Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody.

The botulinum neurotoxins (BoNTs) cause the paralytic human disease botulism and are one of the highest-risk threat agents for bioterrorism. To generate a pharmaceutical to prevent or treat botulism, monoclonal antibodies (mAbs) were generated by phage display and evaluated for neutralization of BoNT serotype A (BoNT/A) in vivo. Although no single mAb significantly neutralized toxin, a combination of three mAbs (oligoclonal Ab) neutralized 450,000 50% lethal doses of BoNT/A, a potency 90 times greater than human hyperimmune globulin. The potency of oligoclonal Ab was primarily due to a large increase in functional Ab binding affinity. The results indicate that the potency of the polyclonal humoral immune response can be deconvoluted to a few mAbs binding nonoverlapping epitopes, providing a route to drugs for preventing and treating botulism and diseases caused by other pathogens and biologic threat agents.

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.

Impact of multiple organ system dysfunction and nosocomial infections on survival of children treated with extracorporeal membrane oxygenation after heart surgery.

OBJECTIVES: To evaluate whether cardiac and noncardiac variables may be used to predict survival in children treated with extracorporeal membrane oxygenation (ECMO) after cardiopulmonary bypass and to determine when to discontinue ECMO support. DESIGN: Retrospective review. SETTING: Neonatal and pediatric intensive care units of Kosair Children's Hospital. PATIENTS: Fifty-nine children treated with ECMO after cardiopulmonary bypass from 1987 through 1996. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Medical, nursing, operative, and perfusion records for each patient were reviewed. The primary outcome measure was survival to hospital discharge. Cardiac and noncardiac variables were recorded at serial times. Nineteen of 59 patients (32%) survived. No cardiac variable was a clinically useful predictor of survival or marker for when to discontinue ECMO. Among the noncardiac variables, progressive multiple organ system dysfunction and development of a nosocomial infection were significantly associated with nonsurvival. No patient with a positive blood culture (n = 3) within the first 24 hrs of ECMO survived, and 21 of 24 children with a positive culture from any site during ECMO died (p = .007). Despite their higher mortality, children with positive cultures were supported with ECMO significantly longer than those with negative cultures (275+/-168 vs. 135+/-108 hrs, respectively; p = .0004). For all patients, the longest duration of ECMO that resulted in survival was 256 hrs. For children with a positive culture, the longest duration of support that resulted in survival was 200 hrs. CONCLUSIONS: Support with ECMO beyond 256 hrs was not associated with survival. Progressive multiple organ system dysfunction and nosocomial infections have a negative impact on survival. Serious consideration should be given to discontinuing ECMO support whenever there is a progressive increase in the number of abnormally functioning organ systems, a nosocomial infection occurs, or native cardiac function has not improved significantly by 250 hrs of ECMO support.

Prognostic value of pre- and postoperative cardiac troponin I measurement in children having cardiac surgery.

The objective of this study was to determine if perioperative elevation of cardiac troponin I (cTnI) predicts mortality in infants and children after surgical correction of congenital heart defects. One hundred infants and children having open heart surgery were studied. Blood samples for cTnI analysis were collected before cardiopulmonary bypass (CPB) and at 4, 8, 12, and 24 h after initiation of CPB. Demographic information, cardiac defect, repair performed, duration of CPB, complications, and outcome were recorded. Cardiac defects were categorized as atrial septal defect (ASD), ventricular septal defect (VSD), hypoplastic left heart syndrome (HLHS), complex, and "other." Baseline cTnI was significantly lower in survivors (mean 0.42 ng/ml, median 0.35 ng/ml) than in nonsurvivors (mean 1.89, median 1.30), p = 0.0001. Baseline cTnI was significantly higher in the HLHS group (mean 1.47, median 1.10) than in all other subgroups (mean 0.62, median 0.35), p 125 ng/ml compared to survivors (2 of 90). Within cardiac defect subgroups, 4 h cTnI was significantly higher in the complex group (mean = 53.51, median = 32.30) than in the ASD (mean = 23.84, median = 19.85) and other (mean = 21.59, median 21.50) subgroups. Perioperative measurement of cTnI identifies children within specific cardiac defect subgroups at risk of mortality after cardiac surgery. We speculate that detection of myocardial injury may decrease mortality and morbidity in children with complicated congenital cardiac lesions by leading to improvements in perioperative management.

