Background. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA/RNA analogues that silence expression of specific genes. We studied whether PPMOs targeted to essential genes in Acinetobacter lwoffii and Acinetobacter baumannii are active in vitro and in vivo.
Methods. PPMOs were evaluated in vitro using minimum inhibitory concentration (MIC) and viability assays, and in vivo using murine pulmonary infection models with intranasal PPMO treatment.
Results. MICs of PPMOs ranged from 0.1 to 64 µM (approximately 0.6–38 µg/mL). The most effective PPMO tested was (RXR)4-AcpP, which is targeted to acpP. (RXR)4-AcpP reduced viability of A. lwoffii and A. baumannii by >103 colony-forming units/mL at 5–8 times MIC. Mice treated with ≥0.25 mg/kg of (RXR)4-AcpP survived longer and had less inflammation and bacterial lung burden than mice treated with a scrambled-sequence PPMO or phosphate-buffered saline. Treatment could be delayed after infection and still increase survival.
Conclusions. PPMOs targeted to essential genes of A. lwoffii and A. baumannii were bactericidal and had MICs in a clinically relevant range. (RXR)4-AcpP increased survival of mice infected with A. lwoffii or A. baumannii, even when initial treatment was delayed after infection. PPMOs could be a viable therapeutic approach in dealing with multidrug-resistant Acinetobacter species.
Acinetobacter; lwoffii; baumannii; MIC; antisense; PPMO; mouse; infection; respiratory infection; phosphorodiamidate morpholino oligomer
In a randomized double-blind trial in healthy Israeli infants in Israel who received the 13-valent or 7-valent pneumococcal conjugate vaccine (PCV13 or PCV7, respectively) at 2, 4, 6, and 12 months, PCV13 significantly reduced nasopharyngeal (NP) colonization of serotypes 1, 6A, 7F, 19A, cross-reacting 6C, and the common PCV7 serotype 19F, from ages 7 to 24 months. No differences were observed between the vaccine groups for serotype 3 or for the remaining common PCV7 serotypes. For serotype 5, too few events were observed to draw an inference. Generally consistent with these findings, PCV13 elicited significantly higher enzyme-linked immunosorbent assay (ELISA) IgG-binding antibody responses than did PCV7 for the additional PCV13 serotypes 1, 3, 5, 6A, 7F, 19A, and for the common serotype 19F, with similar or lower responses for the remaining common serotypes. To further assess immunogenicity and colonization, we conducted a post hoc analysis of PCV13 functional antibody responses measured by opsonophagocytic activity (OPA) assays in a randomly selected subset of subjects. The pattern of functional antibody OPA responses elicited by PCV13 relative to PCV7 was similar to that of the ELISA anticapsular IgG-binding antibody responses described above. In addition, the OPA responses generally correlated positively with IgG responses for all 13 serotypes among the PCV13 recipients and for all 7 common serotypes and the additional serotype 6A but not for 19A or the other serotypes unique to PCV13 among the PCV7 recipients. This post hoc analysis supports an association between serum OPA functional and IgG-binding antibody levels, allowing for a transfer of inferred associations between IgG responses and NP colonization to OPA responses.
Adenitis for which no causative organism can be isolated is a common occurrence in patients with chronic granulomatous disease (CGD). Here we identify Acidomonas methanolica as a pathogen associated with adenitis in a patient with CGD.
The causative pathogen was obtained after prolonged incubation of an excised lymph node in thioglycolate broth. Identification was carried out by sequencing the 16s rRNA. Immunoblots were prepared utilizing protein extracts from the case patient’s A. methanolica isolate, an ATCC type strain of A. methanolica and G. bethesdensis.
Fastidious gram-negative rods grew after prolonged incubation of an excised lymph node in thioglycolate broth. Sequencing of the 16s rRNA identified the organism as A. methanolica. Immunoblot confirmed the pathogen’s role in the patient’s adenitis by showing the patient’s specific immune response to the organism.
