Transcriptional signatures as a disease-specific and predictive inflammatory biomarker for type 1 diabetes.

The complex milieu of inflammatory mediators associated with many diseases is often too dilute to directly measure in the periphery, necessitating development of more sensitive measurements suitable for mechanistic studies, earlier diagnosis, guiding therapeutic decisions and monitoring interventions. We previously demonstrated that plasma samples from recent-onset type 1 diabetes (RO T1D) patients induce a proinflammatory transcriptional signature in freshly drawn peripheral blood mononuclear cells (PBMCs) relative to that of unrelated healthy controls (HC). Here, using cryopreserved PBMC, we analyzed larger RO T1D and HC cohorts, examined T1D progression in pre-onset samples, and compared the RO T1D signature to those associated with three disorders characterized by airway infection and inflammation. The RO T1D signature, consisting of interleukin-1 cytokine family members, chemokines involved in immunocyte chemotaxis, immune receptors and signaling molecules, was detected during early pre-diabetes and found to resolve post-onset. The signatures associated with cystic fibrosis patients chronically infected with Pseudomonas aeruginosa, patients with confirmed bacterial pneumonia, and subjects with H1N1 influenza all reflected immunological activation, yet each were distinct from one another and negatively correlated with that of T1D. This study highlights the remarkable capacity of cells to serve as biosensors capable of sensitively and comprehensively differentiating immunological states.

Prolonged N-myc protein half-life in a neuroblastoma cell line lacking N-myc amplification.

Genomic amplification of the oncogene N-myc is associated with rapid tumor progression and poor prognosis in patients with neuroblastoma (NB). However, 40% of NBs which lack N-myc amplification are also clinically aggressive. Factors other than N-myc copy number must therefore play a role in determining tumor progression in these NBs. We have established an unusual human NB cell line (NBL-S) from the primary tumor of a patient with rapidly progressive disease which lacks N-myc amplification. The doubling time in vitro (48 h) and the time from injection of 2 x 10(7) cells to detectable tumors in nude mice (46 days) in similar to NB cell lines with amplified N-myc. However, karyotype analysis reveals no evidence of double minutes (DMs), homogeneously staining regions (HSRs), or chromosome 1p deletions, features commonly seen in NB cell lines. The cells have the cell surface phenotype typical of N-myc amplified NB (HLA-A,B,C negative and HSAN 1.2 positive), and similar to other NB cell lines, N-myc RNA and protein are expressed. Interestingly, the half-life of the N-myc protein in NBL-S is prolonged (approximately 100 min) compared to the short N-myc protein half-life previously described in N-myc amplified NB cell lines (approximately 30 min). Because N-myc protein is thought to have a regulatory role, prolongation of the half-life of this protein may be an important factor in the regulation of growth in NBs which lack N-myc amplification and rapidly progress.

Differential processing of the two subunits of human choriogonadotropin by granulosa cells. II. In vivo studies.

Using a well characterized preparation of hCG, consisting of a mixture of hCG labeled in the alpha-subunit with 125I and hCG labeled in the beta-subunit with 131I (see preceding paper), the hormone-specific preferential retention by granulosa cells in vivo of the radiolabel originally associated with the beta-subunit of hCG has been confirmed and extended. Additional studies have shown that this retention is peculiar to the granulosa cells. Luteal and interstitial/thecal elements of the ovary failed to show preferential accumulation of the label originally associated with the beta-subunit. Measurement of both radioactivities in crude subfractions of the ovarian tissues revealed that granulosa cells retain the excess of beta-subunit label in a plasma membrane/vesicular component. No such preferential retention of label was seen in any of the subfractions obtained from luteal or interstitial/thecal tissues. The radiolabeled components associated with the granulosa cells were shown to be mainly macromolecular by their precipitability with 13% trichloroacetic acid. Luteal tissue degraded the components associated with each label more rapidly than granulosa cells. In contrast, interstitial/thecal tissue degraded very little of the bound labeled components. The differential processing of individual hCG subunits by granulosa cells was shown not to result from different kinetics of binding of serum-borne hormone by two methods. Thus, changes over time in the ability of circulating hormone to bind to LH receptor in vitro were shown not to be a function of the hCG subunit having the label. Moreover, blockade of further radiolabel uptake by injection of a large excess of unlabeled hCG 30 min after radiolabel administration did not alter the rise in the ratio of beta-subunit label to alpha-subunit label normally observed in granulosa cells. The ability of kidney tissue to accumulate and metabolize hCG also varied with the physiological state. Within the limitations of following the radioiodides added to proteins rather than the peptides themselves, these studies demonstrate that differences exist in the metabolism of hCG by the various target cells of the ovary and that changes in processing occur during luteinization.