The immunogenicity in humans of a botulinum type F vaccine.

A purified monovalent botulinum type F toxoid vaccine was administered to 35 healthy adult volunteers in a phase I clinical trial. Serum samples from the vaccinated volunteers were evaluated for an antibody response at various time intervals over 1 year by mouse bioassay and ELISA. The antibody response was measured for varying doses of vaccine (2, 5, or 10 microg), and after single or multiple (two or three doses @ 10 microg) vaccinations. Six out of 15 (40%) individuals developed antibody titers after receiving a single dose. After two and three vaccinations, there was a 90% (18/20) and 100% (10/10) seroconversion rate, respectively. Eight months after initial injection, 57 and 63% of individuals were antibody positive following two or three vaccinations, respectively. Single vaccinations, at any of the tested dosages, elicited lower, if any, antibody response than did multiple vaccinations. After the third vaccination, ELISA titers positively correlated with mouse neutralization bioassay titers (r(2)=0.86).

Purification, potency, and efficacy of the botulinum neurotoxin type A binding domain from Pichia pastoris as a recombinant vaccine candidate.

Recombinant botulinum neurotoxin serotype A binding domain [BoNT/A(Hc)], expressed in Pichia pastoris, was developed as a vaccine candidate for preventing botulinum neurotoxin type A (BoNT/A) intoxication. After fermentation and cell disruption, BoNT/A(Hc) was purified by using a three-step chromatographic process consisting of expanded-bed chromatography, Mono S cation-exchange chromatography, and hydrophobic interaction chromatography. Two pools of immunogenic product were separated on the Mono S column and processed individually. Both products were more than 95% pure and indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Each protein was assayed for potency in mice at immunogen doses ranging from 2.4 ng to 10 microg, followed by challenge with 1,000 mouse intraperitoneal 50% lethal doses (i.p. LD50) of BoNT/A. The calculated 50% effective dose for both peaks was approximately 0.1 microg/mouse. Peak 1 was evaluated further in a mouse efficacy assay. Mice were injected either once, twice, or three times at five different doses and subsequently challenged with 100,000 mouse i.p. LD50 of BoNT/A. In general, multiple injections protected better than one, with complete or nearly complete protection realized at doses of >/=0.5 microg/mouse. Serum neutralization and ELISA titers were also determined. Tellingly, 82 of 83 mice with antibody titers of >/=1, 600, as measured by ELISA, survived, but only 6 of 42 mice with titers of

Complications associated with severe invasive streptococcal syndrome.

Group A beta-hemolytic streptococcal sepsis may cause life-threatening disease. We describe a child with severe invasive streptococcal syndrome in whom severe respiratory failure and pulmonary pneumatoceles required extracorporeal life support. Physicians should be aware of the full spectrum of pathologic changes and life-threatening complications caused by group A beta-streptococcus.

Severity and carbamazepine level at time of initial poison center contact correlate with outcome in carbamazepine poisoning.