A. methanolica is the second member of the family, Acetobacteaceae to be associated with adenitis in patients with CGD.
Chronic granulomatous disease; Acidomonas methanolica
To realize the full potential of next-generation sequencing, it is important to consider multiple sources of genetic information, including inheritance, association, and bioinformatics. To illustrate the promise of such an approach, we applied our next-generation linkage and association (NGLA) methods to the sequence data of a large 57-member Mexican American family with hypertension. Our results show that OSBPL10--a disease susceptibility gene for dyslipidemia--may also influence systolic blood pressure (SBP). In particular, our NGLA dense single-nucleotide polymorphism (SNP) analysis identified a 2.5-megabase (Mb) region that strongly cosegregates with low SBP (maximum posterior probability of linkage [PPL] = 68%). Furthermore, using the posterior probability of linkage disequilibrium (PPLD), we fine-mapped this region and identified 12 SBP-associated variants (PPLD ranging between 4% and 14%) that comprise a rare, 4-site haplotype. This haplotype extends into the candidate gene, OSBPL10 (oxysterol-binding protein-like 10). In contrast to our NGLA methods, a commonly used filter-based approach identified 23 variants with little evidence for spatial clustering around any particular gene or region of interest.
Impaired signaling in the IFN-γ/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis.
We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections.
PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-γ/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated.
We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-γ–induced gene expression, but we found impaired responses to IFN-γ restimulation.
Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-γ–mediated inflammation.
Signal transducer and activator of transcription 1; IFN-γ; progressive multifocal leukoencephalopathy; Histoplasma capsulatum; Coccidioides immitis; thrush
Vascular endothelial growth factors (VEGFs) have been shown to participate in atherosclerosis, arteriogenesis, cerebral edema, neuroprotection, neurogenesis, angiogenesis, postischemic brain and vessel repair, and the effects of transplanted stem cells in experimental stroke. Most of these actions involve VEGF-A and the VEGFR-2 receptor, but VEGF-B, placental growth factor, and VEGFR-1 have been implicated in some cases as well. VEGF signaling pathways represent important potential targets for the acute and chronic treatment of stroke.
vascular endothelial growth factor; stroke; ischemia; neuroprotection; neurogenesis; angiogenesis
Although the enhanced expression of VEGF in the brains of patients with Alzheimer’s disease (AD) has been reported, the functional significance of VEGF level in the progression of AD is still unclear. We examined the VEGF expression in the hippocampus of patients with AD at different stages of progression by Western blot, and found that VEGF (VEGF189) was barely detectable in normal hippocampus, but significantly increased at the early stage of patients with AD. VEGF189 was decreased with advancing stages of AD. Immunostaining shows that VEGF was significantly increased in the cells in the CA1, CA3 and dentate gyrus regions of hippocampus and the layer III and V of entorhinal cortex of patient with AD, compared to normal brain. Confocal images show that VEGF was predominantly expressed in neurons and astrocyte in the hippocampus and entorhinal cortex of patients with AD. Our data suggest that VEGF level is associated with progressive loss of cognitive function in patients with AD.
VEGF; Alzheimer’s disease; Human; brain; expression
MicroRNAs (miRNAs) can repress multiple targets, but how a single de-balanced interaction affects others remained unclear. We found that changing a single miRNA–target interaction can simultaneously affect multiple other miRNA–target interactions and modify physiological phenotype. We show that miR-608 targets acetylcholinesterase (AChE) and demonstrate weakened miR-608 interaction with the rs17228616 AChE allele having a single-nucleotide polymorphism (SNP) in the 3′-untranslated region (3′UTR). In cultured cells, this weakened interaction potentiated miR-608-mediated suppression of other targets, including CDC42 and interleukin-6 (IL6). Postmortem human cortices homozygote for the minor rs17228616 allele showed AChE elevation and CDC42/IL6 decreases compared with major allele homozygotes. Additionally, minor allele heterozygote and homozygote subjects showed reduced cortisol and elevated blood pressure, predicting risk of anxiety and hypertension. Parallel suppression of the conserved brain CDC42 activity by intracerebroventricular ML141 injection caused acute anxiety in mice. We demonstrate that SNPs in miRNA-binding regions could cause expanded downstream effects changing important biological pathways.
