Nuclear aggregation of huntingtin is not prevented by deletion of chaperone Hsp104.

Polyglutamine expansion causes the disease proteins to aggregate, resulting in stable insoluble aggregates in the nucleus. The in vitro aggregation and cellular toxicity of polyglutamine proteins are reduced by chaperone heat shock proteins (Hsp). In polyglutamine disease animal models, however, polyglutamine inclusions remain in the nucleus despite the suppression of neurodegeneration by Hsp. Studies using yeast genetic approach revealed that the balance of Hsp is important for regulating protein aggregation in the cytoplasm of yeast cells. Here we report that N-terminal fragments of huntingtin with an expanded polyglutamine tract form aggregates only in the cytoplasm of yeast cells and, when tagged with nuclear localization sequences (NLS), are able to aggregate in the nucleus. Deletion of the Hsp104 gene prevents the aggregation of huntingtin in the cytoplasm but is unable to eliminate the aggregation of NLS-tagged huntingtin in the nucleus. The inhibitory effect of Hsp104 deletion on the cytoplasmic aggregation of huntingtin only occurs in viable yeast cells, as aggregates can be formed in Hsp104 deletion cells that have been frozen for 72 h. Fresh cytosolic extracts of the Hsp104 deletion strain inhibit the aggregation of huntingtin in vitro, suggesting that the deletion of Hsp104 may alter the activities of other cytoplasmic factors to inhibit polyglutamine aggregation in the cytoplasm. We propose that the regulatory effects of chaperones may mainly be restricted to the cytoplasm and have much less influence on polyglutamine-containing aggregates in the nucleus.

Nitric oxide and inflammatory bowel disease: evidence for local intestinal production in children with active colonic disease.

BACKGROUND: Active colitis in patients with inflammatory bowel disease is associated with mucosal vasodilation, increased intestinal permeability and abnormal colonic motility. Nitric oxide is a messenger molecule with many functions, including regulation of local blood flow, vasomotor tone, and inflammation. Increased nitric oxide production and inducible nitric oxide synthase activity have been demonstrated in experimental models of colitis. This study was designed to determine the relationship between nitric oxide production and colonic inflammation in children with active colitis and in control subjects and whether expression of inducible nitric oxide synthase protein is demonstrable in the intestinal epithelium of these patients. METHODS: Nitrate + nitrite were measured in urine, stool, and plasma using the Griess assay. Expression of inducible nitric oxide synthase protein in intestinal tissue was determined by immunohistochemical localization. RESULTS: Urinary nitrate + nitrite levels were not significantly different in patients and control subjects. In contrast, stool and plasma nitrate + nitrite concentrations were significantly higher in children with inflammatory bowel disease compared with levels in control children (stool: 162.4 +/- 31.0 mumol/l versus 77.2 +/- 22.1 mumol/l; plasma: 65.2 +/- 9.9 mumol/l versus 38.1 +/- 6.6 mumol/L; p < 0.05). Stool nitrate + nitrite levels significantly correlated with plasma values. Immunohistochemical staining of colonic tissue from children with inflammatory bowel disease demonstrated inducible nitric oxide synthase protein located exclusively in epithelial cells. CONCLUSION: Increased nitric oxide production and enhanced intestinal epithelial cell expression of inducible nitric oxide synthase protein are associated with active colonic inflammation.

Reticuloendothelial system uptake of infused 125I-trypsin in newborn and adult rats.

