Induction of alpha-synuclein aggregation by intracellular nitrative insult.

Brain lesions containing filamentous and aggregated alpha-synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant alpha-synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of alpha-synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of alpha-synuclein. Light microscopy using antibodies against alpha-synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. alpha-Synuclein aggregates were observed in 20-30% of cells expressing wild-type or A53T mutant alpha-synuclein and in 5% of cells expressing A30P mutant alpha-synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing beta-synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing alpha-synuclein. However, alpha-synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote alpha-synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of alpha-synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of alpha-synuclein, indicating that lysosomes are involved in degradation of alpha-synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of alpha-synuclein aggregates.

Posttransplantation plasmacytic proliferations related to Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV), which was originally detected in Kaposi's sarcoma, also has been found in primary effusion lymphomas (PELs) and some cases of multicentric Castleman's disease. We describe two transplant recipients who developed Kaposi's sarcoma and a spectrum of non-neoplastic lymphoproliferative disorders that show pronounced plasmacytic and plasmacytoid features. The first patient had recurrent pleural effusions and Castleman's disease-like changes in lymph nodes. The second patient had systemic lymphadenopathy and hepatosplenomegaly secondary to diffuse infiltration by polyclonal plasma cells and plasmacytoid B lymphocytes that clinically mimicked Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease. In both cases, KSHV DNA was detected by polymerase chain reaction and Southern blotting, and KSHV vIL-6 protein expression was identified in affected tissues by immunohistochemical localization. In contrast, no evidence of KSHV coinfection was detected in any of 31 EBV-related posttransplant lymphoproliferative disorders or 112 non-PEL lymphomas tested. The pathologic findings in these two patients were not representative of malignancy by morphologic, immunophenotypic, or molecular criteria. This study underscores the marked propensity for hematolymphoid proliferations associated with KSHV infections to show plasmacytic features. Additionally, this study describes use of an antibody reactive against KSHV vIL-6 that can readily detect a subpopulation of KSHV-infected hematopoietic cells.

Evidence against KSHV infection in the pathogenesis of multiple myeloma.

Kaposi's sarcoma-associated herpesvirus (KSHV) is likely to play a pathogenic role in Kaposi's sarcoma, body cavity-based primary effusion lymphoma and a subset of Castleman's disease. A recent polymerase chain reaction (PCR)-based study reported an association between KSHV and multiple myeloma (MM). We searched for KSHV infection in MM patients by serology, PCR and immunohistochemistry. In addition, we cultured dendritic and stromal cells from MM patients. KSHV antibodies were universally absent from MM patients (0/25) whereas EBV antibodies were nearly ubiquitous (24/25). All of the bone marrow biopsies (0/16) and negative controls (0/4) were vIL-6 negative. None of the bone marrow aspirates (0/6) or biopsies (0/3), peripheral blood mononuclear cells (0/8), mononuclear apheresis cells (0/5) or dendritic cell cultures (0/5) were positive by PCR. One of the MM stromal cell cultures (1/7) was positive for KSHV DNA by PCR and weakly positive on direct southern hybridization using a probe to the terminal repeat region. However, this same patient was PCR negative using another primer set, KSHV seronegative, and negative for vIL-6 immunostaining. Our results suggest that the KSHV DNA positivity rate among MM patients is much lower than previously reported.

Fever response to acetaminophen in viral vs. bacterial infections.

The effect of acetaminophen on fever in bacterial vs. viral infections was tested in 100 children ages 9 days to 17 years who presented to the Pediatric Service with a rectal or oral temperature of 102 degrees F (38.9 degrees C) or greater. All patients were given acetaminophen, 15 mg/kg, and their temperatures were rechecked at 1 hour. Laboratory tests were ordered at the discretion of the examining physician and usually included viral and bacterial cultures and total white blood cell counts. Sixteen patients had proved viral illnesses and 17 patients had serious bacterial infections. There was a significant difference (P less than 0.02) in the white blood cell count between the two groups, with the higher values in patients with bacterial infections. There was, however, no significant difference in the fever response to acetaminophen between the two groups (P = 0.37). The remaining 67 patients were then placed into one of the two groups based on their clinical illness and outcome. The mean temperature change was then calculated between the two groups, and again the difference was found to be statistically insignificant (P = not significant (t = 0.19]. We conclude that there is no correlation between a child's fever response to acetaminophen and the etiology of the fever.

Antigenuria after immunization with Haemophilus influenzae oligosaccharide CRM197 conjugate (HbOC) vaccine.

