The ability of polymorphonuclear leukocyte priming agents to overcome influenza A virus-induced cell dysfunction.

The major mortality and morbidity resulting from influenza virus infections are due to secondary bacterial infections which occur in association with virus-induced inhibition of polymorphonuclear leukocyte (PMNL) function. The present study was undertaken to determine if compounds which prime PMNL function to subsequent stimulation with N-formylmethionyl-leucylphenylalanine (FMLP) or phorbol 12-myristate 13-acetate (PMA) can overcome influenza A virus (IAV)-induced inhibition of the PMNL chemiluminescence response to these stimuli. Granulocyte-macrophage colony stimulating factor (GM-CSF), guanosine triphosphate (GTP), and 1-oleoyl-2-acetylglycerol (OAG) were able to prime the PMNL response to FMLP and/or PMA and totally or partially overcome IAV-induced PMNL dysfunction in cells stimulated with FMLP or PMA. A direct correlation was found between the extent of PMNL priming due to GM-CSF, GTP, and OAG and the capacity of these compounds to overcome virus-induced PMNL dysfunction. The implications of these findings in regard to the mechanism by which priming agents overcome IAV-induced cell dysfunction and the potential of these compounds as therapeutic agents to treat secondary bacterial infections are discussed.

Influenza A virus alters structural and biochemical functions of the neutrophil cytoskeleton.

Influenza A virus (IAV) has previously been shown to alter chemotactic, oxidative, and secretory functions of polymorphonuclear leukocytes (PMNL). Because of the role of cytoskeletal proteins in these processes, studies were carried out to determine if IAV altered the PMNL cytoskeleton. PMNL were incubated with buffer of IAV, stimulated with f-met-leu-phe (FMLP), fixed and stained with NBD-phallacidin (NBD-Ph) and studied by flow cytometry. Mean F-actin fluorescence was increased 18% in virus treated cells pre-FMLP stimulation and 13% 5 and 10 min post-FMLP (P less than .03); no significant difference in F-actin fluorescence was noted in virus treated PMNL 15-30 s post-FMLP compared to control cells. PMNL exposed to the same conditions were solubilized and actin content was determined following SDS-PAGE of triton insoluble precipitates. Increased actin was recovered from virus treated compared to buffer treated cells before and after FMLP stimulation in the 8,000g precipitates (P less than .001). Immunofluorescent microscopy studies of F-actin distribution were done in PMNL stained with NBD-Ph following FMLP stimulation for 10 min. These studies showed an increased lamellipod F-actin/uropod F-actin ratio in PMNL pre-incubated with IAV compared to controls (4.6 vs. 1.0; P less than .025). Phosphorylation of specific cytoskeletal proteins was examined after immunoprecipitation. IAV alone altered phosphorylation of both vimentin and vinculin, and in stimulated PMNL virus led to decreased phosphorylation of vimentin and vinculin. These data show distributional and biochemical effects of IAV on PMNL cytoskeletal proteins, indicating additional targets for IAV interference in the PMNL signal-transduction-function process.

Serum sickness-like reaction to cefaclor: lack of in vitro cross-reactivity with loracarbef.

OBJECTIVES: A lymphocyte-based in vitro rechallenge technique was used to examine the potential for cross-reactivity between loracarbef and cefaclor in children who had had adverse reactions to cefaclor. STUDY DESIGN: The study cohort included 10 patients (2.2 +/- 1.1 years old) with a serum sickness-like reaction to cefaclor, five patients (4.1 +/- 4.6 years old) with immediate-type hypersensitivity reactions to the drug, and five patients (1.5 +/- 0.9 years old) who had a delayed hypersensitivity reaction to cefaclor without joint involvement (i.e., not a serum sickness-like reaction). Peripheral blood mononuclear cells were isolated from each patient and were exposed to cefaclor, loracarbef, and the metabolites of each generated with phenobarbital-induced murine hepatic microsomes. RESULTS: Among patients with cefaclor-associated serum sickness-like reactions, lymphocyte killing (expressed as percentage cell kill above baseline) in the presence of cefaclor metabolites (83.6% +/- 42.2%) was significantly (p < 0.02) higher than that observed among the patients with either immediate-type (1.1% +/- 2.4%) or delayed hypersensitivity (0%) reactions. In contrast, loracarbef did not produce significant in vitro cytotoxicity among any of the patient subgroups. Our in vitro evidence was supported at therapeutic rechallenge with loracarbef among three children with cefaclor-associated serum sickness-like reactions who tolerated a full course of therapy without adverse reactions. CONCLUSION: The metabolite-mediated cytotoxicity associated with cefaclor among patients who had had serum sickness-like reactions after therapeutic administration of the drug was not shared with loracarbef. The extent to which the apparent lack of in vitro cross-reactivity between cefaclor and loracarbef may be predictive of clinical cross-reactivity is not known.