Detecting gene-gene interaction in complex diseases is a major challenge for common disease genetics. Most interaction detection approaches use disease-marker associations and such methods have low power and unknown reliability in real data. We developed and tested a powerful linkage-analysis-based gene-gene interaction detection strategy based on conditioning the family data on a known disease-causing allele or disease-associated marker allele. We computer-generated multipoint linkage data for a disease caused by two epistatically interacting loci (A and B). We examined several two-locus epistatic inheritance models: dominant-dominant, dominant-recessive, recessive-dominant, recessive-recessive. At one of the loci (A), there was a known disease-related allele. We stratified the family data on the presence of this allele, eliminating family members who were without it. This elimination step has the effect of raising the “penetrance” at the second locus (B). We then calculated the lod score at the second locus (B) and compared the pre- and post-stratification lod scores at B. A positive difference indicated interaction. We also examined if it was possible to detect interaction with locus B based on a disease-marker association (instead of an identified disease allele) at locus A. We also tested whether the presence of genetic heterogeneity would generate false positive evidence of interaction. The power to detect interaction for a known disease allele was 60–90%. The probability of false positives, based on heterogeneity, was low. Decreasing linkage disequilibrium between the disease and marker at locus A decreased the likelihood of detecting interaction. The allele frequency of the associated marker made little difference to the power.
The Burkholderia cepacia complex (BCC) is a group of closely related bacteria that are responsible for respiratory infections in immunocompromised humans, most notably those with cystic fibrosis (CF). We report the genome sequences for Burkholderia cenocepacia ET12 lineage CF isolates K56-2 and BC7.
Neuroglobin is a hypoxia-inducible O2-binding protein with neuroprotective effects in cell and animal models of stroke and Alzheimer’s disease. The mechanism underlying neuroglobin’s cytoprotective action is unknown, although several possibilities have been proposed, including antioxidative and antiapoptotic effects. We used affinity purification-mass spectrometry methods to identify neuroglobin-interacting proteins in normoxic and hypoxic murine neuronal (HN33) cell lysates, and to compare these interactions with those of a structurally and functionally related protein, myoglobin. We report that the protein interactomes of neuroglobin and myoglobin overlap substantially and are modified by hypoxia. In addition, neuroglobin-interacting proteins include partners consistent with both antioxidative and antiapoptotic functions, as well as with a relationship to several neurodegenerative diseases.
hypoxia; ischemia; myoglobin; neuroglobin; proteome
An overview of miRNAs altered in Alzheimer's disease (AD) was established by profiling the hippocampus of a cohort of 41 late-onset AD (LOAD) patients and 23 controls, showing deregulation of 35 miRNAs. Profiling of miRNAs in the prefrontal cortex of a second independent cohort of 49 patients grouped by Braak stages revealed 41 deregulated miRNAs. We focused on miR-132-3p which is strongly altered in both brain areas. Downregulation of this miRNA occurs already at Braak stages III and IV, before loss of neuron-specific miRNAs. Next-generation sequencing confirmed a strong decrease of miR-132-3p and of three family-related miRNAs encoded by the same miRNA cluster on chromosome 17. Deregulation of miR-132-3p in AD brain appears to occur mainly in neurons displaying Tau hyper-phosphorylation. We provide evidence that miR-132-3p may contribute to disease progression through aberrant regulation of mRNA targets in the Tau network. The transcription factor (TF) FOXO1a appears to be a key target of miR-132-3p in this pathway.