Previous studies have demonstrated enhanced intestinal trypsin uptake and decreased liver clearance of trypsin in newborn rats compared to adults. In order to examine the effectiveness of the reticuloendothelial system (RES) in clearing trypsin, bovine trypsin (1.25 mg/100 g body weight) plus trace 125I-trypsin were injected into the portal vein of 2-week-old (n = 57) and adult (n = 44) control rats or following RES stimulation using intraperitoneally injected lipopolysaccharide or RES suppression with intraperitoneally injected oleic acid emulsion. Plasma, liver and spleen 125I activities were assessed at 1, 5 or 15 min following infusion in control, stimulated and suppressed animals. Newborn control rats had significantly increased 125I plasma levels with decreased liver and spleen 125I activity compared to control adults. RES stimulation in the newborns did not lead to any change in liver or plasma levels although splenic values increased while adults had a decrease in liver 125I activity. RES suppression in the newborns led to increased plasma and decreased spleen 125I-trypsin values while adult rat levels were unchanged. The immature reticuloendothelial system in newborns is poorly responsive to RES stimulation although it can be made even further inefficient by RES suppression. The combination of RES immaturity and lack of response to stimulation may make newborns susceptible to proteolytic damage, especially during times of increased systemic levels of proteolytic enzymes.

Esophageal dysmotility in children breast-fed by mothers with silicone breast implants. Long-term follow-up and response to treatment.

Our aims were to determine the long-term clinical and manometric follow-up of 11 children with previously documented esophageal dysmotility, who had been breast-fed by mothers with silicone breast implants, their response to prokinetic agents, and to analyze changes in macrophage activation. Seven of 11 children had subjective clinical improvement. Weight/ height ratios remained the same or improved in 9/11. Biopsies at follow-up endoscopy were either normal or demonstrated mild esophagitis in 8/10. LES and UES pressures and percent propagation were not significantly different at follow-up, while wave amplitude significantly increased. Following intravenous metoclopramide, LES pressure, percent propagation, and wave amplitude significantly increased while UES pressure was unchanged. Urinary neopterin significantly decreased at follow-up, while urinary nitrates were unchanged. Esophageal dysmotility is chronic in this group of children, suggesting persistent autonomic nervous system dysfunction. Prokinetic agents may be useful in long-term management. The decreasing urinary neopterin levels suggest that, ultimately, there may be improvement in esophageal motility.

Increased urinary NO3(-) + NO2- and neopterin excretion in children breast fed by mothers with silicone breast implants: evidence for macrophage activation.

OBJECTIVE: To determine whether children breast fed by mothers with silicone implants (BFSI) have increased urinary excretion of nitric oxide (NO) metabolites and neopterin, whether these are associated with esophageal dysmotility, and whether in vitro incubation of macrophages with silicone increases NO synthesis. METHODS: In a case-control study based on laboratory investigation, 38 BFSI children (17 male, 21 female, mean age 7.1 +/- 3.6 years, range 0.5-16.5) were compared with 30 controls (14 male, 16 female, mean age 8.4 +/- 3.5 years, range 2.5-17). Urinary NO was quantitated using the Griess reaction. Urinary neopterin was determined by radioimmunoassay. Murine macrophages were cultured with or without silicone and NO production assayed. RESULTS: Urinary NO and neopterin were significantly increased in BFSI children compared with controls. There was a significant inverse relationship between urinary neopterin excretion and the severity of esophageal dysfunction. In vitro nitrite production was nearly 60% higher in macrophages grown on silicone compared to other growth conditions. CONCLUSION: BFSI children have evidence of macrophage activation and this is associated with esophageal dysmotility. In vitro data support the proposal that silicone exposure causes macrophage activation.

Lack of autoantibody expression in children born to mothers with silicone breast implants.

OBJECTIVE: We determined systematically the prevalence of autoantibodies in children born to mothers with silicone breast implants and the relationships with clinical symptoms and methods of exposure. METHODS: Autoantibody expression was determined in 80 children born to mothers with silicone implants and in 42 controls. A clinical assessment score was assigned to each patient. Antinuclear antibodies as well as antibodies to mitochondrial, smooth muscle, striational, myocardial, parietal cell, reticulin tissues, or subcellular compartments were measured by indirect fluorescent assay. Antibodies to nRNP (U1-RNP/snRNP); Sm; SS-A; SS-B; Scl-70; thyroid microsome; immunoglobulin (Ig)G, IgM, and IgA antibodies to cardiolipin; and antibodies to native and denatured human types I and II collagen were measured by enzyme-linked immunosorbent assay. Serum complement components C3 and C4 and IgM rheumatoid factor were measured by nephelometry. RESULTS: Autoantibody prevalence was not significantly different between children born to mothers with silicone implants and controls. The presence of autoantibodies was not related to the children's clinical symptoms or to the method of exposure. CONCLUSIONS: Determination of autoantibody production is of limited clinical utility in the evaluation of children born to mothers with silicone breast implants.