Buffy coat transfusions in neonates with sepsis and neutrophil storage pool depletion.

A randomized study was initiated in neonates with neutropenia (absolute peripheral neutrophil count less than 1,500/microL) and suspected bacterial infection. Twenty infants with proven infection were enrolled, nine of whom had depletion of bone marrow stores of maturing neutrophils (less than or equal to 7% metamyelocyte, band and mature forms per 100 nucleated cells). These nine were randomized to receive 15 mL/kg of either buffy coat transfusions (group 2) or plasma and blood products (group 3). The remaining 11 (group 1) were observed. Peripheral neutrophil counts were monitored to determine the neutrophil response to transfusions. There were ten of 11 patients in group 1, two of four in group 2, and two of five in group 3 who lived at least seven days. No complications of transfusion were noted. No difference in the rate of peripheral neutrophil increase was found among the three groups. The study was stopped when it became clear that sufficient numbers of patients could not be entered into the study, in a reasonable period of time, to prove or disprove a clinically significant improvement in outcome. Although in vitro testing of the buffy coat preparations showed normal function in three of four cases, the clinical quality of the buffy coats may have been inadequate because of poor availability of whole fresh blood less than 24 hours old. The role of neutrophil transfusions in these patients remains unclear.

Evaluation of humoral responsiveness in children.

The evaluation of postvaccine antibody responses can provide a reasonably convenient and useful adjunct to the evaluation of possible humoral immune dysfunction syndromes. In patients whose clinical history is suspicious for an antibody deficiency syndrome, such studies can document normal antibody responsiveness. On the other hand the wide age-dependent variation in antibody responses makes careful interpretation necessary in concluding that a patient falls outside the "normal" pattern of antibody responsiveness. Finding a patient well below the mean values for age for multiple vaccines in concert with an appropriate clinical history would strongly suggest an abnormality of immune regulation. At this time the finding of abnormal antibody responses does not always imply that intervention with immunoglobulin therapy is indicated or that it would lead to improvement in clinical symptoms. It might help justify prophylactic antibiotic programs, however, similar to those used for recurrent otitis media. Interpretation of antibody responses in children younger than 2 years of age is probably hazardous since many patients may have relative retardation in their normal development of polysaccharide antibody responsiveness. Such patients usually develop a normal response as they grow older and do not need long term therapy. Until new technologies for measuring immune competence are developed, the above-mentioned measures of humoral immunity, when combined with clinical judgment, should be adequate to guide clinical decision-making in most cases.

Historical cohort evaluation of ribavirin efficacy in respiratory syncytial virus infection.

Evaluation of ribavirin therapy for respiratory syncytial virus infection of the lower respiratory tract is problematic because of multiple risk factors and variable severity of illness in respiratory syncytial virus-infected patients. To address these difficulties we used multivariate analysis and performed a historical concurrent cohort study in two children's hospitals one of which had used ribavirin since licensing and one that had not utilized ribavirin therapy. The medical records of 158 patients who satisfied the American Academy of Pediatrics inclusion criteria for receiving ribavirin were analyzed for three seasons (1988 to 1991). No significant benefit of ribavirin therapy could be appreciated for the whole group in length of stay (median, 5.0 vs. 6.5 days), days on oxygen therapy (median, 5 vs. 3), progression to ventilator status (3.8 vs. 3.9%) or mortality (1.9% vs. 1.9%) for ribavirin treatment vs. supportive care. Multivariate analysis failed to uncover a beneficial effect of ribavirin when all risk factors were considered. No significant differences were noted when ventilated and nonventilated patients were examined separately. Our data raise questions about the efficacy of ribavirin when used in common practice and suggest that further prospective study, with appropriate analysis, is needed to justify the continued widespread use of this drug.