We reviewed data from carbamazepine poisonings reported to the Kentucky Regional Poison Center from January 1986 through March 1992 to identify information available at the time of poison center contact which correlates to outcome. The Spearman rank correlation test was used to describe the relationship between two ordinal variables and interval-level variables. The Kruskal-Wallis test was used to determine the relationship between categorical and ordinal variables. Two way analysis of variance was used to test the effect of routine carbamazepine use on final severity and carbamazepine level of 345 reports involving carbamazepine poisoning; 263 (76%) involved only carbamazepine ingestion and formed the database. One hundred eighty four (70%) carbamazepine ingestions occurred in victims < or = 17 years old, 79 (30%) occurred in adults. Severity assigned at the time of initial poison center contact was significantly correlated with outcome severity for children and adults (r > or = 0.9, p < 0.00001). The amount reported ingested influenced the correlation between initial and final severity; whereas, time elapsed between ingestion and poison center contact did not alter the correlation between initial and final severity. The reason for ingestion was significantly correlated with outcome (p < 0.00001). A significant correlation between outcome and peak carbamazepine level for each age group was observed (pediatric r = 0.5, p < 0.00001, and adult r = 0.4, p = 0.008). Carbamazepine levels > 85 mumol/L (> 20 mcg/mL) were associated with more severe toxicity.

Low-dose beta-agonist continuous nebulization therapy for status asthmaticus in children.

The role of continuous nebulization therapy (CNT) with low-dose beta (beta) agonist was retrospectively reviewed in 7 children admitted to a pediatric intensive care unit for treatment of acute severe asthma. Clinical asthma score (CAS), alveolar-arterial oxygen difference (A-aDO2), PaCO2, heart rate, and respiratory rate were recorded at 0, 12, and 24 hr of CNT. All patients had a decrease in CAS at 12 hr (p < 0.008) and decreased A-aDO2, PaCO2, respiratory rate, and heart rate at 24 hr (p < 0.008). No patient required escalation of dosage or additional intervention such as intravenous beta-agonist therapy or mechanical ventilation. No patient experienced significant cardiovascular toxicity or hypokalemia. We conclude that CNT with low-dose beta agonists should be considered in the initial approach to therapy in children with acute severe asthma.

Influence of low density lipoproteins on vascular smooth muscle cell growth and motility: modulation by extracellular matrix.

Low density lipoproteins (LDL) are thought to play a major role in cardiovascular diseases such as atherosclerosis. Much remains to be done to understand the cellular effects of LDL and how the extracellular matrix (ECM) influences these effects. We found that LDL produced a dose dependent increase in vascular smooth muscle cell (SMC) proliferation. The ECM altered the proliferative response of SMC to LDL: on collagen I there was a 66% inhibition, endothelial cell derived-ECM a 2-fold increase, and collagen IV no difference in proliferation compared to paired controls. LDL affected SMC motility (cell area and shape factor) but the extent and direction of the effect depended on whether the cells were cultured on uncoated or coated dishes. LDL treated cultures had a 5-fold lower migration rate but net movement was not different, suggesting that LDL decreased SMC random movement. There was a dose-dependent accumulation of lipid by SMC incubated with LDL and, subsequently, cytoplasmic lipid droplets were observed. Cells cultured on uncoated plates showed an increased cholesterol content as a function of LDL concentration. In contrast, cells cultured on a collagen IV matrix showed no net change in cholesterol content over the range of LDL concentrations studied. Hence, the uptake of LDL cholesterol appears to be completely inhibited by this matrix. These studies indicate that the influence of LDL on several SMC parameters is modulated by ECM components.

FSH-induced ovulation in intact and hypophysectomized mice.

A single, ovulatory dose of 25 micrograms of a highly purified preparation of ovine FSH caused ovulation in 89% of hypophysectomized and 91% of intact female mice primed 48 h earlier with PMSG; the number of oocytes recovered (29.4 +/- 4.7 and 22 +/- 2.7/mouse ovulating, respectively) compared favourably with the 20.0 +/- 2.9 oocytes per ovulating female recovered from animals that received PMSG + hCG. After oFSH injection, 82% of oocytes released were fertilized and developed to blastocysts. That the trace contamination (less than 0.2%) of the oFSH with oLH was not responsible for the ovulation was shown by the markedly reduced number of oocytes collected from ovulating females that were injected with equivalent low levels of hCG (0.001 micrograms) or oLH (1 microgram) (9.0 +/- 3.3 and 8.0 +/- 3.1, respectively). These results demonstrate that oFSH is as effective as LH in inducing ovulation of competent oocytes in the mouse.