Lymphocyte response to silica among offspring of silicone breast implant recipients.

The current study evaluated immune response to silicon dioxide in children born to women with silicone breast implants. In part one of the study, the T lymphocytes of 21 of 24 such children were significantly stimulated by silicon dioxide (silica). Part two consisted of eleven children, four born preimplantation and seven born postimplantation. None of the preimplant offspring showed T cell responses to silica; five of the seven postimplant children were positive for T cell memory for silica. Part three was a blinded study based on statistically significant differences in T cell stimulation with silicon dioxide between postimplant children and controls. These findings indicate a common immune reaction, that of T cell memory, occurs in mothers and their children born after exposure to silicone mammary implants placed prior to pregnancy. Since not all such children were breast fed the result favors transplacental passage of immunogens such as silicone oligomers or through maternofetal cellular traffic.

Serum α1-Antitrypsin Levels as a Marker of Activity in Pediatric Inflammatory Bowel Disease.

: The purpose of this study was to evaluate the use of the acute-phase serum protein α1-antitrypsin (α1-AT) compared with the erythrocyte sedimentation rate (ESR), platelet count, and hemoglobin level in screening for inflammation in newly diagnosed and known patients with inflammatory bowel disease (IBD). The serum α1-AT level, ESR, platelet count, and hemoglobin values of 154 children who were initially evaluated for possible IBD, as well as of 113 children with known IBD, were compared to evaluate their value as markers of inflammatory activity. Of the 154 children evaluated for IBD, 103 did not have IBD, 28 were diagnosed with Crohn's disease (CD), and 23 were found to have ulcerative colitis (UC). The sensitivity and specificity for CD was for α1-AT, 100%, 92%; for ESR, 68%, 94%; and for UC was for α1-AT, 70%, 92%; for ESR, 61%, 94%. In the 113 children with known IBD (66 CD, 47 UC), the sensitivity and specificity for predicting disease activity was for CD for α1-AT, 94%, 59%; for ESR, 52%, 63%; and for UC was for α1-AT, 79%, 76%; and for ESR, 59%, 82%. The determination of serum α1-AT levels was more sensitive and specific than the ESR in the initial diagnosis of CD and in predicting subsequent disease activity in both CD and UC.

Prolonged medical therapy for severe pediatric ulcerative colitis.

OBJECTIVES: The optimal timing of surgical intervention for severe ulcerative colitis remains uncertain. Numerous reports recommend surgery within 10-14 days if clinical remission is not achieved. We undertook a study to follow the clinical course and long-term follow-up of patients with severe ulcerative colitis treated medically for longer than 14 days (n = 11). METHODS: We performed a retrospective review of all patients admitted to the hospital with a diagnosis of severe ulcerative colitis who were treated for more than 14 days. RESULTS: Nine percent of patients (n = 1) required surgery during their hospitalization. Ninety-one percent of patients (n = 10, mean age 8.6 yr, 7 M, 3 F) treated with medical and nutritional therapy for more than 14 days went into clinical remission. Of these, only 10% (n = 1) ultimately required surgery; 60% remain in clinical remission up to 83 months posthospitalization (mean follow-up, 49.5 months), whereas 30% suffer from mild to moderate colitis (mean follow-up, 26.3 months). CONCLUSIONS: These results do not support the recommendation for colectomy for refractory severe ulcerative colitis if remission is not noted within 2 wk of hospitalization.

Pancolonic disease in cystic fibrosis and high-dose pancreatic enzyme therapy.

We describe a child with cystic fibrosis who was treated with high-dose pancreatic enzyme replacement therapy and who had a prominent ascending colon stricture with submucosal fibrosis. Unlike prior reported cases, this patient's disease was more extensive, involving the entire colon, and was associated with chylous ascites.

