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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Current concepts on pulmonary host defense mechanisms in children.

The respiratory tract is exposed continuously to noxious agents, microbial organisms, particles, and allergens. It has therefore evolved both innate and specific defense mechanisms. The innate host defense mechanisms include components such as collectins, beta-defensins, lactoferrin, and complement, all of which have an important role in modulating the immune response. Immune protection of the lungs by specific antibody is reviewed. The airways are protected by alveolar macrophages, neutrophils, and lymphocytes, and their origins, regulation, functions, and antimicrobial activity are summarized. Antimicrobial peptides and immune-modulating peptides are likely to have a significant therapeutic role for infection and inflammation in the respiratory tract.

Molecular analysis of CCR-3 events in eosinophilic cells.

CCR-3 is a major receptor involved in regulating eosinophil trafficking. Initial analysis of chemokine receptors has demonstrated unique receptor events in different cell types, indicating the importance of investigating CCR-3 events in eosinophilic cell lines. We now report that the eosinophilic cell line, acute myelogenous leukemia (AML) 14.3D10, expresses eosinophil granule proteins and eotaxin, but has no detectable expression of eosinophil chemokine receptors. Treatment of the cell line with butyric acid and IL-5 results in a dose-dependent synergistic induction of CCR-3 and, to a lesser extent, CCR-1 and CCR-5. Interestingly, using a luciferase reporter construct under the control of the hCCR-3 promoter, the uninduced and induced cells display high, but comparable, levels of promoter activity. Differentiated AML cells developed enhanced functional activation, as indicated by adhesion to respiratory epithelial cells and chemokine-induced transepithelial migration. Chemokine signaling did not inhibit adenylate cyclase activity even though calcium transients were blocked by pertussis toxin. Additionally, chemokine-induced calcium transients were inhibited by pretreatment with PMA, but not forskolin. Eotaxin treatment of differentiated AML cells resulted in marked down-modulation of CCR-3 expression for at least 18 h. Receptor internalization was not dependent upon chronic ligand exposure and was not accompanied by receptor degradation. Thus, CCR-3 is a late differentiation marker on AML cells and uses a signal transduction pathway involving rapid and prolonged receptor internalization, calcium transients inhibitable by protein kinase C but not protein kinase A, and the paradoxical lack of inhibition of adenylate cyclase activity.

The relative strengths of SR protein-mediated associations of alternative and constitutive exons can influence alternative splicing.

We have characterized the functional role of SR protein-mediated exon/exon associations in the alternative splicing of exon 5 of chicken cardiac troponin T (cTnT). We have previously shown that SR proteins can promote the association of the alternative exon 5 with the flanking constitutive exon 6 of this pre-mRNA. In this study, we have shown that when exons 2, 3, and 4 of the cTnT pre-mRNA are spliced together, the composite exon 2/3/4 contains an additional SR protein binding site. Furthermore, we have found that SR proteins can also promote interactions between the pairs of exons 2/3/4-5 and 2/3/4-6. We then asked whether the SR protein binding sites in these exons play a role in cTnT alternative splicing in vivo. We found that the SR protein binding sites in exons 2/3/4 and 6 promote exon 5 skipping, and it has previously been shown that the SR protein binding site in exon 5 promotes exon 5 inclusion. Consistent with these results, we find that the SR protein-mediated association of exon 2/3/4 with 6 is preferred over associations involving exon 5, in that exons 2/3/4 and 6 are more efficient than exon 5 in competing an SR protein-mediated exon/exon association. We suggest that the relative strengths of SR protein-mediated associations of alternative and constitutive exons play a role in determining alternative splicing patterns.

Interleukin-1 receptor antagonist inhibits interleukin-8 expression in A549 respiratory epithelial cells infected in vitro with a replication-deficient recombinant adenovirus vector.

