Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase

BACKGROUND: Commercially available assays for IgE antibody provide results in international units per milliliter for many allergen extracts, but this is not easily achieved with purified or novel allergens. OBJECTIVE: To develop assays for IgE antibody suitable for purified or novel allergens by using a commercially available immunosorbent. METHODS: Streptavidin coupled to a high-capacity immunosorbent (CAP) was used to bind biotinylated purified allergens from mite (Der p 1 and Der p 2), cat (Fel d 1), and dog (Can f 1). Assays for IgE antibody to these allergens were performed on sera from children (asthma and control) as well as adults with atopic dermatitis. RESULTS: The results were validated by serial dilution of sera with high and low levels of IgE antibody and were quantitated in international units per milliliter by using a standard curve. Values for IgE antibody to Der p 1, Der p 2, and Fel d 1 correlated with values obtained with the allergen extracts (r2 = 0.80, 0.84, and 0.95, respectively; P < .001 in each case). Furthermore, the values for IgE antibody in sera from children with high exposure to mite and cat allergens demonstrated 10-fold higher levels of IgE antibody to Der p 1 and Der p 2 than to Fel d 1 (P < .001). CONCLUSION: The streptavidin immunosorbent technique provides a new method for quantifying IgE antibody to purified proteins. The results provide evidence about the high quantities of IgE antibody to purified inhalant allergens in patients with atopic dermatitis. In addition, the results demonstrate major differences in IgE antibodies specific for mite and cat allergens among children with high exposure to both allergens.     

Neu–Laxova syndrome presenting prenatally with increased nuchal translucency and cystic hygroma: The utility of exome sequencing in deciphering the diagnosis

Neu–Laxova syndrome (NLS) is a lethal autosomal recessive microcephaly syndrome associated with intrauterine growth restriction (IUGR) and multiple congenital anomalies. Clinical features include central nervous system malformations, joint contractures, ichthyosis, edema, and dysmorphic facial features. Biallelic pathogenic variants in either the PHGDH or PSAT1 genes have been shown to cause NLS. Using exome sequencing, we aimed to identify the underlying genetic diagnosis in three fetuses (from one family) with prenatal skin edema, severe IUGR, micrognathia, renal anomalies, and arthrogryposis and identified a homozygous c.1A>C (p.Met1?, NM_006623.3) variant in the PHGDH gene. Loss of the translation start codon is a novel genetic mechanism for the development of NLS. Prenatal diagnosis of NLS is challenging and few reports describe the fetal pathology. Fetal neuropathologic examination revealed: delayed brain development, congenital agenesis of the corticospinal tracts, and hypoplasia of the hippocampus, cerebellum and brainstem. Each pregnancy also showed increased nuchal translucency (NT) or cystic hygroma. While NLS is rare, it may be a cause of recurrent increased NT/cystic hygroma. This finding provides further support that cystic hygroma has many different genetic causes and that exome sequencing may shed light on the underlying genetic diagnoses in this group of prenatal patients.     

Discriminatory power and reproducibility of novel DNA typing methods for Mycobacterium tuberculosis complex strains

In recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110 restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods for Mycobacterium tuberculosis were investigated using the strain collection of the interlaboratory study of Kremer et al. This strain collection contains 90 M. tuberculosis complex and 10 non-M. tuberculosis complex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods for M. tuberculosis and that VNTR typing is the epidemiological typing method of choice for the near future.     

Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of β-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities. Received: 9 January / 5 March 1998     

Neuropsychological functions,sleep, and mental health in adults with Klinefelter syndrome

A few studies have examined neuropsychological functions, sleep, and mental health combined in Klinefelter syndrome (KS; 47,XXY). We investigated neuropsychological functions with standard tests, sleep with actigraphy, and self‐reported mental health in 30 men with KS (Mean age = 36.7 years) compared to 21 controls (Mean age = 36.8 years). Men with KS scored significantly lower on mental speed, attention span, working memory, inhibition, and set‐shifting tests, as well as overall IQ (mean effect size difference Cohen's d = 0.79). Men with KS had significantly longer night wakes, with no differences in other sleep variables (mean d = 0.34). Men with KS reported poorer mental health than controls (mean d = 1.16). Regression analyses showed neuropsychological functions explained variance in some sleep domains for men with KS but not for controls. Neuropsychological functions explained variance in some mental health domains for controls. For men with KS, however, verbal IQ was the only significant predictor of mental health. Altogether, men with KS display problems in neuropsychological functions and mental health but do not appear different from controls on most sleep parameters. Our findings indicate that relations between neuropsychological functions, sleep, and mental health differ between men with KS and controls.     