Sclerodermalike esophageal disease in children breast-fed by mothers with silicone breast implants.

OBJECTIVE: To determine whether breast-fed children of mothers with silicone implants are at increased risk for the development of sclerodermalike esophageal involvement compared with children not exposed to silicone implants. DESIGN: Case-series [corrected]. SETTING: Referral-based pediatric gastroenterology clinic. PATIENTS: Eleven children (mean age, 6.0 years; range, 1.5 to 13 years; six boys and five girls) referred for abdominal pain who were born to mothers who had silicone breast implants (eight breast-fed children and three bottle-fed) were compared with 17 patients (mean age, 10.7 years; range, 2 to 18 years; 11 boys and six girls) with abdominal pain who were not exposed to silicone implants. METHODS: All children underwent esophageal manometry and upper intestinal endoscopy with esophageal biopsy and were tested for antinuclear antibody and autoantibodies to Scl-70, centromere, ribonucleoprotein, Sm, Ro, La, and phospholipid. RESULTS: Six of the eight breast-fed children from mothers with silicone implants had significantly abnormal esophageal motility with nearly absent peristalsis in the distal two thirds of the esophagus and decreased lower sphincter pressure. Upper esophageal pressures and motility were normal. Compared with controls, the breast-fed children had significantly decreased lower sphincter pressure and abnormal esophageal wave propagation. These manometric abnormalities were not seen in the three bottle-fed children. There was no difference in the expression of autoantibodies in the breast-fed children compared with the bottle-fed children or controls. CONCLUSIONS: A relationship appears to exist between breast-feeding by mothers with silicone implants and abnormal esophageal motility. Studies evaluating larger numbers of children are needed to determine the extent of the risk.

Intraarticular alpha 2-macroglobulin complexes and proteolytic activity in children with juvenile rheumatoid arthritis.

In juvenile rheumatoid arthritis (JRA), it is likely that the release of proteolytic enzymes from activated synovial fluid neutrophils overwhelms the major protease inhibitor, alpha 2-macroglobulin (alpha 2-MG), and leads to cartilage destruction. Due to the unique nature of the alpha 2-MG-protease complex, proteolytic function is maintained until the complex is cleared. In this study, we sought to determine the concentration of alpha 2-MG-protease complexes in synovial fluid of patients with JRA, the proteolytic activity found in their synovial fluid, and whether the alpha 2-MG complexes are associated with increased proteolytic activity. The JRA patients' synovial fluids had complex levels of 217.0 +/- 192.2 nmol/L--significantly elevated compared with plasma values (p < 0.001) and with control synovial fluid (p < 0.05). Elastase activity (almost entirely neutrophil elastase) was detected in all JRA synovial fluid samples (mean 2.9 +/- 2.6 mg/L) and significantly correlated with alpha 2-MG-complex values (r = 0.67, p < 0.01). Synovial fluid tryptic activity was detectable in all JRA patients but did not significantly correlate with alpha 2-MG complexes (r = 0.53, p > 0.05). Seventy-four percent of total elastase activity and 41% of total tryptic activity were contained in the alpha 2-MG-complex fractions. We suggest that the increased concentration of synovial fluid alpha 2-MG complexes with retained elastase activity contributes to continued proteolysis and joint destruction and may affect the subsequent disease course through its role as a modulator of IL-6.

Essential fatty acid deficiency associated with the use of a medium-chain-triglyceride infant formula in pediatric hepatobiliary disease.

Serum phospholipid fatty acid patterns were determined by gas chromatography in four infants with hepatobiliary disease receiving a formula with a high content of medium-chain-triglyceride (MCT) oil. All four infants demonstrated signs of essential fatty acid deficiency, characterized by decreased arachidonic acid and increased palmitoleic and oleic acids. All had substantial concentrations of the pathologic triene 5,8,11-eicosatrienoic acid. Three of four had decreased linoleic acid concentrations and abnormal ratios of triene to tetraene (5,8,11-eicosatrienoic acid: arachidonic acid), greater than 0.38. One patient may have experienced growth failure due to abnormal essential fatty acid status. Infants with the potential for fat malabsorption should only receive MCT-oil feedings with well above the generally recommended requirements for linoleic acid (3% of total caloric intake).