Alzheimer's disease; hippocampus; prefrontal cortex; microRNA; miR-132-3p
Background. Granulibacter bethesdensis is a recently described member of the Acetobacteraceae family that has been isolated from patients with chronic granulomatous disease (CGD). Its pathogenesis, environmental reservoir(s), and incidence of infection among CGD patients and the general population are unknown.
Methods. Detected antigens were identified by mass spectroscopy after 2-dimensional electrophoresis and immunoaffinity chromatography. The prevalence of Granulibacter immunoreactivity was assessed through immunoblotting and enzyme-linked immunosorbent assay (ELISA).
Results. Methanol dehydrogenase (MDH) and formaldehyde-activating enzyme were recognized during analysis of sera from infected patients. Unique patterns of immunoreactive bands were identified in Granulibacter extracts, compared with extracts of other Acetobacteraceae species. By use of criteria based on these specific bands, specimens from 79 of 175 CGD patients (45.1%) and 23 of 93 healthy donors (24.7%) reacted to all 11 bands. An ELISA that used native MDH to capture and detect immunoglobulin G was developed and revealed high-titer MDH seroreactivity in culture-confirmed cases and 5 additional CGD patients. Testing of samples collected prior to culture-confirmed infection demonstrated instances of recent seroconversion, as well as sustained seropositivity. Infection of CGD mice with G. bethesdensis confirmed acquisition of high-titer antibody-recognizing MDH.
Conclusions. These serologic tests suggest that Granulibacter immunoreactivity is more common among CGD patients and, perhaps, among healthy donors than was previously suspected. This finding raises the possibility that clinical presentations of Granulibacter infection may be underappreciated.
Despite enormous data collection and analysis efforts, the genetic
influences on common epilepsies remain mostly unknown. We propose that reasons
for the lack of progress can be traced to three factors: (1) A reluctance to
consider fine-grained phenotype definitions based on extensive and carefully
collected clinical data; (2) the pursuit of genetic analysis methods that are
popular but poorly conceived and are inadequate to the task of resolving the
problems inherent in common disease studies; (3) preconceived ideas about the
genetic mechanisms that cause epilepsy (which we have discussed elsewhere). We
propose a paradigm for finding epilepsy-related loci and alleles that has proven
successful in other common diseases.
Exome; Genome-wide association; Linkage analysis; Phenotype; Single nucleotide polymorphisms; Whole genome sequencing
Postlicensure surveillance of pneumonia incidence can be used to estimate whether pneumococcal conjugate vaccines (PCVs) affect incidence. We used Poisson regression models that control for baseline seasonality to determine the impact of PCVs and the possible effects of variations in virus activity in Israel on these surveillance estimates. PCV was associated with significant declines in radiologically confirmed alveolar pneumonia (RCAP) among patients <6 months, 6–17 months, and 18–35 months of age (–31% [95% CI –51% to –15%], –41% [95% CI –52 to –32%], and –34% [95% CI –42% to –25%], respectively). Respiratory syncytial virus (RSV) activity was associated with strong increases in RCAP incidence, with up to 44% of cases attributable to RSV among infants <6 months of age and lower but significant impacts in older children. Seasonal variations, particularly in RSV activity, masked the impact of 7-valent PCVs, especially for young children in the first 2 years after vaccine introduction.
pneumococcal conjugate vaccines; pneumonia; RSV; influenza; regression model; surveillance; viruses; Israel; respiratory syncytial virus; alveolar pneumonia
Vascular endothelial growth factor (VEGF) and neuroglobin (Ngb) participate in neuronal responses to hypoxia and ischemia, but the relationship between their effects, if any, is unknown. To address this issue, we measured Ngb levels in VEGF-treated mouse cerebrocortical cultures and VEGF levels in cerebrocortical cultures from Ngb-overexpressing transgenic mice. VEGF stimulated Ngb expression in a VEGFR2/Flk1 receptor-dependent manner, whereas Ngb overexpression suppressed expression of VEGF. These findings provide further insight into hypoxia-stimulated neuronal signaling pathways.