Respiratory syncytial virus infections in the pediatric intensive care unit: clinical characteristics and risk factors for adverse outcomes.

OBJECTIVES: To describe the clinical characteristics of infants admitted to a pediatric intensive care unit (PICU) with respiratory syncytial virus (RSV) infection, including the prevalence of indications for RSV passive antibody prophylaxis (as currently recommended by the American Academy of Pediatrics), and to identify risk factors that predict adverse outcomes among this population. DESIGN: Retrospective medical record review. SETTING: Tertiary care PICU. PATIENTS: Children <2 yrs of age admitted to PICU for the management of RSV disease during the 1994-95, 1995-96, and 1996-97 RSV seasons. MEASUREMENTS AND MAIN RESULTS: The medical records of 89 infants were reviewed. Of these, 55% were born before 36-wks gestation, 14% had chronic lung disease that required medical therapy within the previous 6 months, and 30% met at least one indication for RSV passive antibody prophylaxis. Seven infants had congenital heart disease, five had upper airway abnormalities, and six had various noncardiac congenital malformations. Logistic regression was used to determine which characteristics were associated with prolonged durations (>75th percentile) of mechanical ventilation, PICU stay, and hospital stay. Prolonged mechanical ventilation was associated with congenital heart disease (p = 0.014), chronic lung disease (p = 0.007), and noncardiac congenital malformations (p = 0.022). Only congenital heart disease was associated with prolonged PICU stay (p = 0.004) or prolonged hospital stay (p = 0.006). All of the infants with airway abnormalities had prolonged ventilator days, PICU days, and hospital days. Currently recommended indications for RSV passive antibody prophylaxis were not predictive of prolonged ventilation, PICU stay, or hospital stay. CONCLUSIONS: A minority of infants admitted to our PICU for severe RSV disease meet currently recommended indications for RSV passive antibody prophylaxis. Risk factors that predict prolonged durations of ventilation, PICU stay, or hospital stay among this population include congenital heart disease, chronic lung disease, upper airway abnormalities, and noncardiac congenital malformations.

Immunoprecipitation of the simian virus 40 late transcription complex with antibody against T-antigen.

Tumor antigen (T-antigen) of simian virus 40 (SV40) has been shown to have a number of regulatory roles in both viral replication and early viral transcription. However, the nature of its role on late viral transcription remains unclear. We have analyzed for the presence of T-antigen on SV40 late viral transcription complexes which exhibit RNA polymerase II extension activity in vitro. Nuclear extract or glycerol gradient-isolated transcription complexes were treated with either polyclonal or monoclonal antibodies, and the amount of extension activity that could be immunoprecipitated was determined. Anti-T antibody derived from hamster ascites as well as the anti-T monoclonal antibodies PAb 102, 109, 416, and 419 all precipitated 12-29% of viral transcription complex activity. Immunoprecipitation resulted in significant enrichment of transcription complex activity relative to bulk minichromosomes, indicating a preferential association of T-antigen with the late viral transcription complex. This is the first direct demonstration of the presence of T-antigen on the SV40 late transcription complex. Furthermore, the immunoprecipitated transcription complexes exhibited a salt dependence of their in vitro extension activity which was distinct from that of the total complex population, indicating that T-antigen is present on a specific subclass of transcription complexes.

Molybdate permits resolution of untransformed glucocorticoid receptors from the transformed state.

The presence of 10 mM sodium molybdate has a profound effect on the physical behavior of glucocorticoid receptors submitted to ammonium sulfate precipitation or to DEAE-cellulose chromatography. Molybdate inhibits the inactivation of the unoccupied receptor and prevents the transformation of the steroid-bound receptor that occurs when rat liver cytosol is precipitated with ammonium sulfate. The transformed glucocorticoid . receptor complex is precipitated at 30 to 35% ammonium sulfate, whereas the unoccupied receptor and the untransformed steroid-bound receptor are precipitated at 45 to 55% of ammonium sulfate saturation. The untransformed steroid . receptor complex precipitated at 45 to 55% ammonium sulfate saturation in the presence of molybdate can undergo temperature-mediated transformation when it is redissolved in buffer without molybdate. The presence of molybdate in both the loading buffer and eluting gradient during DEAE-cellulose chromatography prevents the transformation of steroid-bound receptor to a less negatively charged, DNA-binding state which otherwise occurs during the chromatographic procedure. In the presence of molybdate, DEAE-chromatography yields a 33-fold purification of the untransformed steroid . receptor complex.

Increased frequency of the tumor necrosis factor-alpha-308 A allele in adults with human immunodeficiency virus dementia.

