Comparison of pneumococcal vaccination response in children with sickle cell disease: HbSS and HbSC.

INTRODUCTION: Sickle cell disease (SCD) children are at increased risk of invasive pneumococcal disease and rely on penicillin prophylaxis and vaccination for infection prevention. Post-vaccination antibody levels in SCD may wane overtime. HbSC are believed to have better immunological response than HbSS. OBJECTIVE: To compare antibody response to 23-valent pneumococcal polysaccharide vaccine (PPSV-23) between HbSS and HbSC. METHODS: Patients with HbSS (n=33) and HbSC (n=11), aged 7-18 years, were prospectively recruited. Luminex pneumococcal antibody levels were measured for 23-serotypes, after two PPSV-23 doses. RESULTS: Absolute median titer for 20 of the 23 serotypes was higher in HbSC than HbSS and significantly higher for serotypes 22 (3.9 vs. 1.6mcg/ml; p=0.039) and 43 (2.9 vs. 0.8mcg/ml; p=0.007). HbSC mounted a better immune anti-pneumococcal response compared to HbSS (≥1.3mcg/ml) for 18 of 23 serotypes, albeit not significant for any of the serotypes. More HbSC (64%) than HbSS (42%) were good vaccine responders (p=0.303). Two of 21 (10%) good vaccine responders and nine of 23 (39%) poor vaccine responders SCD participants subsequently developed acute chest syndrome or pneumonia (p=0.036). None of the HbSC patients developed ACS after receiving PPSV-23. HbSS poor vaccine responders were at increased future recurrence risk for ACS (p=0.003), pneumonia (p=0.036) or both (p=0.011), compared to good vaccine responders. CONCLUSION: HbSC possess better pneumococcal vaccine response than HbSS. Poor vaccine response is concerning for future acute pulmonary events. Current vaccination strategy for SCD sub-types are lacking, therefore further study to evaluate utility of vaccine boosters is necessary.

A wire-flooring model for inducing lameness in broilers: evaluation of probiotics as a prophylactic treatment.

Bacterial chondronecrosis with osteomyelitis (BCO) is the most common cause of lameness in commercial broilers. Bacteria entering the blood via translocation from the respiratory system or gastrointestinal tract spread hematogenously to the proximal epiphyseal-physeal cartilage of rapidly growing femora and tibiae, causing BCO. We tested the hypothesis that rearing broilers on wire flooring should increase the incidence of BCO by persistently imposing additional torque and shear stress on susceptible leg joints. We also tested the hypothesis that probiotics might attenuate bacterial translocation and thereby reduce the incidence of BCO. In 5 independent experiments using 4 commercial lines, broilers grown on wire flooring developed lameness attributable predominately to BCO. The fastest-growing birds were not necessarily the most susceptible to lameness on wire flooring, nor did the genders differ in susceptibility in the 2 experiments that included both male and female broilers. The pathogenesis of BCO is not instantaneous, and accordingly, many broilers that did not exhibit lameness, nevertheless, did possess early pathognomonic lesions. These subclinical lesions were equally likely to develop in the right or left leg. The lesion status of the proximal femoral head did not determine the lesion status of the ipsilateral or contralateral proximal tibial head and vice versa. Broilers reared on wire flooring consistently had higher incidences of lameness than hatch-mates reared on wood-shavings litter. Adding probiotics to the diet beginning at 1 d of age consistently reduced the incidence of lameness for broilers reared on wire flooring. These experiments indicate that probiotics administered prophylactically may constitute an alternative to antibiotics for reducing lameness attributable to BCO. Rearing broilers on wire flooring provides an important new research model for investigating the etiology, pathogenesis, and treatment strategies for BCO.

Double-strand breaks and tumorigenesis.

The establishment of connections between biochemical defects and clinical disease is a major goal of modern molecular genetics. In this review, we examine the current literature that relates defects in the two major DNA double-strand-break repair pathways--homologous recombination and nonhomologous end-joining--with the development of human tumors. Although definitive proof has yet to be obtained, the current literature is highly suggestive of such a link.

Risk of leukaemia among children living near the Solway coast of Dumfries and Galloway Health Board area, Scotland, 1975-2002.