Protective effect of BCG against tuberculous meningitis and miliary tuberculosis: a meta-analysis.

The protective effect of BCG against tuberculosis (TB) estimated in randomized controlled trials and observational studies ranges from negative to close to a 100%. One of the many explanations offered for this is that different immunological mechanisms may be associated with protective effect against different forms and sites of disease. In this investigation, we recalculated vaccine protective effect separately for pulmonary disease and for meningeal/miliary disease in randomized controlled trials and case-control studies, tested for heterogeneity in site-specific estimates of protective effect and calculated a summary measure when appropriate. We found protective effect against pulmonary disease to be heterogeneous to a statistically significant degree, and thus we did not calculate a summary measure of protection. Protective effect against meningeal and miliary TB was higher than against pulmonary disease and, except for a single study with two cases only, appeared to be homogenous. Summary BCG protective effect against miliary or meningeal TB in randomized controlled trials was 86% (95% confidence interval [CI] 65, 95) and in case-control studies 75% (95% CI: 61, 84). The fact that protective effect appeared to be homogeneous against meningitis and miliary TB but not against pulmonary disease may result from the fact that patients with meningitis are on average younger and thus less likely to have been exposed to atypical bacteria; to a waning of the protective effect of BCG; or from the diversity of mechanisms of pathogenesis of pulmonary disease, which can originate from reinfection, reactivation or primary progression.

Advocating for children's health at the state level: lessons learned.

This article documents the successful creation and promotion of a bill to fund a nurse home visitation program for high-risk mothers in Arkansas. It illustrates several key factors in successful advocacy by pediatricians working in an academic setting: a realistic time commitment; a community needs assessment, data assimilation, and review of existing resources; the identification and incorporation of stakeholders; a narrow focus on the area of greatest need; the backing of political partners; and favorable opportunities to advance child health issues.

A number-needed-to-treat analysis of the use of respiratory syncytial virus immune globulin to prevent hospitalization.

OBJECTIVES: To estimate how many infants in selected high-risk subgroups would require treatment with respiratory syncytial virus immune globulin (RSV-IG) to avoid 1 hospital admission and to determine whether this is economically justified. DESIGN: Cost-benefit analysis. Data from 3 randomized controlled trials of RSV-IG are used to estimate the number needed to treat to prevent 1 hospital admission for respiratory syncytial virus infection. The threshold number needed to treat is computed according to a formula incorporating costs and benefits of RSV-IG prophylaxis. Estimates of the willingness to pay were obtained from a sample of 39 health care providers (35 physicians and 4 nurses). MAIN OUTCOME MEASURES: The number needed to treat to prevent 1 hospital admission for respiratory syncytial virus infection. The threshold number needed to treat that would balance costs with benefits. RESULTS: More than 16 (95% confidence interval, 12.5-23.8) infants would need to be treated with RSV-IG to avoid 1 hospital admission for respiratory syncytial virus infection, ranging from 63 for premature infants without chronic lung disease to 12 (confidence interval, 6.3-100.0) for infants with bronchopulmonary dysplasia. A sensitivity analysis of the costs and values of hospital admission for respiratory syncytial virus infection and RSV-IG treatment resulted in a weak recommendation against the treatment of infants with bronchopulmonary dysplasia and strong recommendations that the costs and risks of RSV-IG treatment outweigh the benefits for the combined sample of infants and premature infants without lung disease. CONCLUSIONS: The number-needed-to-treat procedures offer a method to assess evidence of treatment effects and decision rules for whether to accept treatment recommendations. Under plausible assumptions, treatment with RSV-IG is not recommended for infants without lung disease. Institutions can examine cost and benefit assumptions that best fit their own practice setting.

Vero cytotoxin-producing Escherichia coli in a study of infectious intestinal disease in England.