Genetic polymorphisms in the regulatory regions of various cytokine genes influence the amount of cytokine produced in response to inflammatory stimuli. To determine whether such polymorphisms might play a role in human immunodeficiency virus (HIV) dementia, a disease process in which tumor necrosis factor (TNF)-alpha is believed to play a role, we analyzed HIV-infected adults with and without dementia and control populations for a polymorphic site located in the promoter region of the gene coding for TNF-alpha. The presence of the A allele at the TNF-alpha-308 site was overrepresented among adults with HIV dementia compared to those without dementia (0.28 vs 0.07; OR 5.5; 95% CI 1.8-17.0) and a healthy control population (0.28 vs 0.11). The increased frequency of the A allele in HlV-infected adults with dementia suggests that this locus may play a role in the pathophysiology of dementia and suggests a genetic predisposition for the development of HIV dementia.

Septic shock and respiratory failure in community-acquired pneumonia have different TNF polymorphism associations.

Genetic factors are likely to contribute to the variable presentation of community-acquired pneumonia (CAP). The purpose of this prospective cohort study was to determine whether the LTalpha+250 (TNFbeta+250) and TNFalpha-308 gene polymorphisms are associated with different presentations of CAP. Septic shock (SS) was defined using American College of Chest Physicians/Society of Critical Care Medicine (ACCP-SCCM) criteria. Type I respiratory failure (T1RF) was defined as an O(2) saturation on room air of < 90% with a normal PCO(2). A total of 280 patients were genotyped; 31 had SS, 80 had T1RF. Genotype proportions are given in the order of AA/GA/ GG. The proportion of patients in each genotype developing SS was as follows: LTalpha+250 0.19/0.07/0.09 (p = 0.01 AA versus non-AA); TNFalpha-308 0.16/0.06/0.12 (p = NS). Carrying at least one AA (tumor necrosis factor [TNF] high secretor) genotype had an 18.0% risk of SS versus 6.8% (p = 0.006). GG homozygotes (TNF low secretors) at both loci had only a 2.9% risk of SS. Septic shock was associated with the LTalpha+250:TNFalpha-308 A:G haplotype but not the A:A haplotype, suggesting that LTalpha+250 is a marker, rather than a causative polymorphism. Carriage of the G:G haplotype had a significant protective effect against the development of septic shock (p = 0.011). T1RF was not associated with LTalpha+250 AA genotype. In the absence of septic shock, there was a significant trend to greater T1RF in patients with LTalpha+250 GG (TNFalpha hyposecretor) genotype (p = 0.03). Our finding of different genotype associations for SS and T1RF has important implications for immunotherapy in both CAP and sepsis, as well as for the definition of the systemic inflammatory response syndrome (SIRS).

Plastic bronchitis occurring late after the Fontan procedure: treatment with aerosolized urokinase.

OBJECTIVE: To describe the use of aerosolized urokinase in a patient with plastic bronchitis after a Fontan procedure. DESIGN: Case report. SETTING: Pediatric intensive care unit in a university-affiliated children's hospital. PATIENTS: Report of one patient with acute respiratory failure secondary to plastic bronchitis. INTERVENTIONS: Aerosolized urokinase, multiple bronchoscopies, corticosteroids, mucolytics, bronchodilators, and atrial pacing. MEASUREMENTS AND MAIN RESULTS: Airway obstruction secondary to recurring casts improved with the treatments. Histologic analysis of the casts demonstrated less fibrin after treatments with aerosolized urokinase. No adverse events were noted. CONCLUSIONS: The addition of aerosolized urokinase to this patient's treatment regimen helped to resolve life-threatening airway obstruction secondary to fibrin casts.

Prevalence of retinal hemorrhages in pediatric patients after in-hospital cardiopulmonary resuscitation: a prospective study.