OBJECTIVE: To investigate allegations of an excess risk of leukaemia among children living near the Solway Firth coast of Dumfries and Galloway Health Board area in Scotland, UK. METHODS: Incident cases of childhood leukaemia (International Classification of Diseases, 10th revision, C91-C95, patients aged 0-14 years) for two almost equal calendar periods of diagnosis (1975-89 and 1990-2002) were selected from the Scottish Cancer Registry database and allocated to predetermined study areas, on the basis of proximity of residence to the Solway coast. Expected numbers of childhood leukaemia cases for the study areas were calculated by applying Scotland's age-specific, sex-specific and calendar period-specific rates to estimates of the person-years at risk in each study area. The ratios of observed to expected cases or standardised incidence ratios (SIRs) were calculated overall and for each sex and calendar period category. Exact 95% confidence intervals (CIs) for the SIRs were calculated assuming a Poisson distribution for the observed number of cases of childhood leukaemia. RESULTS: No statistically significantly increased SIRs were found in boys, girls or both combined for any of the areas or periods of diagnosis studied. For the total period of observation (1975-2002), and the more immediate coastal area studied, the SIR for both sexes combined was 1.22 (95% CI 0.53 to 2.40). CONCLUSION: No statistically significant evidence was found of an excess risk of childhood leukaemia in the vicinity of the Solway Firth coast of Dumfries and Galloway Health Board area in Scotland.

Severe Hepatopulmonary Syndrome in a Child with Caroli Syndrome.

Hepatopulmonary Syndrome (HPS) is a potential complication of chronic liver disease and is more commonly seen in the adult population. Caroli Syndrome is a rare inherited disorder characterized by intrahepatic ductal dilation and liver fibrosis that leads to portal hypertension. In children with liver disease, HPS should be considered in the differential diagnosis of prolonged, otherwise unexplained, hypoxemia. The presence of HPS can improve patient priority on the liver transplantation wait list, despite their Pediatric End-Stage Liver Disease (PELD) score. We present a 6-year-old girl with Caroli Syndrome and End-Stage Renal Disease who presented with persistent hypoxemia. The goal of this report is to increase awareness of HPS in children.

Infection of A549 cells with a recombinant adenovirus vector induces ICAM-1 expression and increased CD-18-dependent adhesion of activated neutrophils.

A significant number of pulmonary exacerbations in patients with cystic fibrosis (CF) and asthma are associated with respiratory virus infections. The molecular mediators of this process are beginning to be understood. Viral infection of respiratory epithelial cultures in vitro leads to the production of intercellular adhesion molecule-1 (ICAM-1) (a ligand for inflammatory cell adhesion and activation) and a number of proinflammatory cytokines. Human gene therapy vectors derived from human adenoviruses (AV) are currently under evaluation for CF transmembrane regulator (CFTR) gene delivery to the airway epithelium of CF patients. However, studies in animal models using these AV vectors demonstrate pulmonary inflammation following AV exposure. Using an in vitro model, we examined the hypothesis that exposure of respiratory epithelial cells to AV vectors results in upregulation of ICAM-1 gene expression. Infections were performed using a replication-deficient, first-generation AV vector. A549 cells (a human pulmonary adenocarcinoma cell line) were exposed to AV at multiplicity of infection of 50-150 plaque-forming units/cell (resulting in > 90% of cells expressing the reporter gene by 48 hr following exposure). Measurements of ICAM-1 expression were made at time intervals following virus exposure using enzyme immunoassay, flow cytometry, and Northern blot analysis. Cell-bound ICAM-1 was significantly increased 96 hr following vector exposure, two to four times control, p < 0.001). The AV-exposed A549 cells also supported increased levels of adhesion of activated neutrophils 96 hr following AV exposure (four times control, p < 0.001) that was blocked by antibody to CD18. AV exposure of A549 monolayers increases expression of biologically active ICAM-1. Strategies to minimize host cellular proinflammatory responses to the replication-deficient AV vectors may improve their safety for gene therapy.

Electron spin resonance detection of oxygen-centred radicals in murine macrophages stimulated with bacterial endotoxin.

The production of oxygen radicals by Bacille-Calmette-Guerin primed mouse macrophages stimulated with bacterial endotoxin has been investigated. Superoxide radicals were spin-trapped in this system with dimethylpyrroline-N-oxide after a lag period of 20-40 minutes. The electron spin resonance signals due to the superoxide radical adduct could be inhibited by superoxide dismutase but not by catalase.

Comparison of the fine structure of cortical and collicular terminals in the rat medial geniculate body.

