Identification of a Campylobacter jejuni protein that cross-reacts with cholera toxin

The question of whether Campylobacter jejuni produces a cholera toxin-like toxin (CTLT) has been controversial. The objective of this study was to identify the factor that cross-reacts with CT from C. jejuni. Filtrates of C. jejuni grown in four different liquid media reported to promote CTLT production were tested by Chinese hamster ovary (CHO) cell elongation assay and for reactivity with CT antibody using GM1 ganglioside enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Protein sequence was determined by matrix-assisted laser desorption ionization-time of flight (MALDI TOF-TOF). Filtrates from seven reference strains reported to produce CTLT and from 80 clinical strains were negative in the CHO cell assay, but those from three reference strains and 16 clinical strains were positive by GM1 ELISA. All strains tested, including C. jejuni NCTC 11168, which does not contain a CT gene homologue, possessed a 53-kDa protein which reacted with CT antibody by immunoblotting. This band was identified as the major outer membrane protein, PorA, of C. jejuni. CT antibody reacted by immunoblotting with a recombinant PorA, but antibody to the recombinant PorA did not react with CT. Our results indicate that C. jejuni does not produce a functional CTLT, but the reactivity of PorA with CT antibody would lead to the erroneous conclusion that C. jejuni produces a functional CTLT.     

Stable micro-dystrophin gene transfer using an integrating adeno-retroviral hybrid vector ameliorates the dystrophic pathology in mdx mouse muscle

The ability to transfer the dystrophin gene stably to the skeletal muscle of DMD patients is a major confounding issue in establishing an effective gene therapy for this disease. To overcome this problem, we have examined the ability of muscle fibres from mdx mice to act as in situ factories of retroviral vector production. Tibialis anterior (TA) muscles from 4-week-old mdx mice were injected with an adenoviral vector expressing LacZ within a retroviral expression cassette (AdLZIN). Retroviral vector production was induced by the inclusion of two additional adenoviral vectors expressing retroviral gag-pol (AdGagPol) and 10A1 env genes (Ad10A1). Upon introduction of infected muscles into cell culture, colonies of beta-galactosidase-expressing myotubes formed only in cultures where the muscle was injected with AdLZIN, AdGagPol and Ad10A1, but not from muscle injected with AdLZIN only. Muscles from mdx/nude mice producing retroviral vector displayed a 4.6-fold increase in beta-galactosidase-positive myofibres after 1 month, compared with contralateral muscle in the same animal injected with AdLZIN and AdGagPol only. By constructing a hybrid adeno-retroviral vector expressing a truncated micro-dystrophin construct (AdmicroDyIN), we were able to partially correct the mdx dystrophic phenotype. AdmicroDyIN-mediated expression of micro-dystrophin in mdx TA muscle restored the formation of the dystrophin-associated glycoprotein complex and significantly reduced the level of muscle degeneration over uninjected controls. By stimulating in situ production of retroviral vector expressing micro-dystrophin, we achieved 92%+/-6% transduction of myofibres in the TA muscle by 4 weeks. Strikingly, by 3 months post injection, micro-dystrophin was still expressed to high levels in nearly all the myofibres of the TA muscle. By comparison, there was a pronounced drop in the levels of micro-dystrophin expressed by muscles injected with AdmicroDyIN only. Finally, using a novel PCR approach, we detected reverse-transcribed, integrated proviral sequences in TA muscle genomic DNA by 4 weeks post injection, the levels of which were found to increase after 3 months.     

Genetic enhancement of cognition in a kindred with cone-rod dystrophy due to RIMS1 mutation

Background

The genetic basis of variation in human cognitive abilities is poorly understood. RIMS1 encodes a synapse active‐zone protein with important roles in the maintenance of normal synaptic function: mice lacking this protein have greatly reduced learning ability and memory function.

Objective

An established paradigm examining the structural and functional effects of mutations in genes expressed in the eye and the brain was used to study a kindred with an inherited retinal dystrophy due to RIMS1 mutation.

Materials and methods

Neuropsychological tests and high‐resolution MRI brain scanning were undertaken in the kindred. In a population cohort, neuropsychological scores were associated with common variation in RIMS1. Additionally, RIMS1 was sequenced in top‐scoring individuals. Evolution of RIMS1 was assessed, and its expression in developing human brain was studied.