Kv1 channels selectively prevent dendritic hyperexcitability in rat Purkinje cells

Purkinje cells, the sole output of the cerebellar cortex, encode the timing signals required for motor coordination in their firing rate and activity pattern. Dendrites of Purkinje cells express a high density of P/Q-type voltage-gated calcium channels and fire dendritic calcium spikes. Here we show that dendritic subthreshold Kv1.2 subunit-containing Kv1 potassium channels prevent generation of random spontaneous calcium spikes. With Kv1 channels blocked, dendritic calcium spikes drive bursts of somatic sodium spikes and prevent the cell from faithfully encoding motor timing signals. The selective dendritic function of Kv1 channels in Purkinje cells allows them to effectively suppress dendritic hyperexcitability without hindering the generation of somatic action potentials. Further, we show that Kv1 channels also contribute to dendritic integration of parallel fibre synaptic input. Kv1 channels are often targeted to soma and axon and the data presented support a major dendritic function for these channels.     

Murine model of pulmonary anthrax: kinetics of dissemination, histopathology, and mouse strain susceptibility

Bioweapons are most often designed for delivery to the lung, although this route is not the usual portal of entry for many of the pathogens in the natural environment. Vaccines and therapeutics that are efficacious for natural routes of infection may not be effective against the pulmonary route. Pulmonary models are needed to investigate the importance of specific bacterial genes in virulence, to identify components of the host immune system that are important in providing innate and acquired protection, and for testing diagnostic and therapeutic strategies. This report describes the characteristics of host and Bacillus anthracis interactions in a murine pulmonary-infection model. The infective dose varied depending on the route and method of inoculation. The germination process in the lung began within 1 h of inoculation into the lung, although growth within the lung was limited. B. anthracis was found in the lung-associated lymph nodes approximately 5 h after infection. Minimal pneumonitis was associated with the lung infection, but significant systemic pathology was noted after dissemination. Infected mice typically succumbed to infection approximately 3 to 4 days after inoculation. The 50% lethal doses differed among inbred strains of mice, but within a given mouse strain, neither the age nor the sex of the mice influenced susceptibility to B. anthracis.     

Peptide-based approaches to treat asthma, arthritis, other autoimmune diseases and pathologies of the central nervous system

In this review we focus on peptide- and peptidomimetic-based approaches that target autoimmune diseases and some pathologies of the central nervous system. Special attention is given to asthma, allergic rhinitis, osteoarthritis, and Alzheimer's disease, but other related pathologies are also reviewed, although to a lesser degree. Among others, drugs like Diacerhein and its active form Rhein, Pralnacasan, Anakinra (Kineret), Omalizumab, an antibody "BION-1", directed against the common beta-chain of cytokine receptors, are described below as well as attempts to target beta-amyloid peptide aggregation. Parts of the review are also dedicated to targeting of pathologic conditions in the brain and in other tissues with peptides as well as methods to deliver larger molecules through the "blood--brain barrier" by exploring receptor-mediated transport, or elsewhere in the body by using peptides as carriers through cellular membranes. In addition to highlighting current developments in the field, we also propose, for future drug targets, the components of the inflammasome protein complex, which is believed to initiate the activation of caspase- 1 dependent signaling events, as well as other pathways that signal inflammation. Thus we discuss the possibility of targeting inflammasome components for negative or positive modulation of an inflammatory response.     

Persistent organochlorines, sedentary occupation, obesity and human male subfertility

BACKGROUND: Studies have suggested that the quality of human semen has been declining over recent decades, presumably because of lifestyle or environmental factors. METHODS: Polychlorinated biphenyls and organochlorine pesticides were analysed in the plasma of 25 men with poor semen quality, 20 men with normal semen quality and idiopathic subfertility and 27 men with normal semen quality and female factor subfertility. Samples of seminal fluid were also analysed to assess the relationship between the levels in blood and semen. RESULTS: The results indicate no difference in the levels of organochlorines between the groups. The levels of organochlorines in seminal fluid were proportional to the levels in plasma, but approximately 40 times lower. Men with poor semen quality were three times more likely to be obese than men with normal semen quality. There was also a significant negative correlation between semen quality parameters and body mass index among men with normal semen quality. The prevalence of sedentary work was lowest among men with the best semen quality. CONCLUSIONS: Poor semen quality was found to be associated with sedentary work and obesity but not with plasma levels of persistent organochlorines. More research is needed to assess whether sedentary lifestyle and obesity are causal factors in the decline of semen quality.