We tested the urine of 30 infants 6 weeks to 7 months of age after they received standard 10-micrograms (0.5-ml) doses of HbOC (HibTITER) Haemophilus influenzae b (Hib) conjugate vaccine for the presence of Hib antigenuria using a commercially available latex particle agglutination assay (Directigen). Urines were collected within 1 hour, from 1 to 3 hours, at 24 hours and at 3, 6 and 9 days after vaccine administration and reactions were quantitated from 0 to 3+. In contrast to previous studies in older children which showed little or no antigenuria following HbOC vaccination, our study shows that in infants intense Hib antigenuria is evident within 2 to 3 hours and persists 3 days after vaccine administration and that less intense antigenuria may be detected in some infants for several days. With efficacious vaccines now being used in 2- to 6-month-old infants, invasive Hib disease may soon be limited to infants of this age just before their seroconversion. It should be recognized that antigenuria occurs for several days after vaccination with Hib conjugate vaccines and that it could be erroneously interpreted as evidence of invasive Hib infection.

Antimicrobial treatment of occult bacteremia: a multicenter cooperative study.

This prospective multicenter study was conducted to define more clearly clinical and laboratory criteria that predict a strong probability of occult bacteremia and to evaluate the effect of empiric broad spectrum antimicrobial treatment of these children. Children 3 to 36 months old with fever > or = 40 degrees C (104 degrees F) or, > or = 39.5 degrees C (103 degrees F) with white blood cells (WBC) > or = 15 x 10(9)/liter, and no focus of infection had blood cultures obtained and were randomized to treatment with oral amoxicillin/potassium clavulanate or intramuscular ceftriaxone. Sixty of 519 (11.6%) study patients had positive blood cultures: Streptococcus pneumoniae, 51; Haemophilus influenzae b, 6; Neisseria meningitidis, 2; and Group B Streptococcus, 1. Subgroups of high risk were identified as fever > or = 39.5 degrees C and WBC > or = 15 x 10(9)/liter, 55 of 331 or 16.6% positive with increasing incidence of positive culture with increasing increments of degrees of leukocytosis to WBC > or = 30 x 10(9)/liter where 9 of 21 or 42.9% were positive. Subgroups of significantly lower risk were identified as fever > or = 39.5 degrees C and WBC < 15 x 10(9)/liter, 5 of 182 or 2.7% positive and those with WBC < 10 x 10(9)/liter, 0 of 99 or 0.0% positive. Children with positive cultures who received ceftriaxone were nearly all afebrile after 24 hours whereas a significant number who received amoxicillin/potassium clavulanate remained febrile. In the 459 culture-negative children more amoxicillin/potassium clavulanate-treated children developed diarrhea and had less improvement in clinical scores after 24 hours than ceftriaxone-treated children.(ABSTRACT TRUNCATED AT 250 WORDS)

Prospective randomized double blind placebo-controlled evaluation of azithromycin for treatment of cat-scratch disease.

OBJECTIVE: To determine the efficacy of azithromycin in the treatment of patients with typical cat-scratch disease. DESIGN: Prospective, randomized, double blind, placebo-controlled clinical trial. SETTING: Large military medical center and its referring clinics. PATIENTS: Active duty military members and their dependents with laboratory-confirmed, clinically typical cat-scratch disease. INTERVENTION: Study participants assigned by randomization to treatment with oral azithromycin or placebo for 5 days. OUTCOME MEASURES: Lymph node volume was calculated using three dimensional ultrasonography at entry and at weekly intervals. The ultrasonographer was blinded to the treatment groups. Endpoint evaluations were predetermined as time in days to 80% resolution of the initial total lymph node volume. RESULTS: Demographic and clinical data showed that the azithromycin and placebo treatment groups were comparable at entry although the placebo group tended to be older. Eighty percent decrease of initial lymph node volume was documented in 7 of 14 azithromycin-treated patients compared with 1 of 15 placebo-treated controls during the first 30 days of observation (P = 0.026). After 30 days there was no significant difference in rate or degree of resolution between the two groups. CONCLUSIONS: Treatment of patients with typical cat-scratch disease with oral azithromycin for five days affords significant clinical benefit as measured by total decrease in lymph node volume within the first month of treatment.

Decline of erythromycin resistance of group A beta-hemolytic streptococci in Japan. Comparison with worldwide reports.

