Detection of Active Infection in Nonhuman Primates with Lyme Neuroborreliosis: Comparison of PCR, Culture, and a Bioassay

Ideally a diagnosis of infection of the central nervous system (CNS) is made by culture of the etiologic pathogen, but Borrelia burgdorferi, the causative agent of Lyme neuroborreliosis (LNB), is rarely cultured from the cerebrospinal fluid (CSF). PCR and measurement of specific antibody in the CSF also have their limitations. The role of available assays for LNB has not been studied carefully in a comparative investigation. There is a need to assess the reliability of assays and to increase the ability to document active infection in the CNS. The recent development of the nonhuman primate (NHP) model of LNB allowed us to address this need in a faithful model of human LNB. In this study we compared the abilities of PCR and culture to detect the presence of spirochetes in the CSF and brain tissue of infected NHPs and related these measures of infection to the development of anti-B. burgdorferi antibody. We also tested a bioassay, the mouse infectivity test (MIT), in this model. Fourteen of 16 CSFs from four NHPs were positive by at least one of these techniques. Detection of spirochetes in the CSF by PCR, the MIT, and culture was inversely related to the concomitant presence of anti-B. burgdorferi antibody intrathecally. The performance of any particular test was associated with the strength of the host immune response. In early CNS infection, when anti-B. burgdorferi antibody had not yet appeared, or in immunocompromised hosts, the MIT compared favorably to culture and PCR for infected NHPs; antibody in the CSF was the most useful assay for immunocompetent NHPs.     

Conditional dendritic spike propagation following distal synaptic activation of hippocampal CA1 pyramidal neurons

The perforant-path projection to the hippocampus forms synapses in the apical tuft of CA1 pyramidal neurons. We used computer modeling to examine the function of these distal synaptic inputs, which led to three predictions that we confirmed in experiments using rat hippocampal slices. First, activation of CA1 neurons by the perforant path is limited, a result of the long distance between these inputs and the soma. Second, activation of CA1 neurons by the perforant path depends on the generation of dendritic spikes. Third, the forward propagation of these spikes is unreliable, but can be facilitated by modest activation of Schaffer-collateral synapses in the upper apical dendrites. This 'gating' of dendritic spike propagation may be an important activation mode of CA1 pyramidal neurons, and its modulation by neurotransmitters or long-term, activity-dependent plasticity may be an important feature of dendritic integration during mnemonic processing in the hippocampus.     

Comparison of the smoking behaviour and attitudes of smokers who attribute respiratory symptoms to smoking with those who do not.

General practitioners' (GPs') advice against smoking helps smokers to stop; unfortunately, GPs cannot predict which patients will quit following advice. This postal questionnaire survey suggests that where smokers attribute their respiratory symptoms to smoking, they are eight times (95% confidence interval [CI] = 3.0-23.3) more likely to believe that their health will improve if they stop smoking and six times (95% CI = 1.4-23.3) more likely to intend to stop smoking.     

Detection of N-myc gene amplification by fluorescence in situ hybridization. Diagnostic utility for neuroblastoma.

We assessed fluorescence in situ hybridization (FISH) as an alternative to Southern blot analysis for determination of N-myc gene amplification in neuroblastoma. In the 44 pediatric solid tumor cell lines examined (20 neuroblastomas), the mean number of N-myc copies determined by FISH correlated closely with Southern blot results. There was wide intercellular variability in gene copy number in tumors that had evidence of amplification; however, tumors judged to be non-amplified completely lacked any cells with high N-myc copy number. FISH provided reliable estimates of N-myc amplification in 12 clinical samples even when the percentage of tumor was low. The other advantages of FISH over Southern blot analysis were speed and technical simplicity, ability to discern heterogeneous gene amplification among tumor cells in the same specimen, and capacity to determine the source of the amplified N-myc signal, whether extrachromosomal double-minute chromosomes, expanded intrachromosomal regions, or chromosome 2 aneuploidy. We conclude that FISH would refine the analysis of N-myc amplification in neuroblastoma and thus improve the assignment of patients to prognostic groups based on this unfavorable risk factor.     

Molecular Manipulations of Extracellular Superoxide Dismutase: Functional Importance for Learning

Extracellular superoxide dismutase (EC-SOD) controls the availability of extracellular superoxide (O ₂ ^- ), which is important for a variety of physiological pathways, including the primary means of inactivating nitric oxide (NO). The role of EC-SOD in neurobehavioral function has been until now unexplored. In the current studies, the phenotypic expression of genotypic alterations of EC-SOD production in mice were characterized for spatial learning and memory. Dramatic impairments in spatial learning in the win-shift 8-arm radial maze were seen in both EC-SOD knockout mice and EC-SOD overexpressing mice. The EC-SOD overexpressing mice were further characterized as having significant deficits in a repeated acquisition task in the radial-arm maze, which permitted the dissociation of long and short-term learning. Long-term learning was significantly impaired by EC-SOD overexpression, whereas short-term learning was not significantly affected by EC-SOD overexpression. NO systems have been shown to be importantly involved in learning and memory. This may be important in the current studies because EC-SOD has primary control over the inactivation of NO. We found that EC-SOD overexpressing mice were resistant to the cognitive effects of L-NAME (N^G-nitro-L-arginine methyl ester hydrochloride), an NO synthase inhibitor. Decreased NO catabolism in these mice may have served to counter the effects of NOS inhibition by L-NAME. The current finding that EC-SOD levels that were either higher or lower than controls impaired learning demonstrates that the proper control of brain extracellular (O ₂ ^- ) may be more vital than merely reduction of brain extracelluar (O ₂ ^- ) in maintaining adequate learning function.     

Characteristics of bovine alveolar macrophage elastase

Lavage of isolated bovine lung lobes was used to retrieve pulmonary alveolar macrophages (PAM). Nominal yields of 72 million viable PAM were routinely obtained using 500 ml of calcium- and magnesium-free phosphate-buffered saline. Bovine PAM readily attached to glass coverslips and within 24 hours, provided cell monolayers consisting exclusively of macrophages. Bovine PAM synthesized and secreted a calcium-dependent elastase in serum-free media. Optimal proteolytic activity using a radiolabeled elastin substrate was observed at pH 7.6. The elastase was insensitive to synthetic peptide chloromethyl ketone elastase inhibitors and to the serine protease inhibitor, phenylmethylsulfonyl fluoride. Enzyme activity, however, was effectively inhibited by the metal chelator, ethylenediaminetetraacetic acid, or suppressed by inhibition of protein synthesis with cycloheximide.     

Kabuki syndrome with midgut malrotation and hyperinsulinemic hypoglycemia: A rare co‐occurrence from Thailand

Kabuki syndrome (KS) is a rare heterogeneous phenotypic genetic syndrome, characterized by hypotonia, developmental delay and/or intellectual disability with typical facial features. It is challenging to diagnose KS in newborn and young infant. We report a Thai girl who presented with two rare co‐occurrence phenotypes, hyperinsulinemic hypoglycemia and midgut malrotation. She had not have distinctive facial dysmorphism during neonatal period. At 4 months of age, she had poor weight gain with some facial features suggestive KS. Singleton whole exome sequencing (WES) was carried out followed by Sanger sequencing of the supposed variant. The result indicated a novel de novo heterozygous KMT2D mutation, c.15364A>T (p.Lys5122*), confirming KS. Our patient revealed rare clinical manifestations from the diverse population and address the benefit of WES in establishing early diagnosis of KS before typical facial gestalt exhibited, which allows timely and appropriate management to maximize developmental achievement.