Vascular endothelial growth factor; neuroglobin; hypoxia
Clinical studies suggest a correlation between changes in activity of the contralesional cerebral cortex and spontaneous recovery from stroke, but whether this is a causal relationship is uncertain.
Young adult Sprague-Dawley male rats underwent unilateral or bilateral permanent distal middle cerebral artery occlusion (dMCAO). Infarct volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 hr after dMCAO, and functional outcome was assessed 1–28 days after dMCAO using the ladder rung walking and limb placing tests.
Infarct volume was unchanged, but functional neurological deficits were reduced 1 day after bilateral compared to unilateral dMCAO.
Activity in the contralesional cerebral cortex may inhibit functional motor recovery acutely after experimental stroke.
Stroke; ischemia; recovery; rat
Burkholderia multivorans is a Gram-negative bacterium and a member of the Burkholderia cepacia complex, which is frequently associated with respiratory infections in people with cystic fibrosis (CF) and chronic granulomatous disease (CGD). We are reporting the genome sequences of 4 B. multivorans strains, 2 from CF patients and 2 from CGD patients.
Vascular endothelial growth factor-B (VEGF-B) protects against experimental stroke, but the effect of stroke on VEGF-B expression is uncertain.
We examined VEGF-B expression by immunohistochemistry in the ischemic border zone 1–7 days after middle cerebral artery occlusion in rats.
VEGF-B immunoreactivity in the border zone was increased after middle cerebral artery occlusion and was associated with neurons and macrophages/microglia, but not astrocytes or endothelial cells.
These findings provide additional evidence for a role of VEGF-B in the endogenous response to cerebral ischemia.
Vascular endothelial growth factor-B (VEGF-B); Stroke; Ischemia
The more than 120 genotypes of human enteroviruses (HEVs) reflect a wide range of evolutionary divergence, and there are 23 currently classified as human enterovirus C species (HEV-C). Two new HEV-C (EV-C117 and EV-C118) were identified in the Community-Acquired Pneumonia Pediatric Research Initiative (CAP-PRI) study, and the present paper describes the characterisation of the complete genome of one EV-C117 strain (LIT22) and two EV-C118 (ISR38 and ISR10) strains. The EV-C117 and EV-C118 5′UTR sequences were related to those of EV-C104, EV-C105 and EV-C109, and were slightly shorter than those of other HEV A-D species. Similarity plot analyses showed that EV-C117 and EV-C118 have a P1 region that is highly divergent from that of the other HEV-C, and phylogenetic analyses highly supported a monophyletic group consisting of EV-C117, EV-C118, EV-C104, EV-C105 and EV-C109 strains. Phylogenetic, Simplot and Bootscan analyses indicated that recombination was not the main mechanism of EV-C117 and EV-C118 evolution, thus strengthening the hypothesis of the monophyletic origin of the coding regions, as in the case of other HEV-C. Phylogenetic analysis also revealed the emergence of a new group within HEV-C that is divided into two subgroups. Nucleotide and amino acid identity in VP1 sequences have been established as useful criteria for assigning new HEV types, but analysis of the complete P1 region improves resolution.
Neurobrucellosis is an uncommon complication of pediatric brucellosis. Acute meningitis and encephalitis are the most common clinical manifestations, however symptoms may be protean and diagnosis requires a high index of suspicion in patients from endemic areas. Diagnosis is often based on neurological symptoms, serology, and suggestive brain imaging because cerebrospinal fluid culture yields are low. Two cases of pediatric neurobrucellosis with unusual clinical and radiologic findings are presented.
The new enterovirus C strain EV-C118 belongs to the human enterovirus C species of the Picornaviridae family. We report the complete genome sequence of this strain, which was identified in respiratory specimens of two children hospitalized in Israel because of acute otitis media and community-acquired pneumonia who were enrolled in the Community-Acquired Pneumonia Pediatric Research Initiative (CAP-PRI) study.