treatments (five pigs per treatment). Treatments administered by i.m. injection were as follows: 1) 1 mL of sterile saline solution (Sal); and 2) 1 mL of 200 mg Fe as Fe-dextran (Fe). Pigs were bled (d 0 and 13) to determine hemoglobin (Hb), hematocrit (Hct), transferrin (Tf), and plasma Fe (PFe), and then killed (d 13) to determine spontaneous and 2-mercaptoethanol (2-ME)-inducible IRP RNA binding activity in liver and liver and whole-body mineral concentrations. Contemporary pigs (n = 5; 2.2 kg; age = 14 +/- 2 h) were killed at d 0 to establish baseline (BL1) measurements. In Exp. 2, pigs (six pigs per treatment; 6.5 kg; age = 19 +/- 3 d) were fed a basal diet (Phase 1 = d 0 to 7; Phase 2 = d 7 to 21; Phase 3 = d 21 to 35) supplemented with 0 or 150 mg/kg of Fe as ferrous sulfate and killed at d 35 (18.3 kg; age = 54 +/- 3 d). In addition, pigs (n = 5; 5.9 kg; age = 19 +/- 3 d) were killed at the start of Exp. 2 to establish baseline (BL2) measurements, and liver samples were collected and analyzed for IRP RNA binding activity. In Exp. 1, no difference (P = 0.482) was observed in ADG. On d 13, Fe-treated pigs had greater (P = 0.001) Hb, Hct, and PFe and less (P = 0.002) Tf than Sal-treated pigs. Whole-body Fe concentration was greater (P = 0.002) in Fe- vs. Sal-treated pigs. Treated pigs (Fe or Sal) had greater (P = 0.006) whole-body Cu and less (P = 0.002) whole-body Ca, Mg, Mn, P, and Zn concentrations than BL1. Liver Fe concentration was greater (P = 0.001) in Fe- vs. Sal-treated pigs, but liver Fe concentration of Sal-treated pigs was less (P = 0.001) than that of BL1 pigs. Sal-treated pigs had greater (P = 0.004) spontaneous IRP binding activity than Fe-treated pigs. In Exp. 2, spontaneous and 2-ME inducible IRP binding activities were greater (P = 0.013 and 0.005, respectively) in pigs fed diets containing 0 vs. 150 mg of added Fe/kg of diet. Moreover, pigs fed either treatment for 35 d had greater (P = 0.001) 2-ME inducible IRP binding activity than BL2 pigs. Results indicate that IRP binding activity is influenced by Fe supplementation. Subsequently, other indicators of Fe status are affected via the role of IRP in posttranscriptional expression of Fe storage and transport proteins.

Iron overload impairs pro-inflammatory cytokine responses by Kupffer cells.

AIM: The aim of the present study was to determine the effect of chronic iron overload on Kupffer cell cytokine production. METHODS: Kupffer cells were isolated from rats that were fed either a control or iron-supplemented diet for 12 months. Cytokine mRNA and protein levels were determined by using a ribonuclease protection assay and ELISA, respectively. RESULTS: Baseline levels of tumor necrosis factor-alpha, transforming growth factor-beta1, interleukin-6 and granulocyte macrophage colony stimulating factor were similar in iron-loaded and control Kupffer cells. Following the addition of lipopolysaccharide to control cells, tumor necrosis factor-alpha, interleukin-1alpha and interleukin-6 mRNA levels increased. Tumor necrosis factor-alpha mRNA and protein levels were reduced by 40 and 60%, respectively, in iron-loaded cells compared with controls following the addition of lipopolysaccharide. Interleukin-6 mRNA levels in iron-loaded Kupffer cells were also reduced. Granulocyte macrophage colony stimulating factor mRNA levels remained unchanged in controls, but were significantly elevated in iron-loaded cells. Tumor growth factor-beta1 mRNA and protein levels were similar in control and iron-loaded cells. CONCLUSION: Deposition of iron in Kupffer cells in chronic dietary iron overload results in an impaired pro-inflammatory cytokine response to lipopolysaccharide. Our observations may have relevance to the altered immune function observed in chronic iron-overload syndromes.

Arterial lines: an analysis of good practice.

The use of arterial line monitoring in critically ill children provides an indispensable foundation of critical care. The information provided by their use ensures accurate monitoring and blood gas analysis and subsequent modifications to prescribed treatment. The invasive nature of intra-arterial catheters may lead to complications of haemorrhage, thrombosis and infection. The value of arterial cannulation in relation to alternative less invasive methods of monitoring is discussed. The literature reveals that in many areas nurses are failing to unite theory and practice. This has implications for the safety of children and the accountability of nurses caring for them.