Results

Affected individuals showed significantly enhanced cognitive abilities across a range of domains. Analysis suggests that factors other than RIMS1 mutation were unlikely to explain enhanced cognition. No association with common variation and verbal IQ was found in the population cohort, and no other mutations in RIMS1 were detected in the highest scoring individuals from this cohort. RIMS1 protein is expressed in developing human brain, but RIMS1 does not seem to have been subjected to accelerated evolution in man.

Conclusions

A possible role for RIMS1 in the enhancement of cognitive function at least in this kindred is suggested. Although further work is clearly required to explore these findings before a role for RIMS1 in human cognition can be formally accepted, the findings suggest that genetic mutation may enhance human cognition in some cases.A genetic contribution to variation in human intelligence is well established, but the identities of the genes responsible remain elusive. Many mutations are associated with impaired cognition:¹ no definite genetic causes of enhanced cognition are established,² and there are no known cognition‐enhancing “gain‐of‐function” mutations in genes otherwise associated with cognitive impairment. Therapeutic possibilities deriving from the discovery of any such genes or variants are potentially important: cognitive decline reduces the quality of life,³ and low intelligence test scores are associated with increased morbidity and shorter life‐span.⁴ Accelerated evolution of genes subserving neurodevelopment figures in molecular explanations of the advance of the human nervous system: many of the identified genes regulate brain size and behaviour, some encoding critical synaptic proteins.⁵To identify genes influencing human brain development and function, including cognitive function, we use a paradigm evaluating cerebral structure and function in individuals with known mutations in genes co‐expressed in the lineage‐sharing eye and brain, ascertained by their obvious ocular phenotype, but in whom a neurological phenotype was not fully appreciated. Using this paradigm, we demonstrated roles for the genes PAX6, PITX2, SOX2 and OTX2 in human brain development, cognitive function and memory.^{6,7,8,9,10,11}We now report on the functional and structural effects of mutation in the eye‐ and brain‐expressed gene RIMS1, through the study of individuals from a family already reported to have retinal dystrophy caused by RIMS1 mutation.^12,13 To our knowledge, this is the only family so far reported with such a mutation: the eye phenotype is homogeneous in the family, and has been documented in detail.¹³ The orthologous murine Rim1α encodes a synaptic active‐zone protein necessary for preserving the normal probability of synaptic neurotransmitter release and for long‐term presynaptic potentiation.^14,15Rim1α is also expressed in retinal ribbon synapses.¹⁶ Mice lacking Rim1α protein show severely impaired learning and memory.¹⁷ In our kindred, RIMS1 mutation (Arg844His) causes a late‐onset dominantly inherited cone–rod dystrophy (CORD7; OMIM 603649), leading to varying degrees of visual loss starting from the third decade onwards.¹³ Because RIMS1/Rim1α is also expressed in the brain, we hypothesised that this RIMS1 mutation would produce a structural and functional neurological phenotype.     

The chemokine SDF-1, stromal cell-derived factor 1, attracts early stage B cell precursors via the chemokine receptor CXCR4

In the bone marrow, progenitor (pro-) and precursor (pre-) B cells depend on close contact with stromal cells for growth and maturation. Stromal cell-derived factor 1 (SDF-1), also known as pre-B cell growth-stimulating factor, is produced by bone marrow stromal cells and was reported to act together with interleukin-7 as co-mitogen for pre-B cells. SDF-1 was recently shown to be a chemokine which is chemotactic for different types of leukocytes and acts via the chemokine receptor CXCR4. Using sorted B220⁺ bone marrow cells and several B cell lines characteristic for different stages of B lymphopoiesis, we now show that SDF-1 is a potent attractant for pro- and pre-B cells, but is inactive on B cells at later stages of development. In early B cell precursors, SDF-1 induced intracellular Ca²⁺ mobilization and in vitro migration with a potency and efficacy similar to that observed for chemokines acting on blood leukocytes. These responses were mediated via CXCR4 as they could be inhibited by an anti-receptor antibody. SDF-1 is the first chemokine shown to act on early-stage B cell precursors. Mice lacking SDF-1 die perinatally and show a severe deficiency in B lymphopoiesis. We propose that SDF-1 released from the stromal cells exerts its critical hematopoietic function by selectively attracting and confining early B cell precursors within the bone marrow microenvironment that provides the necessary factors for growth and differentiation.     

