Genetic analysis of self-associating immunoglobulin G rheumatoid factors from two rheumatoid synovia implicates an antigen-driven response.

Although much has been learned about the molecular basis of immunoglobulin M (IgM) rheumatoid factors (RFs) in healthy individuals and in patients with mixed cryoglobulinemia and rheumatoid arthritis, little is known about the genetic origins of the potentially pathogenic IgG RFs in the inflamed rheumatoid synovia of patients. Recently, we generated from unmanipulated synovium B cells several hybridomas that secreted self-associating IgG RFs. To delineate the genetic origins of such potentially pathogenic RFs, we adapted the anchored polymerase chain reaction to rapidly clone and characterize the expressed Ig V genes for the L1 and the D1 IgG RFs. Then, we identified the germline counterparts of the expressed L1 IgG RF V genes. The results showed that the L1 heavy chain was encoded by a Vh gene that is expressed preferentially during early ontogenic development, and that is probably located within 240 kb upstream of the Jh locus. The overlap between this RF Vh gene and the restricted fetal antibody repertoire is reminiscent of the natural antibody-associated Vh genes, and suggests that at least part of the "potential pathogenic" IgG RFs in rheumatoid synovium may derive from the "physiological" natural antibody repertoire in a normal immune system. Indeed, the corresponding germline Vh gene for L1 encodes the heavy chain of an IgM RF found in a 19-wk-old fetal spleen. Furthermore, the comparisons of the expressed RF V genes and their germline counterparts reveal that the L1 heavy and light chain variable regions had, respectively, 16 and 7 somatic mutations, which resulted in eight and four amino acid changes. Strikingly, all eight mutations in the complementarity determining regions of the V gene-encoded regions were replacement changes, while only 6 of 11 mutations in the framework regions caused amino acid changes. Combined with L1's high binding affinity toward the Fc fragment, these results suggest strongly that the L1 IgG RF must have been driven by the Fc antigen

Neurochemical coding compared between varicose axons and cell bodies of myenteric neurons in the guinea-pig ileum

This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/.The discrete functional classes of enteric neurons in the mammalian gastrointestinal tract have been successfully distinguished on the basis of the unique combination of molecules and enzymes in their cell bodies (“chemical coding”). Whether the same chemical coding exists in varicose axons of different functional classes has not been systematically tested. In this study, we quantified the coexistence of markers that define classes of nerve cell bodies in the myenteric plexus of the guinea-pig ileum, in varicose axons of the same neurons. Profound differences between the combinations of immunohistochemical markers in myenteric nerve cell bodies and in their varicosities were identified. These discrepancies were particularly notable for classes of neurons that had previously been classified as cholinergic, based on immunoreactivity for choline acetyltransferase (ChAT) in their cell bodies. To detect cholinergic varicose axons of enteric neurons in this study, we used antiserum against the vesicular acetylcholine transporter (VAChT). ChAT-immunoreactivity has been reported to be consistently co-localized with 5-hydroxytryptamine (5-HT) in interneuronal cell bodies, yet only 29 ± 5% (n = 4) of 5-HT-immunoreactive varicosities contained vesicular acetylcholine transporter (VAChT). Somatostatin coexists with ChAT-immunoreactivity in a class of descending interneuron but only 21 ± 1% (n = 4) of somatostatin-immunoreactive varicosities were VAChT-immunoreactive. Comparable discrepancies were also noted for non-cholinergic markers. The results suggest that chemical coding of cell bodies does not necessarily reflect chemical coding of varicose axon terminals and that the assumption that nerve cell bodies that contain ChAT are functionally cholinergic may be questionable.Australian National Health & Medical Research Counci

Sensory innervation of the guinea pig colon and rectum compared using retrograde tracing and immunohistochemistry.