An investigation of infectious intestinal disease in England included examination of feces for Vero cytotoxin-producing Escherichia coli (VTEC). Using DNA probe hybridization 27 VTEC strains were identified, 12 were from cases, and of these three belonged to serogroup O157. The remaining 15 strains were isolated from controls. The strains were confirmed biochemically as E. coli, they were serotyped and characterized according to their toxin production, the presence of sequences encoding intimin (eae) and enterohemolysin was determined and resistance to antimicrobial agents was determined. Six of the nine cases with non-O157 VTEC were less than 16 years old, only two of the 15 controls were under 16. Infection with more than one micro-organism was also considered.

Transneuronal labeling of spinal interneurons and sympathetic preganglionic neurons after pseudorabies virus injections in the rat medial gastrocnemius muscle.

The distribution of retrogradely and transneuronally labeled neurons was studied in CNS of rats 4 days after injections of the Bartha strain of pseudorabies virus (PRV) into the medial gastrocnemius (MG) muscle. Tissue sections were processed for immunohistochemical detection of PRV. Retrogradely labeled cells were identified in the ipsilateral MG motor column in the caudal L4 and the L5 spinal segments. In order to evaluate the efficacy of PRV retrograde cell body labeling, the number of PRV retrogradely labeled neurons in the MG motor column was compared to the number labeled with two conventional retrograde cell body markers--Fluoro-Gold and cholera toxin-HRP. A ratio of 1:3 representing medium-sized (less than 30 microns) versus large neurons (greater than 30 microns) was found in the Fluoro-Gold dye experiments; a 1:2 ratio was seen in the PRV experiments. In contrast, when cholera toxin-HRP was used as a retrograde marker, mainly large neurons were labeled; the medium-to-large cell body ratio was 1:10 suggesting cholera toxin-HRP may have a greater affinity for the terminals of alpha-motoneurons as opposed to gamma-motoneurons. Transneuronally labeled cells were identified in the L1-L6 spinal gray matter, intermediolateral cell column (T11-L2), lateral spinal nucleus and medial part of lamina VII in C4 and C5 spinal segments, brainstem (caudal raphe nuclei, rostral ventrolateral medulla, A5 cell group, paralemniscal nucleus, locus coeruleus, subcoeruleus nucleus, red nucleus) and paraventricular hypothalamic nucleus. In the L5 spinal cord, transneuronally labeled neurons were seen in the ipsilateral spinal laminae I and II and bilaterally in spinal laminae IV-VIII, and X. Similar results were obtained in rats that had chronic unilateral L3-L6 dorsal rhizotomies indicating most of the labeling was due to retrograde transneuronal cell body labeling. In order to determine whether PRV was transported into the spinal cord by the dorsal root axons, the ipsilateral dorsal root ganglia (DRGs) were examined for PRV immunoreactivity; none was found. However, using the polymerase chain reaction, viral DNA was shown to be present in the ipsilateral DRGs indicating that some of spinal cord cell body labeling may have resulted from anterograde transneuronal labeling, as well.

Human leucocyte antigen (HLA) and malaria morbidity in a Gambian community.

Human leucocyte antigen (HLA) class I and class II typing was performed on 177 children in a rural area of The Gambia who were followed for 2 years in a longitudinal study of malaria morbidity. A comparison was made between those who experienced an episode of clinical malaria in one or both years and those who showed no evidence of infection in either year. No convincing association was found between morbidity and class I phenotype. An overall association of morbidity with the distribution of class II haplotypes was seen, but association with individual DR-DQ haplotypes were not conclusive.

Aflatoxin exposure, malaria and hepatitis B infection in rural Gambian children.