Neurons throughout the rat medial geniculate body, including the dorsal and ventral divisions, display a variety of responses to auditory stimuli. To investigate possible structural determinants of this variability, measurements of axon terminal profile area and postsynaptic dendrite diameter were made on inferior colliculus and corticothalamic terminal profiles in the medial geniculate body identified by anterograde tracer labeling following injections into the inferior colliculus or cortex. Over 90% of the synapses observed were axodendritic, with few axosomatic synapses. Small (<0.5 microm(2)) and large (>1.0 microm(2)) collicular profiles were found throughout the medial geniculate, but were smaller on average in the dorsal division (0.49+/-0.49 microm(2)) than in the ventral division (0.70+/-0.64 microm(2)). Almost all corticothalamic profiles were small and ended on small-caliber dendrites (0.57+/-0.25 microm diameter) throughout the medial geniculate. A few very large (>2.0 microm(2)) corticothalamic profiles were found in the dorsal division and in the marginal zone of the medial geniculate. GABA immunostaining demonstrated the presence of GABAergic profiles arising from cells in the inferior colliculus. These profiles were compared with GABAergic profiles not labeled with anterograde tracer, which were presumed to be unlabeled inferior colliculus profiles or thalamic reticular nucleus profiles. The distributions of dendritic diameters postsynaptic to collicular, cortical and unlabeled GABAergic profiles were compared with dendritic diameters of intracellularly labeled medial geniculate neurons from rat brain slices. Our results demonstrate a corticothalamic projection to medial geniculate body that is similar to other sensory corticothalamic projections. However, the heterogeneous distributions of excitatory inferior collicular terminal sizes and postsynaptic dendritic diameters, along with the presence of a GABAergic inferior collicular projection to dendrites in the medial geniculate body, suggest a colliculogeniculate projection that is more complex than the ascending projections to other sensory thalamic nuclei. These findings may be useful in understanding some of the differences in the response characteristics of medial geniculate neurons in vivo.

Embryonic neuronal death due to neurotrophin and neurotransmitter deprivation occurs independent of Apaf-1.

Apoptotic protease-activating factor-1 (Apaf-1), dATP, and procaspase-9 form a multimeric complex that triggers programmed cell death through the activation of caspases upon release of cytochrome c from the mitochondria into the cytosol. Although cell death pathways exist that can bypass the requirement for cytochrome c release and caspase activation, several gene knockout studies have shown that the cytochrome c-mediated apoptotic pathway is critical for neural development. Specifically, the number of neuronal progenitor cells is abnormally increased in Apaf-1-, caspase-9-, caspase-3-deficient mice. However, the role of the cytochrome c cell death pathway for apoptosis of postmitotic, differentiated neurons in the developing brain has not been investigated in vivo. In this study we investigated embryonic neuronal cell death caused by trophic factor deprivation or lack of neurotransmitter release by analyzing Apaf-1/tyrosine kinase receptor A (TrkA) and Apaf-1/Munc-18 double mutant mice. Histological analysis of the double mutants' brains (including cell counting and terminal (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining) reveals that neuronal cell death caused by these stimuli can proceed independent of Apaf-1. We propose that a switch between apoptotic programs (and their respective proteins) characterizes the transition of a neuronal precursor cell from the progenitor pool to the postmitotic population of differentiated neurons.

Meningococcal infection and proteolytic control.

Cascade enzyme inhibitors (C1-esterase inhibitor, C3b inactivator, antithrombin III) and other major proteolytic enzyme inhibitors (alpha 1 trypsin inhibitor, alpha 1 chymotrypsin inhibitor, inter-alpha-trypsin inhibitor, alpha 2 macroglobulin) as well as C3 and alpha 1 acid glycoprotein, have been examined in the sera of Nigerian patients suffering from meningococcal infection of varied severity. Patients with meningococcaemia had lower serum concentrations of important inhibitors than did patients with localised meningitic infection. Within the coccaemic group, those who died had the lowest values, notably of antithrombin III and alpha 2 macroglobulin (and also of C3). The clinical end-result of meningococcal infection may be related to the degree of disequilibrium of the linked system of proteolytic control induced by the meningococcal endotoxin.

Leukotriene B4 formation: human neutrophil-airway epithelial cell interactions.

Leukotriene B4 (LTB4) is a potent inflammatory mediator involved in the pathogenesis of many pulmonary diseases. Although the neutrophil is the predominant source of LTB4, other cells can also interact with neutrophils and increase LTB4 formation. In this study, we investigated whether human neutrophil-airway epithelial cell interactions can increase LTB4 formation. Neutrophils were cocultured with transformed airway epithelial cells (9HTEo- cells), and LTB4 and leukotriene A4 (LTA4) degradation product release was measured by high-performance liquid chromatography and ultraviolet spectrometry. When stimulated with the calcium ionophore A-23187, neutrophil-9HTEo- cell cocultures released more LTB4 and less LTA4 degradation products in a time- and dose-related manner than did neutrophils alone. This increase in LTB4 release involved the metabolism of neutrophil-derived LTA4 to LTB4 by 9HTEo- cells and was affected by the neutrophil-to-epithelial cell ratio. Enhanced LTB4 release required proximity between neutrophils and 9HTEo- cells but not specific cell-cell adhesion. Our data demonstrate that human neutrophil-airway epithelial cell interactions can increase LTB4 formation through transcellular arachidonic acid metabolism.