OBJECTIVE: To determine the current prevalence of erythromycin resistance and penicillin tolerance of group A beta-hemolytic streptococci (GABHS) in Japan. RESEARCH DESIGN: One hundred four isolates of GABHS from the civilian community in Tokyo and 101 isolates from the military population at the US Air Force Base in Yokota, Japan, were tested for erythromycin susceptibility and penicillin susceptibility and tolerance. SETTING: US Army Medical Center. RESULTS: Of the Japanese civilian isolates, two were moderately susceptible and none were resistant to erythromycin; of the military isolates, none were moderately susceptible and one was resistant, for an overall resistance rate of 0.49%. All isolates were exquisitely susceptible to penicillin, and no evidence of penicillin tolerance was found. CONCLUSIONS: Comparison with similar studies world-wide shows that erythromycin susceptibility of GABHS in Japan is now among the lowest in the world, while just over a decade ago it was the highest. These observations mandate constant monitoring of erythromycin resistance of GABHS wherever this drug is used to treat patients with infections due to this organism.

Treatment of Candida arthritis with flucytosine and amphotericin B.

A premature infant with disseminated Candida tropicalis infection with arthritis and osteomyelitis is presented. Although the organism was susceptible to amphotericin B, synovial fluid cultures were still positive for yeast after 2 weeks of intravenous amphotericin B therapy. The addition of oral flucytosine (25 mg/kg four times daily) resulted in sterilization of the synovial fluid within 4 days. Simultaneous serum and synovial levels of flucytosine were 47.5 micrograms/ml and 39.6 micrograms/ml, respectively. This is the first documentation in the medical literature of intraarticular levels of flucytosine, and provides further rationale for the use of flucytosine in addition to amphotericin B in patients with Candida arthritis.

Failure of intrapartum antibiotics to prevent culture-proved neonatal group B streptococcal sepsis.

Early-onset group B streptococci (GBS-EOS) sepsis may be prevented by intrapartum antibiotics administered for GBS maternal colonization, premature labor, or prolonged rupture of membranes. We sought to identify cases of neonatal GBS sepsis after apparent failure of intrapartum chemotherapy and to determine the factors associated with failure of intrapartum antibiotics in these cases. We identified 96 GBS blood culture-positive infants at five military medical centers from 1987 to 1990. Eighteen (18.7%) of these infants had mothers who had received intrapartum antibiotics; 16 of 18 cases were early-onset disease, 15 of which initially had symptoms at less than 1 hour of age. Two infants had late-onset disease develop at 3 weeks of age. At least one perinatal risk factor (prematurity, prolonged rupture of membranes > 12 hours, maternal fever) was present in each of the 16 cases. Indications for intrapartum antibiotics were suspected chorioamnionitis (13 cases), GBS colonization and prolonged rupture of membranes or prematurity (3), and GBS colonization alone (2). Maternal antibiotics included ampicillin (14 cases), cephadyl (1), vancomycin (1), clindamycin (1), and gentamicin alone (1). The median number of doses of ampicillin before delivery was 1 (range, 1 to 21), which was administered at a median of 4 hours (range, 1 to 84) before birth. The mean dose of ampicillin was 1.8 gm/dose (range, 1 to 2 gm/dose). Two of 16 (12.5%) infants with GBS-EOS died as a result of GBS sepsis. In our population of neonates with GBS-EOS, 18.4% (16 of 87) of the infants had positive blood cultures despite intrapartum antibiotics. Intrapartum antibiotics may fail to prevent GBS sepsis in a number of infants born to mothers colonized with GBS or to those with acute chorioamnionitis.

Topically induced diphenhydramine toxicity.

We report the case of a 2 1/2-year-old child who manifested acute anticholinergic toxicity after the applications of a topical calamine-antihistamine lotion. This mechanism of diphenhydramine toxicity is uncommon, with only a few other case reports noted in the literature. This case is also intriguing in that this child had an underlying varicella illness with fever that tended to obscure the picture. This report describes the characteristic history and physical examination pertinent to anticholinergic toxicity, varicella complication considerations, and case management.

Restructuring an academic pediatric inpatient service using concepts developed by hospitalists.

In an effort to increase continuity of inpatient care by attending physicians, decrease use of hospital resources, and improve medical education without compromising quality of care, an inpatient pediatric ward service was restructured by using concepts developed by hospitalists. This reorganization reduced the number of yearly inpatient attendings, added a consistent 14-day call schedule, and eliminated attending outpatient responsibilities during their ward service. The restructured ward service attending acted as the attending of record for all general and specialty patients, excluding hematology/oncology, with the subspecialists fulfilling a consultant role. To evaluate the impact of this restructuring, a baseline year of the traditional ward service (TWS) was compared with a subsequent year of the restructured ward service (RWS). Our goal was to evaluate the impact of this new system on average costs of hospitalization, length of stay, resource utilization, inpatient mortality, and 7- and 31-day readmission rates. Hospital costs per patient were lower by 13% (p=0.018) in the restructured system. Average lengths of stay in an observation bed were significantly lower on the RWS (p=0.007), but there was no significant difference in admission length of stay. There was decreased resource utilization for laboratory and radiology tests (p<0.01) on the restructured service. Readmission rates were not significantly changed, and satisfaction among attendings, housestaff, students, and patients was uniformly high in both groups. A reorganized academic pediatric medical service, which allows specific attendings to focus on inpatient care and teaching, can decrease hospital resource utilization without compromising the quality of patient care or medical education.