Isolation and primary culture of rat Kupffer cells.

The aim of this study was to describe a reproducible method for the isolation, purification and primary culture of rat Kupffer cells. Kupffer cells were isolated following sequential pronase/collagenase digestion of the liver and enrichment of a non-parenchymal cell fraction by a single-density gradient centrifugation step using 30% metrizamide. Kupffer cells were isolated and further purified from this cell fraction by centrifugal elutriation. Kupffer cells were isolated at 1017 g at 48-110 mL/min. All Kupffer cell fractions exhibited phagocytosis of 3 microm latex beads. Kupffer cell fractions isolated at 48 and 60 mL/min were predominantly ED2 negative while later fractions (80-110 mL/min) were ED2 positive. Kupffer cells were adherent in culture after 2 h. This method for Kupffer cell isolation resulted in a yield of 80-120 x 10(6) Kupffer cells per liver.

Nuclear hormone receptors and adipogenesis.

Adipocytes provide a model cell system for exploring the complexities of nuclear hormone receptor transcriptional regulation. Adipocytes produce lipid and cholesterol metabolites which can serve as activating ligands for many of the classic and "orphan" nuclear hormone receptors. At the same time, nuclear hormone receptors directly control adipocyte commitment. The recently described nuclear hormone receptor coactivators and corepressors provide an additional level of complexity to this system. This review emphasizes available in vitro and in vivo models and discusses them in the context of current controversies and future experimental directions.

Gadolinium chloride suppresses hepatic oval cell proliferation in rats with biliary obstruction.

Liver injury due to bile duct ligation (BDL) is histologically characterized by cholestasis, bile ductular proliferation, hepatocellular damage, portal fibrosis, and ultimately biliary cirrhosis. Stem cells within the liver may act as progenitor cells for small epithelial cells termed oval cells that can differentiate into bile duct cells or hepatocytes, whereas myofibroblasts are the principal source of collagen production in fibrosis. The aims of this study were to determine 1) whether BDL induces oval cell proliferation and 2) whether blockade of Kupffer cells affects oval cell proliferation, bile duct proliferation, and myofibroblast transformation in experimental biliary obstruction. Male Sprague-Dawley rats were divided into two groups to receive either a single dose of gadolinium chloride (a selective Kupffer cell blocking agent) or vehicle. One day later, the gadolinium- and vehicle-treated groups were further subdivided to receive either BDL or sham operation. The rats were sacrificed on day 7 postoperatively. Serum was collected for measurement of aspartate aminotransferase, gamma-glutamyl transpeptidase, and bilirubin levels. Liver tissue was taken for evaluation of fibrosis, bile ductular cells, oval cells, and myofibroblasts. BDL resulted in elevated aspartate aminotransferase, gamma-glutamyl transpeptidase, and bilirubin in serum, and gadolinium pretreatment did not modify these effects. BDL induced marked oval cell proliferation, which was completely prevented by gadolinium pretreatment. Gadolinium did not affect the induction of bile duct expansion or myofibroblasts after BDL. We conclude that experimental biliary obstruction induces oval cell proliferation, which can be prevented by gadolinium pretreatment. This suggests that bile ductular proliferation and myofibroblast transformation are not mediated by Kupffer cells and that ductular proliferation can proceed in the absence of oval cells. Alternatively, gadolinium may directly affect oval cell proliferation after BDL.

CAAT/enhancer binding proteins directly modulate transcription from the peroxisome proliferator-activated receptor gamma 2 promoter.

The CCAAT/enhancer binding proteins (C/EBPs) and the peroxisome proliferator-activated receptors (PPARs) together regulate adipogenesis. The current work uses co-transfection studies to examine the C/ EBP dependence of PPAR gamma 2 transcription. Both C/ EBP alpha and C/EBP delta expression vectors activated transcription from a PPAR gamma 2 promoter/luciferase expression vector by 5-6 fold in UMR106 cells. The simultaneous transfection of the C/EBP homologous protein (CHOP) (also known as growth arrest DNA damage protein 153 or gadd153) inhibited this C/EBP-dependent activation in a concentration dependent manner. The CHOP protein is known to heterodimerize with other C/EBP proteins to form transcriptionally inactive complexes. Mutation of the two C/EBP DNA recognition elements at -340 bp and -327 bp within the PPAR gamma 2 promoter reduced the inductive effects of both C/EBP alpha and C/EBP delta. These findings demonstrate that proteins within the C/EBP family directly modulate transcription from the PPAR gamma 2 promoter.