The ability of smooth and rough strains of Streptococcus pneumoniae to activate human complement by the alternative pathway.

Three capsulate pneumococcal strains of serotypes 1, 2 ans 3, and one non-capsulate strain of serotype 47, were found to activate human complement by the alternative pathway to a similar extent over the concentration range examined. Nevertheless, the capsulate strains, in contrast to the non-capsulate, are known to require complement attachment for phagocytosis and it is therefore postulated that the toxic by-products released cause the wave of oedema characteristic of pneumococcal lobar pneumonia.

Sensitivity to endotoxin is induced by increased membrane fatty-acid unsaturation and oxidant stress.

The mechanisms modulating host susceptibility to endotoxin are unknown. Evidence suggests that endotoxin pathophysiology is mediated in part by oxidative reactions that lead to tissue damage and organ failure. The proposition is that conditions which favour oxidation sensitise the host to endotoxin. Central to this hypothesis is that an increase in the polyunsaturated fatty-acid composition of membrane phospholipids enhances susceptibility because such fatty acids are easily oxidised to produce mediators of the endotoxic crisis. Cytokines, such as tumour-necrosis factor and interferon-gamma, may be ultimately responsible for orchestrating these changes and thereby modify the host response to endotoxin.

Decreased pulmonary clearance of S. pneumoniae following influenza A infection in mice.

In children, the incidence of complicated pneumonias (including empyemas and lung abscesses) associated with Streptococcus pneumoniae infection has increased in recent years. In many cases, these complicated pneumonias followed flu-like illnesses. To determine mechanisms behind this association, a murine model of sequential pulmonary infection has been developed. BALB/cJ mice infected with influenza A had mild pulmonary inflammation that resolved within 5-7 days. Seven days following their initial 'treatment' (mock infection or influenza exposure), mice were challenged with 10(6) cfu of S. pneumoniae, and their lungs were harvested at intervals for analysis. Lungs of influenza-exposed mice demonstrated greater colony counts 24 and 48 h following S. pneumoniae exposure compared to control mice. In addition, neutrophil numbers were significantly increased in the influenza/S. pneumoniae sequentially-infected animals compared to S. pneumoniae infection alone (1.4+/-0.6 x 10(6) vs. 0.06+/-0.07 x 10(6) cells, P < 0.05, 24 h). Influenza-exposed animals had greater levels of IL-1beta and TNF-alpha in lung homogenates following S. pneumoniae inoculation. These data demonstrate that mice exposed to influenza have enhanced inflammatory responses and increased bacterial burden following S. pneumoniae exposure than do control mice. This model will be useful in defining mechanisms behind the enhanced susceptibility to S. pneumoniae that occurs after influenza exposure.

Infection of cultured human tracheal epithelial cells by human parainfluenza virus types 2 and 3.

Despite growing information of the effects of human respiratory virus infection on airway physiology, little information is available on the mechanisms of pathology and pathophysiology in these infections. The human respiratory pathogens, parainfluenza virus types 2 and 3 (hPIV2, hPIV3, respectively), clinically cause laryngotracheobronchitis (infection of the large proximal airways). In order to examine the pathobiology of these viruses in airway cells of human origin, we exposed primary cultures of human tracheal epithelial cells. Primary cultures of human tracheal epithelial cells were readily infected by these agents: cells exposed to hPIV2 and hPIV3 expressed viral antigens (demonstrated by indirect immunofluorescence assay), produced infectious virus, and demonstrated cytopathic effects (including early syncytium formation). Peak viral titers of 2 x 10(7) plaque-forming units per milliliter were obtained, similar to titers from permissive CV-1 cells. Trypan blue staining and direct cell counts demonstrated no difference in the viability of the control and infected cells until the infected cells began to detach from the culture substrate. However, infected cells release significantly more LDH than control cells by 48 h following infection at a multiplicity of infection of 1 virus/target cell. This system provides a model for studying the effects of infection of the human tracheal epithelium by human respiratory viral pathogens without confounding interactions with other cell and tissue types.

Detection of enhanced neutrophil adhesion to parainfluenza-infected airway epithelial cells using a modified myeloperoxidase assay in a microtiter format.