Aflatoxin-albumin adduct levels were measured in serum samples obtained from a group of Gambian children. The relationships between exposure to aflatoxin and the prevalence of malaria, between exposure and humoral and cellular responses in vitro to defined malaria antigens and, amongst children with evidence of exposure to hepatitis B infection, between aflatoxin and carriage of the hepatitis B surface antigen (HBsAg), were assessed. Aflatoxin-albumin adduct was found in nearly all serum samples collected during a survey performed at the end of the dry season and levels of adduct were generally high (up to 720 pg aflatoxin-lysine equivalent/mg albumin). Higher levels of aflatoxin-albumin adduct were detected in Wollof children than in children of other ethnic groups and marked variation in mean adduct levels between villages was observed. Aflatoxin-albumin adduct levels were higher in children who were HbsAg positive and in children with Plasmodium falciparum parasitaemia than in controls. However, levels of adduct had no consistent effect on either malaria-specific antibody responses, lymphoproliferative responses in vitro, or morbidity from malaria during the subsequent rainy season. Much lower levels of aflatoxin-albumin adduct were detected in repeat samples obtained at the end of the rainy season. There was poor correlation between dry and rainy season levels of adduct in individual children. We have shown that Gambian children are exposed to high levels of aflatoxin. The seasonal variation of aflatoxin-albumin adduct and marked fluctuation of adduct with time in individual children need to be considered in the future planning of epidemiological studies using this marker of exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene specific assay to differentiate strains of pseudorabies virus.

The need for compatibility between Pseudorabies vaccination and disease eradication measures has caused the production and release of diverse Pseudorabies virus (PRV) vaccine strains with altered genetic makeups due to the deletion of specific genes. These genes code for antigens used as differential serologic markers. By use of polymerase chain reaction (PCR), it is possible to determine, in a rapid and sensitive way, if a given PRV strain has a "wildtype" genotype, or if instead it carries a deletion for a specific gene. A sequence of 217 bp was selected as an amplification target within the gene of the essential glycoprotein 50 (gp50). Another sequence of 173 bp was selected in the joint area of glycoprotein 63 (gp63) and glycoprotein I (gI) genes. Under optimal amplification conditions, the simultaneous use of both PCR tests allowed us to differentiate specifically gI negative strains from several other wild type PRV strains, utilizing cell culture-propagated virus, acutely and latently infected neural tissue of mice and pigs as source of DNA targets. This kind of test will be useful for the rapid identification of PRV strains detected in tissues from individual animals, especially in cases of single reactors occurring in vaccinated herds. At the same time, the gene-defined PCR test will be useful for the evaluation of vaccines in their ability to prevent latency, by permitting unequivocal differentiation between vaccine and challenge virus strains.

Impact of dietary yogurt on immune function.

Studies of the effects of yogurt on immunity and atopic diseases have suggested improvements in cytokine (interleukin-2 and interferon-gamma) responses and clinical scores in patients with allergic rhinitis. This study compares prospectively immune parameters of participants who received 16 oz of yogurt versus 16 oz of milk/day in a randomized cross-over design. Yogurt that contained live, active Lactobacillus bulgaricus and Streptococcus thermophilus or 2% milk was consumed for one month each. Twenty otherwise healthy adults with atopic histories documented by skin testing were enrolled. Immune studies were performed at the beginning and end of the two 1-month study phases, separated by a 2-week washout period. These studies included measurements of cellular, humoral, and phagocytic function. No adverse events were noted in either group. No significant improvements in any immune parameter were noted. The consumption of yogurt that contained the live active bacteria L bulgaricus and S thermophilus does not appear to enhance immune function in atopic individuals at the dosage and duration used in this study.

Does clinical experience with a treatment regimen affect survival of lung cancer patients? An analysis based on consecutive randomized trials of the Medical Research Council in small cell and non-small cell tumours. Medical Research Council Lung Cancer Working Party.

This paper investigates the influence on patient survival of an individual clinician's experience with a particular treatment regimen and the collective experience of the centre through which the patient is recruited to a particular clinical trial. Data were available from two series of randomized trials, one series in small cell and the other in non-small cell lung cancer, which were conducted by the Medical Research Council Lung Cancer Working Party. Successive small cell trials used the same chemotherapy regimen and successive non-small cell trials the same radiotherapy regimen. We found no evidence that either the degree of experience of individual clinicians with the regimen or the participation in terms of number of patients recruited to centres influenced patient survival. There is a strong suggestion, in non-small cell lung cancer that, as clinicians' experience with the regimen extended, they became increasingly likely to admit patients in poor condition to the trials. We stress how important it is to take account of patient prognostic characteristics in such an analysis, as we found that unadjusted comparisons suggested negative influences of increasing experience with the regimen. The implications for routine audit are self evident, in particular that it is not a substitute for the randomized controlled trial.