False-negative urine latex particle agglutination testing in neonates with group B streptococcal bacteremia. A function of improper test implementation?

Although blood cultures remain the most specific indicator of Group B streptococcus (GBS) sepsis, a potentially life-threatening infection in neonates, test results may not be available for 24 to 48 hours. Detection of GBS antigen in the urine by latex particle agglutination (LPA) may speed diagnosis. This study analyzed the sensitivity of the GBS urine LPA assay under clinical conditions. The urine of neonates with early-onset GBS bacteremia was analyzed for GBS antigen over a three-year period at six military medical centers. Overall, 53.5% (38/71) of infants with positive blood cultures had a positive urine LPA test. Only one medical center routinely followed manufacturer's recommendations to concentrate urine specimens before testing. These data suggest that the sensitivity for the urine LPA assay, when performed on unconcentrated urine, is lower than previously reported. Clinicians should insist that the laboratory maximize sensitivity by concentrating urine prior to GBS LPA testing.

Comparative study of clinical evaluation of heart murmurs by general pediatricians and pediatric cardiologists.

In a study to compare the clinical diagnostic skills of academic general pediatricians and academic pediatric cardiologists in the evaluation of heart murmurs, a total of 128 patients (aged 1 month to 18 years) newly referred to a university pediatric cardiology clinic were evaluated by one of three general pediatricians and one of four pediatric cardiologists. The murmurs were clinically classified as innocent, pathologic, or possibly pathologic. The classification was revised after the review of electrocardiogram (EKG) and chest radiograph (CXR), if indicated. The definitive diagnosis was ascertained by echocardiography (94 normal, 34 abnormal). The general pediatricians identified as many pathologic heart murmurs as the pediatric cardiologists (27/34 vs. 29/34), with no difference in sensitivity, 79% vs. 85% (p = 0.53). The similarity in sensitivity could be because the general pediatricians were more cautious in the classification of heart murmurs and had classified more innocent heart murmurs as pathologic than the pediatric cardiologists (13/39 vs. 3/23), 41% vs. 13% (p = 0.02). The pediatric cardiologists correctly identified more innocent murmurs than general pediatricians (52/94 vs. 72/94), with a better specificity, 55% vs. 76% (p = 0.001); however, the accuracy of prediction of innocence was similar for both groups (52/59 vs. 72/77), 88% vs. 93% (p = 0.36). The revision of diagnosis with review of EKG and CXR was more often misleading than helpful for either group. Academic general pediatricians would identify most of the pathologic murmurs and are no more likely than an academic pediatric cardiologist to misclassify a pathologic heart murmur as innocent.

Wine as a digestive aid: comparative antimicrobial effects of bismuth salicylate and red and white wine.

OBJECTIVE: To test whether red and white wines are as potent as bismuth salicylate against the bacteria responsible for traveller's diarrhoea to try to explain wine's legendary reputation as a digestive aid. DESIGN: Red and white wine, bismuth salicylate, two solutions containing ethanol (diluted absolute ethanol and tequila), and sterilised water were tested against suspensions of salmonella, shigella, and Escherichia coli to determine relative antibacterial activity. Suspensions of 10(7) colony forming units of shigella, salmonella, and E coli were added to the test solutions and plated on standard nutrient agar at 0, 10, 20, 30, 60, and 120 minutes and 24 hours. Dilutions of wine and bismuth salicylate were then tested with E coli as the test bacterium, and the experiment repeated. MAIN OUTCOME MEASURES: Exposure times necessary for eradication of organisms for the different solutions; decreases in colony counts at the different exposure times for dilutions of wine and bismuth salicylates. RESULTS: Undiluted wine and bismuth salicylate were both effective in reducing the number of viable organisms (by 10(5)-10(6) colony forming units) after 20-30 minutes. Dilutions of wine were much more effective in decreasing colony counts than were similar dilutions of bismuth salicylate. CONCLUSION: The antibacterial property of wine is largely responsible for wine's reputation as a digestive aid.