Despite growing evidence that respiratory virus infections precipitate episodes of airway obstruction and airway hyper-responsiveness in young children and in asthma, little information is available on the mechanisms by which virus infections alter the airway physiology. Airway inflammatory changes (including influx of inflammatory cells such as neutrophils) have been described during episodes of airway hyper-responsiveness in both animal models and human subjects. Neutrophil damage to several cell types has been shown to require adhesion as a primary step. In order to examine the potential interactions between virus-infected airway epithelial cells and neutrophils, we have studied the ability of neutrophils to adhere to virus-infected airway epithelial cell cultures. Neutrophil adherence was determined indirectly, using myeloperoxidase as a marker for adherent neutrophils in an assay system described here. Airway epithelial cell cultures (both primary human tracheal epithelial cells, and two permanent cell lines, A549 and BEAS-2B) were grown in 96-well tissue culture plates and infected with human parainfluenza virus type 2. Infected airway epithelial cell cultures supported significantly enhanced levels of neutrophil adherence (up to 50-75% of neutrophils added to the wells) compared to uninfected control cultures. Moreover, this adherence occurred in a virus dose-dependent fashion, with increasing levels of adherence noted at increasing viral multiplicities of infection. The assay system described allows the detection of small numbers of adherent neutrophils (as few as 1000 neutrophils) in a 96-well format.

Occurrence of respiratory syncytial virus subtypes in hospitalized children in Cleveland, Ohio from 1985 to 1988.

In order to determine the frequency of occurrence of the two respiratory syncytial virus (RSV) subtypes in hospitalized children in Cleveland, Ohio, we analyzed clinical isolates obtained during three consecutive winter epidemic seasons between 1985 and 1988. RSV was recovered from the frozen clinical specimens of 197 patients: 176 subtype A, and 21 subtype B. Subtype A predominated during all three epidemic seasons, ranging from 83 to 94% of isolates. We surveyed the clinical records of 16 children with subtype B, and 101 children with subtype A infections, hospitalized at the University Hospitals of Cleveland during these winter epidemics and found no differences in age, sex, race, or clinical spectrum of severity of disease caused by the two subtypes. In contrast to previously reported data, subtype A predominated in each of the winter seasons studied within this community. We conclude that both subtypes circulate concurrently within the community during the winter. In hospitalized children both subtypes appear to cause a similar spectrum of disease. Both the concurrent circulation of RSV subtypes and the similar spectrum of illness pose for important considerations in the development of effective vaccines against this common respiratory agent in children.

SIRT1 deacetylase promotes acquisition of genetic mutations for drug resistance in CML cells.

BCR-ABL transforms bone marrow progenitor cells and promotes genome instability, leading to development of chronic myelogenous leukemia (CML). The tyrosine kinase inhibitor imatinib effectively treats CML, but acquired resistance can develop because of BCR-ABL mutations. Mechanisms for acquisition of BCR-ABL mutations are not fully understood. Using a novel culture model of CML acquired resistance, we show that inhibition of SIRT1 deacetylase by small molecule inhibitors or gene knockdown blocks acquisition of BCR-ABL mutations and relapse of CML cells on tyrosine kinase inhibitors. SIRT1 knockdown also suppresses de novo genetic mutations of hypoxanthine phosphoribosyl transferase gene in CML and non-CML cells upon treatment with DNA damaging agent camptothecin. Although SIRT1 can enhance cellular DNA damage response, it alters functions of DNA repair machineries in CML cells and stimulates activity of error-prone DNA damage repair, in association with acquisition of genetic mutations. These results reveal a previously unrecognized role of SIRT1 for promoting mutation acquisition in cancer, and have implication for targeting SIRT1 to overcome CML drug resistance.

Role of SUMO:SIM-mediated protein-protein interaction in non-homologous end joining.

Although post-translational modifications by the small ubiquitin-like modifiers (SUMO) are known to be important in DNA damage response, it is unclear whether they have a role in double-strand break (DSB) repair by non-homologous end joining (NHEJ). Here, we analyzed various DSB repair pathways upon inhibition of SUMO-mediated protein-protein interactions using peptides that contain the SUMO-interaction motif (SIM) and discriminate between mono- and SUMO-chain modifications. The SIM peptides specifically inhibit NHEJ as shown by in vivo repair assays and radio-sensitivity of cell lines deficient in different DSB repair pathways. Furthermore, mono-SUMO, instead of SUMO-chain, modifications appear to be involved in NHEJ. Immunoprecipitation experiments also showed that the SIM peptide interacted with SUMOylated Ku70 after radiation. This study is the first to show an important role for SUMO:SIM-mediated protein-protein interactions in NHEJ, and provides a mechanistic basis for the role of SIM peptide in sensitizing genotoxic stress of cancer cells.