In an earlier study, we showed that a recombinant adenovirus vector with deletions in the E1 and E3 regions of the viral genome (AV1LacZ4) induces expression of interleukin (IL)-8 in A549 cells (a human respiratory cell line). IL-8 can be induced through several pathways, including activation by IL-1. We tested the hypothesis that the induction of IL-8 by the AV1LacZ4 adenovirus is accomplished by means of the IL-1/IL-8 activation pathway, which could be blocked by IL-1 receptor antagonist (IRAP). Viral infections of A549 cells were performed at a multiplicity of infection (MOI) of 50 in the presence and absence of IRAP (50 ng/ml). A549 cells were also stimulated with tumor necrosis factor (TNF)-alpha (100 ng/ml), a known stimulant of IL-8, in the presence and absence of IRAP. IL-8 expression was evaluated by Northern blot analysis and enzyme-linked immunosorbent assay. Levels of IL-8 protein and messenger RNA (mRNA) were greater in the infected cells than in the uninfected ones at 24, 48, and 96 h (P < 0.01). Virus-infected cells treated with IRAP expressed 75% less IL-8 mRNA and protein (P < 0.01) than did untreated cells, whereas IRAP pretreatment of TNF-alpha-stimulated cells did not affect IL-8 production. IL-1 production by the virus-infected cells was detectable by concentration of the supernatants and reverse transcription-polymerase chain reaction. We conclude that IL-8 is produced by virus vector-infected cells, partly through IL-1 activation that can be downregulated by IRAP.

Induction of CD95 (Fas) and apoptosis in respiratory epithelial cell cultures following respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection is associated with epithelial cell death and vigorous inflammation. In mouse models, and in immunosuppressed patients, CD8(+) T cells are necessary for RSV clearance. In vitro, RSV has been shown to induce expression of several proteins on the respiratory epithelial cell, including RSV proteins, ICAM-1, and MHC class I, that can potentially interact with CD8(+) T cells in initiating apoptosis of the target cell. One mechanism of T-cell-directed cell death is the interaction of FasL on the CD8(+) T lymphocytes and Fas expressed on the target cell. In order to determine the ability of RSV to induce Fas on the respiratory epithelium, we studied the RSV infection of a human respiratory epithelial cell line (A549) in vitro. Fas mRNA and protein levels are increased two-to-fourfold following RSV infection, and transcriptional upregulation of Fas was demonstrated using promoter/reporter gene constructs. RSV infection directly resulted in cellular apoptosis, and the frequency of apoptotic cells was further increased by cross-linking with antibodies to Fas. These data demonstrate that RSV infection induces cellular apoptosis and suggest that interactions of surface Fas with T cells may further augment this process in vivo.

Immunological adjuvance of metabolic origin: oxidative stress, postulated impaired function of thiol proteases and immunogenicity.

Autoimmune disease may sometimes arise because of dysfunction of thiol proteases, which are vulnerable to oxidation of their sulphydryl groups. This may be initially signalled by hyperinsulinaemia, regarded here as a telltale phenomenon of oxidative stress and indicating difficulty in protein catabolism. Initial immunogenic sensitization may take place when antigen processing is altered by a metabolic process, which has locally overwhelmed the antioxidant systems and led to diminished thiol-protease digestion and to the repeated survival in critical cells of immunogenic peptide fragments. From this it follows that an immunological host may be either prejudiced towards or against tolerance by agents, respectively, which stabilize or destabilize antioxidant homeostasis.

Surfactant protein-A-deficient mice are susceptible to Pseudomonas aeruginosa infection.