Pre-operative radionuclide localization of intracerebral lesions: a five-year experience.

Intracerebral lesions demonstrated by computerized tomography usually require histological confirmation to determine subsequent management. Tissue samples are generally obtained by craniotomy or burr hole biopsy; either procedure can prove negative if a lesion is small, deep, or very superficial. Pre-operative imaging and localization reduce biopsy failures. Before the introduction of this straight forward radionuclide technique, our biopsy success rate using conventional localization methods was 88%. In a 5-year period, 200 patients underwent pre-operative radionuclide localization, with an improvement in the overall biopsy success rate to 92.7% (95.5% for lesions which took up radionuclide). Patients have benefitted from reduced operating time and improved post-operative recovery rates. About 85% of all intracerebral lesions may be expected to accumulate radionuclide. However in our series, 93.2% were sufficiently well visualized for a siting marker to be placed with confidence. Within this group, low grade astrocytomas (Kernohan Grades I and II) showed a predictably low incidence of imaging (30.8%). For the majority of lesions which present difficulties in biopsy due to size or site, the radionuclide method is a simple procedure which increases the chance of obtaining positive tissue with the minimum of surgical intervention.

99Tcm-nanocolloid imaging in inflammatory bowel disease.

We have used 99Tcm-labelled nanocolloid in an attempt to locate areas of inflamed bowel wall or abscesses in five patients with ulcerative colitis and nine with Crohn's disease. The scintigraphic findings were evaluated by comparison with those of recent barium studies and, in three patients, with surgical findings at laparotomy. It proved difficult to localize segments of inflamed bowel accurately with 99Tcm-nanocolloid because of the accumulation of radioactivity in the gut lumen, especially 2 or more hours after injection. However, there was little uptake of the labelled nanocolloid by areas of inflamed gut wall in the period before 2 h. When 99Tcm-nanocolloid scans were compared with 111In-WBC scans in eight patients who had both investigations, 99Tcm-nanocolloid scintigraphy was considerably less sensitive than 111In-WBC scintigraphy. One abscess was located correctly; the other was obscured by nearby bladder and bone marrow radioactivity. We conclude that 99Tcm-nanocolloid scanning is neither sensitive nor reliable enough for assessing the location of inflamed bowel wall or the presence of abscess in patients with inflammatory bowel disease.

Characterization of antibody response to porcine reproductive and respiratory syndrome virus ORF5 product following infection and evaluation of its diagnostic use in pigs.

The sensitivity and specificity of recombinant open reading frame 5 products used in the Western blotting assay for confirmation of porcine reproductive and respiratory syndrome virus (PRRSV) serologic status were evaluated. The recombinant antigen-based assays were specifically compared with a commercial enzyme-linked immunosorbent assay (ELISA) for PRRSV antibodies using 1) PRRSV antibody-negative reference sera (n = 30), 2) naturally infected pig sera (n = 40), 3) sequential sera obtained from 24 experimentally infected pigs, and 4) sera submitted to 3 state diagnostic laboratories (n = 200). The recombinant antigen assay yielded an average increased sensitivity of 10% over the commercial PRRSV ELISA. The negative controls (group 1 sera) showed no difference between the 2 assays. This comparison confirmed that the recombinant antigen-specific assay was more sensitive than the commercial ELISA and is well suited for routine confirmation of the presence of PRRSV antibodies.

Investigation of sites of pseudorabies virus latency, using polymerase chain reaction.

Pseudorabies virus (PRV) latency was investigated, using polymerase chain reaction (PCR). A PCR protocol was developed that specifically amplified a 217-base pair sequence within the gene encoding the essential glycoprotein gp50 of PRV. Using this PCR procedure, the gp50 sequence was amplified from tissues of pigs infected with various doses of PRV (Becker strain). At postinoculation day 64, viral isolation was performed on nasal swab specimens and homogenates of tonsils and trigeminal nerve ganglia obtained from 11 PRV-convalescent, seropositive pigs. Results were negative in all cases. By use of PCR, 11 of 11 pigs had PRV-positive trigeminal nerve ganglia and brain stem, 10 of 11 pigs had PRV-positive tonsils, and 9 of 11 pigs had PRV-positive olfactory bulbs.