Liver uptake of trypsin-inhibitor complexes; differences between suckling and adult rats.

Previous studies have demonstrated increased intestinal trypsin uptake in newborn rats compared to adults. The mechanisms that protect tissues against proteolytic damage by trypsin include trypsin-inhibitor binding and subsequent liver uptake. In order to examine these mechanisms, bovine trypsin (1.25 mg/100 g body weight) plus trace 125I-trypsin were injected into the portal vein of 2-week-old and adult rats. Liver, kidney and plasma 125I activity were assessed at 1, 5 or 15 min following infusion and the different trypsin inhibitor fractions were separated and examined for 125I activity. Newborn rats had significantly increased plasma levels of 125I-trypsin and significantly decreased liver 125I levels 1 min after infusion compared to adults. In addition, the slow decline in liver 125I activity seen in the adult rats did not occur in the newborns. The pattern of trypsin-inhibitor binding was not significantly different at 1, 5 and 15 min and there were no differences at these time intervals between newborns and adults. We suggest that the newborn liver is less effective in clearing infused trypsin leading to increased plasma levels, and this increased plasma trypsin concentration subsequently leads to increased liver levels of 125I-trypsin. The increased trypsin levels in the liver may predispose newborns to protease-induced liver damage.

Manometric variations following spiral myotomy for long-gap esophageal atresia.

We have previously demonstrated that a spiral myotomy and delayed definitive procedure is a viable alternative for esophageal reconstruction in long-gap esophageal atresia. In this study we sought to determine whether this procedure leads to esophageal motility disturbances and to compare the manometric findings with controls as well as with those seen in children with esophageal atresia and primary anastomosis. Six beagles had esophageal transection and a spiral myotomy, one had esophageal transection without a myotomy, and two served as normal controls. Following esophageal reconstruction, esophageal manometry was studied in all dogs using a standard pull-through technique. We found that the three control dogs all had similar manometric findings with normal peristalsis. In contrast, the dogs with a spiral myotomy all had propagation of waves in the myotomized segment but termination of waves at the anastomotic site. There was delayed velocity through the myotomized segment and retrograde peristalsis distally. Finally, upper esophageal sphincter pressure was elevated, while lower esophageal sphincter pressure was similar to that in the normal dogs. These findings are similar to those described in children with primary anastomosis and suggest that (a) spinal myotomy is a good alternative to other esophageal replacement options in patients with long-gap esophageal atresia and that (b) the motility dysfunction observed in children with esophageal atresia following primary anastomosis may be secondary to the disruption of the vagus nerve and that may be part of the congenital abnormality or secondary to surgical trauma.

The role of alpha-macroglobulin in experimental intestinal ischemia.

Intestinal ischemia can lead to mucosal damage and presumably increased intestinal permeability. alpha-Macroglobulin (alpha-MG) is a plasma protein responsible for the inhibition and clearance of systemic proteases, including those derived from the intestinal lumen. In this study, we sought to determine the relationship between intestinal ischemia and alpha-MG in dogs using a nonsurgical model of ischemia. In addition, we sought to detect plasma alpha-MG complexes after ischemic injury. We found that alpha-MG levels were significantly reduced 24 h after induction of occlusion compared with those values obtained immediately after vascular occlusion. The onset of the decline in alpha-MG coincided with the onset of full-thickness intestinal damage, alpha-MG levels in the control animals were unchanged. However, alpha-MG complexes were not elevated in the experimental dogs after ischemia. We suggest that the decline in alpha-MG levels reflects a response to excessive protease uptake across a damaged intestine, leading to increase formation of alpha-MG-protease complexes, which are then rapidly cleared. The absence of detectable systemic complexes suggests that in ischemia, clearance is effective in removing the complexes from the bloodstream, alpha-MG appears to play an important role in protecting against proteolytic damage in prolonged intestinal ischemia.