To determine the role of surfactant protein-A (SP-A) in host defense, the murine SP-A locus was targeted by homologous recombination to produce mice lacking SP-A. SP-A-/- and wild-type mice were infected with mucoid Pseudomonas aeruginosa by intratracheal instillation. Pulmonary bacterial loads were greater in SP-A-/- than in wild-type mice, with increased numbers of mucoid P. aeruginosa in lung homogenates at 6 and 24 h after infection. Pulmonary infiltration with polymorphonuclear leukocytes (PMN) was similar in both groups; however, an earlier influx of PMN into the lung occurred in the SP-A-/- mice. The number of bacteria phagocytosed by alveolar macrophages was decreased in the SP-A-/- mice at 1 h after infection. Superoxide-radical generation by PMN was similar for the SP-A-/- and wild-type mice, but nitrite levels were increased in SP-A-/- mice. Concentrations of tumor necrosis factor-alpha, interleukin-6, and macrophage inflammatory protein-2 (proinflammatory cytokines) were greater in bronchoalveolar lavage fluid at 2 h after infection in SP-A-/- mice. SP-A plays an important role in the pathogenesis of mucoid P. aeruginosa infection in the lung in vivo by enhancing macrophage phagocytosis and clearance of bacteria, and by modifying the inflammatory response.

The heat shock response inhibits RANTES gene expression in cultured human lung epithelium.

The chemokine RANTES is thought to be involved in the pathophysiology of inflammation-associated acute lung injury. Although much is known regarding signals that induce RANTES gene expression, relatively few data exist regarding signals that inhibit RANTES gene expression. The heat shock response, a highly conserved cellular defense mechanism, has been demonstrated to inhibit a variety of lung proinflammatory responses. We tested the hypothesis that induction of the heat shock response inhibits RANTES gene expression. Treatment of A549 cells with TNF-alpha induced RANTES gene expression in a concentration-dependent manner. Induction of the heat shock response inhibited subsequent TNF-alpha-mediated RANTES mRNA expression and secretion of immunoreactive RANTES. Transient transfection assays involving a RANTES promoter-luciferase reporter plasmid demonstrated that the heat shock response inhibited TNF-alpha-mediated activation of the RANTES promoter. Inhibition of NF-kappaB nuclear translocation with isohelenin inhibited TNF-alpha-mediated RANTES mRNA expression, indicating that RANTES gene expression is NF-kappaB dependent in A549 cells. Induction of the heat shock response inhibited degradation of the NF-kappaB inhibitory protein, I-kappaBalpha but did not significantly inhibit phosphorylation of I-kappaBalpha. We conclude that the heat shock response inhibits RANTES gene expression by a mechanism involving inhibition of NF-kappaB nuclear translocation and subsequent inhibition of RANTES promoter activation. The mechanism by which the heat shock response inhibits NF-kappaB nuclear translocation involves stabilization of I-kappaBalpha, without significantly affecting phosphorylation of I-kappaBalpha.

Inhibition of TNF-alpha-induced NF-kappaB activation and IL-8 release in A549 cells with the proteasome inhibitor MG-132.

The working hypothesis of the studies described herein was that inhibition of proteasome-mediated IkappaB degradation would inhibit TNF-alpha-induced nuclear factor-kappaB (NF-kappaB) activation, interleukin-8 (IL-8) gene transcription, and IL-8 protein release in A549 cells. Mutational analysis of the 5' flanking region of the IL-8 gene confirmed that an intact NF-kappaB site is necessary for TNF-alpha-induced IL-8 gene transcription. The addition of TNF-alpha to A549 cells resulted in rapid loss of IkappaB from the cytoplasm of cells, associated with a corresponding increase in NF-kappaB-binding activity in nuclear extracts from the cells. However, pretreatment of the cells with the proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132, 10 microM) reversed the effects of TNF-alpha on IL-8 release from A549 cells (as determined with an enzyme-linked immunosorbent assay [ELISA]) and on IL-8 gene transcription (as determined with reporter-gene assays). MG-132 reversed the effects of TNF-alpha on IkappaB degradation as determined by Western blot analysis. IkappaB phosphorylation and ubiquination were not altered by MG-132, which implies that the effects of MG-132 were secondary to proteasome inhibition. MG-132 also reversed the increase in NF-kappaB binding in nuclear extracts from TNF-alpha-treated cells. These studies show that inhibition of proteasome-mediated IkappaB degradation results in inhibition of TNF-alpha induced IL-8 production in A549 cells by limiting NF-kappaB-mediated gene transcription.