OBJECTIVE: Child abuse occurs in 1% of children in the United States every year; 10% of the traumatic injuries suffered by children under 5 years old are nonaccidental, and 5% to 20% of these nonaccidental injuries are lethal. Rapid characterization of the injury as nonaccidental is of considerable benefit to child protection workers and police investigators seeking to safeguard the child care environment and apprehend and prosecute those who have committed the crime of child abuse. Physically abused children present with a variety of well-described injuries that are usually easily identifiable. In some cases, however, particularly those involving children with the shaken baby syndrome, obvious signs of physical injury may not exist. Although external signs of such an injury are infrequent, the rapid acceleration-deceleration forces involved often cause subdural hematomas and retinal hemorrhages, hallmarks of the syndrome. Frequently, retinal hemorrhages may be the only presenting sign that child abuse has occurred. Complicating the interpretation of the finding of retinal hemorrhages is the belief by some physicians that retinal hemorrhages may be the result of chest compressions given during resuscitative efforts. The objective of this study is to determine the prevalence of retinal hemorrhages after inpatient cardiopulmonary resuscitation (CPR) in pediatric patients hospitalized for nontraumatic illnesses in an intensive care unit. DESIGN: Prospective clinical study. SETTING: Pediatric intensive care unit. PATIENTS: Forty-three pediatric patients receiving at least 1 minute of chest compressions as inpatients and surviving long enough for a retinal examination. Patients were excluded if they were admitted with evidence of trauma, documented retinal hemorrhages before the arrest, suspicion of child abuse, or diagnosis of near-drowning or seizures. All of the precipitating events leading to cardiopulmonary arrest occurred in our intensive care unit, eliminating the possibility of physical abuse as an etiology. INTERVENTIONS: None. MEASUREMENTS: Examination of the retina was performed by one of two pediatric ophthalmologists within 96 hours of CPR. The chart was reviewed for pertinent demographic information; the platelet count, prothrombin time, and partial thromboplastin time proximate to the CPR were recorded if they had been determined. RESULTS: A total of 43 pediatric patients hospitalized with nontraumatic illnesses survived 45 episodes of inpatient CPR. The mean age was 23 months (range, 1 month to 15.8 years), and 84% of the patients were under 2 years old. The majority of the patients (44%) were admitted to the intensive care unit after surgery for congenital heart disease, and another 21% were admitted for respiratory failure. The mean duration of chest compressions was 16.4 minutes +/- 17 minutes with 58% lasting between 1 and 10 minutes. Five patients had chest compressions lasting >40 minutes, and two patients had open chest cardiac massage. All patients survived their resuscitative efforts. Ninety-three percent of patients had an elevated prothrombin time and/or partial thromboplastin time while 49% were thrombocytopenic. Sixty-two percent of the patients had low platelet counts and an elevated prothrombin time and/or partial thromboplastin time. Small punctate retinal hemorrhages were found in only one patient. CONCLUSIONS: Retinal hemorrhages are rarely found after chest compressions in pediatric patients with nontraumatic illnesses, and those retinal hemorrhages that are found appear to be different from the hemorrhages found in the shaken baby syndrome. Despite the small number of patients in this prospective study, we believe that these data support the idea that chest compressions do not result in retinal hemorrhages in children with a normal coagulation profile and platelet count. A larger number of patients should be evaluated in a prospective multi-institutional study to achieve statistical significance

Routine chest radiographs in pediatric intensive care units.

OBJECTIVES: To determine whether interventions were performed based on portable routine morning chest x-rays (CXRs) in pediatric intensive care unit (PICU) patients and to identify patient subgroups for whom the routine CXR is most useful. DESIGN: Prospective multiinstitutional study. Setting. PICUs of 15 tertiary care hospitals. Patients. PICU patients who received a routine morning CXR were included in the study. OUTCOME MEASURES: Recorded data included: weight, diagnosis, presence of active cardiopulmonary problems, length of stay, and number and type of devices. The number and types of interventions based on the interpretation of the CXR were recorded. RESULTS: Five hundred twelve routine CXRs were evaluated. The majority of the routine chest radiographs were obtained on patients who were admitted for cardiovascular disease (195/512; 38%) or respiratory failure (186/512; 36%), and 465/512 of the routine CXRs (91%) were performed on patients with one or more devices. Two hundred thirty-one of the 512 routine CXRs (45%) resulted in 1 or more interventions. One hundred fifty-five of the 284 routine CXRs (55%) obtained in children /=40 kg, respectively. The frequency of interventions increased from 19% in children with no devices to >50% in children with 2 or more devices. One or more interventions were performed in 27% of routine CXRs when no active cardiopulmonary problems were present, compared with 51% of routine CXRs when active cardiopulmonary problems were present. Diagnosis and length of intensive care unit stay at the time the routine CXR was obtained did not affect the percentage of CXRs that resulted in interventions. CONCLUSIONS: Routine CXRs are more likely to result in interventions in the smaller, critically ill child with one or more devices and if active cardiopulmonary problems are present.

Increased frequency of alleles associated with elevated tumor necrosis factor-alpha levels in children with Kawasaki disease.

Genetic polymorphisms influence the magnitude of the cytokine response after an inflammatory stimulus. To determine whether such polymorphisms might play a role in Kawasaki disease (KD), we analyzed white and Japanese children with KD and control populations for two polymorphic loci in which the A allele is associated with high tumor necrosis factor-alpha secretion. The lymphotoxin-alpha+250 A/A genotype was overrepresented among white children with KD compared with controls (0.59 versus 0.36; p = 0.013). The tumor necrosis factor-alpha-308 A/G genotype was overrepresented among whites with KD who had coronary artery abnormalities compared with those with normal echocardiograms (0.36 versus 0.09; p = 0.044). No significant difference was seen at either locus between Japanese children with KD and Japanese controls. The increased frequency of the high secretor alleles in white children with KD suggests that these loci may be related to susceptibility to KD and to outcome after disease.