Long-term outcomes of conventional therapy for infantile spasms.

Infantile spasms (IS) is an age-specific epilepsy which responds to anticonvulsant therapy but has a generally poor prognosis for normal psychomotor development. The subgroup of infants with a cryptogenic aetiology or whose therapy is initiated promptly is thought to have a more favourable prognosis. We retrospectively reviewed 28 infants with IS treated between 1990 and 1996 with adrenocorticotropic hormone (ACTH), valproic acid (VPA), or both, in order to correlate therapeutic response with long-term outcome. Mean age at onset of treatment was 6.4 months, with 57% of patients started within 1 month of IS appearance. IS was considered cryptogenic in 39%. The majority of infants responded to ACTH or VPA with a reduction in spasms of 75% or more. Total remission of seizures occurred in 52%. Death occurred in eight patients; mean duration of follow-up for survivors was 55 months. All subgroups based on age, aetiology, or treatment had poor outcomes, commonly with residual epilepsy, cerebral palsy or mental retardation. Conventional treatment for IS, even when initially successful in reducing spasms, is inadequate when viewed from a long-term developmental perspective, suggesting the need for novel innovative approaches for treating IS.

Thrombosis in children receiving L-asparaginase. Determining patients at risk.

PURPOSE: A prospective study of coagulation in 15 children who received L-asparaginase, vincristine, or prednisone plus or minus an anthracycline as part of a treatment program for leukemia or leukemia-lymphoma syndrome was conducted. PATIENTS AND METHODS: One patient developed a central nervous system thrombosis. RESULTS: The inhibitors of coagulation, including antithrombin-III, protein C, protein S, and plasminogen, were decreased in many individuals, but were not significantly different in the patient who had experienced the thrombotic event. Platelet aggregations to low molar ADP were performed in four patients, and in three patients showed a hyperaggregable pattern. CONCLUSIONS: The patient with thrombosis developed a transient acquired type II pattern on multimeric analysis of the von Willebrand factor, which was not seen in the other individuals studied.

Pulmonary eosinophilic response to respiratory syncytial virus infection in mice sensitized to the major surface glycoprotein G.

To investigate the contribution of immunity to individual respiratory syncytial (RS) virus proteins to the augmentation of pulmonary pathology, mice were scarified with recombinant vaccinia viruses (rVV) expressing individual RS virus proteins. The pulmonary response to infection with RS virus was monitored by bronchoalveolar lavage (BAL). In mice vaccinated with the major surface glycoprotein (G), 14-25% of BAL cells were eosinophils; these comprised less than 3% of BAL cells from other groups of mice after RS virus challenge. Mice sensitized to the G or fusion (F) proteins developed lung haemorrhage and those sensitized to G, F or nucleoprotein (N) showed pulmonary polymorphonucleocyte efflux. To investigate the concomitant changes in local T-cell subsets, BAL cells were stained with mAbs to CD4, CD8, CD45RB, alpha beta and gamma delta T cell receptor (TCR) proteins. Three colour flow cytometry showed that most cells were CD3+CD4+ alpha beta+gamma delta+ or CD3+CD8+ alpha beta+gamma delta-, although some CD4-CD8-SIg- cells were also identified. Most of these 'null' cells lacked CD3, but CD3+ null cells from rVV-G or -F primed mice bore either alpha beta and gamma delta TCR in approximately equal numbers. The intensity of staining for CD45RB declined rapidly after infection with RS virus on both CD4 and CD8 cells. The rate of loss of CD45RB on CD4 T cells was accelerated by prior sensitization with rVV-G, consistent with conversion to helper T cell subsets producing eosinophil-promoting cytokines. The eosinophilic reaction to RS virus infection therefore specifically reflects sensitization to G protein, but sensitization to other proteins can also cause distinct pathological effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of continuous and intermittent Factor VIII concentrate therapy in hemophilia A.

Comparison of continuous versus intermittent (every 12 h bolus) infusion of a high-purity Factor VIII concentrate in 12 severe hemophilia A patients undergoing surgical procedures indicated that a higher (50 u/dl versus 35 u/dl) minimal plasma level was seen with the same amount of product when given continuously. A continuous VIII concentrate infusion of 2 u/kg/h produces a mean VIIIc level of about 50 u/dl or, alternatively, about 0.04 u/kg/h raises the VIIIc level by 1 u/dl (1%). None of the patients showed any abnormal bleeding. Higher plasma levels of VIIIc were noted by the two-stage VIIIc assay than by the one-stage VIIIc assay. Fibrinogen levels were normal or only slightly elevated, and the monomer test was consistently negative.