Rapid Detection of Rare Deleterious Variants by Next Generation Sequencing with Optional Microarray SNP Genotype Data

Autozygosity mapping is a powerful technique for the identification of rare, autosomal recessive, disease‐causing genes. The ease with which this category of disease gene can be identified has greatly increased through the availability of genome‐wide SNP genotyping microarrays and subsequently of exome sequencing. Although these methods have simplified the generation of experimental data, its analysis, particularly when disparate data types must be integrated, remains time consuming. Moreover, the huge volume of sequence variant data generated from next generation sequencing experiments opens up the possibility of using these data instead of microarray genotype data to identify disease loci. To allow these two types of data to be used in an integrated fashion, we have developed AgileVCFMapper, a program that performs both the mapping of disease loci by SNP genotyping and the analysis of potentially deleterious variants using exome sequence variant data, in a single step. This method does not require microarray SNP genotype data, although analysis with a combination of microarray and exome genotype data enables more precise delineation of disease loci, due to superior marker density and distribution.     

Oncogenic S1P signalling in EBV‐associated nasopharyngeal carcinoma activates AKT and promotes cell migration through S1P receptor 3

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein–Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine‐1‐phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV‐encoded latent genes (EBNA1, LMP1, and LMP2A) can up‐regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is overexpressed in EBV‐positive NPC patient‐derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P‐induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Sulfolipid deficiency does not affect the virulence of Mycobacterium tuberculosis H37Rv in mice and guinea pigs

Lipids that are found only in the cell envelope of pathogenic mycobacteria, such as those containing multiple methyl-branched fatty acids, have long been thought to play a role in pathogenesis. Among these complex lipids, sulfolipids have been the most extensively studied over the last 50 years. The numerous biological effects exhibited by purified sulfolipids on phagocytic cells led to the idea that these molecules are probably important virulence factors facilitating the intracellular survival of Mycobacterium tuberculosis. However, definitive evidence to support this concept has been lacking. The recent construction of an isogenic sulfolipid-deficient mutant of M. tuberculosis H37Rv (Sirakova et al., J. Biol. Chem. 276:16833-16839, 2001) has for the first time provided the opportunity to directly assess the contribution of these complex lipids to pathogenesis. In the present study, we show that against all expectations, sulfolipid deficiency does not significantly affect the replication, persistence, and pathogenicity of M. tuberculosis H37Rv in mice and guinea pigs or in cultured macrophages.     

Architecture of secondary lymphoid tissue in sheep experimentally challenged with scrapie

Scrapie is a transmissible spongiform encephalopathy in which there is an accumulation of the abnormal form of the prion protein, PrPsc, in the lymphoreticular system and nervous system. There is a particular accumulation of PrPsc on follicular dendritic cells within the germinal centre of B-cell follicles. Because accumulation of PrPsc in the nervous system leads to neuronal cell loss we have examined PrPsc accumulation in the prescapular and mesenteric lymph nodes in relation to lymph node architecture of scrapie-challenged sheep. We demonstrate that an accumulation of PrPsc in the lymph node fails to result in gross defects in the microanatomy and phenotype of T- and B-cell areas in the lymph nodes.     

Acute exercise decreases airway inflammation, but not responsiveness, in an allergic asthma model

Previous studies have suggested that the asthmatic responses of airway inflammation, remodeling, and hyperresponsiveness (AHR) are interrelated; in this study, we used exercise to examine the nature of this interrelationship. Mice were sensitized and challenged with ovalbumin (OVA); mice were then exercised via running on a motorized treadmill at a moderate intensity. Data indicate that, within the lungs of OVA-treated mice, exercise attenuated the production of inflammatory mediators, including chemokines KC, RANTES, and MCP-1 and IL-12p40/p80. Coordinately, OVA-treated and exercised mice displayed decreases in leukocyte infiltration, including eosinophils, as compared with sedentary controls. Results also show that a single bout of exercise significantly decreased phosphorylation of the NFkappaB p65 subunit, which regulates the gene expression of a wide variety of inflammatory mediators. In addition, OVA-treated and exercised mice exhibited decreases in the levels of Th2-derived cytokines IL-5 and IL-13 and the prostaglandin PGE(2), as compared with sedentary controls. In contrast, results show that a single bout of exercise had no effect on AHR in OVA-treated mice challenged with increasing doses of aerosolized methacholine (0-50 mg/ml) as compared with sedentary mice. Exercise also had no effect on epithelial cell hypertrophy, mucus production, or airway wall thickening in OVA-treated mice as compared with sedentary controls. These findings suggest that a single bout of aerobic exercise at a moderate intensity attenuates airway inflammation but not AHR or airway remodeling in OVA-treated mice. The implication of these findings for the interrelationship between airway inflammation, airway remodeling, and AHR is discussed.