Embargoed until 2 April 2017 as per publisher's policy

Failure to activate the IFN-beta promoter by a paramyxovirus lacking an interferon antagonist

It is generally thought that pathogen-associated molecular patterns (PAMPs) responsible for triggering interferon (IFN) induction are produced during virus replication and, to limit the activation of the IFN response by these PAMPs, viruses encode antagonists of IFN induction. Here we have studied the induction of IFN by parainfluenza virus type 5 (PIV5) at the single-cell level, using a cell line expressing GFP under the control of the IFN-β promoter. We demonstrate that a recombinant PIV5 (termed PIV5-VΔC) that lacks a functional V protein (the viral IFN antagonist) does not activate the IFN-β promoter in the majority of infected cells. We conclude that viral PAMPs capable of activating the IFN induction cascade are not produced or exposed during the normal replication cycle of PIV5, and suggest instead that defective viruses are primarily responsible for inducing IFN during PIV5 infection in this syste

Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-LeX identified in a previous glycan microarray screen

Between-days intra-rater reliability with a hand held myotonometer to quantify muscle tone in the acute stroke population

A myotonometer can objectively quantify changes in muscle tone. The between-days intra-rater reliability in a ward setting for the acute stroke population remains unknown. This study aimed to investigate the device’s between-days intra-rater reliability when used in a ward setting for acute stroke participants. Muscle tone of biceps brachii, brachioradialis, rectus femoris, and tibialis anterior was recorded in the ward at bedside by one physiotherapist on two consecutive days. This study included participants who were within 1 month of their first stroke occurrence. Participants who were medically unstable or who suffered from brain stem injury were excluded. Reliability was assessed by the intraclass correlation coefficient (ICC), standard error of measurement (SEM), smallest real difference (SRD), and the Bland-Altman limits of agreement. The results indicated excellent between-days intra-rater reliability (ICC > 0.75). SEM and SRD show small differences between measurements. The Bland-Altman analysis indicated a tendency of overestimation of the rectus femoris. MyotonPRO demonstrated acceptable reliability when used in a ward setting in those patients with acute stroke. However, results should be interpreted with caution, due to the limitations of the study and the varying level of consistency observed between different muscles

Simple, Robust, and Plasticizer-Free Iodide-Selective Sensor Based on Copolymerized Triazole-Based Ionic Liquid

Novel solid-contact iodide-selective electrodes based on covalently attached 1,2,3 triazole ionic liquid (IL) were prepared and investigated in this study. Triazole-based IL moieties were synthesized using click chemistry and were further copolymerized with lauryl methacrylate via a simple one-step free radical polymerization to produce a "self-plasticized" copolymer. The mechanical properties of the copolymer are suitable for the fabrication of plasticizer-free ion-selective membrane electrodes. We demonstrate that covalently attached IL moieties provide adequate functionality to the ion-selective membrane, thus achieving a very simple, one-component sensing membrane. We also demonstrate that the presence of iodide as the counterion in the triazole moiety has direct influence on the membrane's functionality. Potentiometric experiments revealed that each electrode displays high selectivity toward iodide anions over a number of inorganic anions. Moreover, the inherent presence of the iodide in the membrane reduces the need for conditioning. The nonconditioned electrodes show strikingly similar response characteristics compared to the conditioned ones. The electrodes exhibited a near Nernstian behavior with a slope of -56.1 mV per decade across a large concentration range with lower detection limits found at approximately 6.3 × 10(-8) M or 8 ppb. These all-solid-state sensors were utilized for the selective potentiometric determination of iodide ions in artificial urine samples in the nanomolar concentration range

Neurochemical characterization of extrinsic nerves in myenteric ganglia of the guinea pig distal colon

"This is the peer reviewed version of the following article: [Chen, B. N., Sharrad, D. F., Hibberd, T. J., Zagorodnyuk, V. P., Costa, M. and Brookes, S. J.H. (2015), Neurochemical characterization of extrinsic nerves in myenteric ganglia of the guinea pig distal colon. J. Comp. Neurol., 523: 742–756. doi: 10.1002/cne.23704], which has been published in final form at [http://dx.doi.org/10.1002/cne.23704]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. http://olabout.wiley.com/WileyCDA/Section/id-820227.html#terms"Extrinsic nerves to the gut influence the absorption of water and electrolytes and expulsion of waste contents, largely via regulation of enteric neural circuits; they also contribute to the control of blood flow. The distal colon is innervated by extrinsic sympathetic and parasympathetic efferent and spinal afferent neurons, via axons in colonic nerve trunks. In the present study, biotinamide tracing of colonic nerves was combined with immunohistochemical labeling for markers of sympathetic, parasympathetic and spinal afferent neurons to quantify their relative contribution to the extrinsic innervation. Calcitonin gene-related peptide, vesicular acetylcholine transporter and tyrosine hydroxylase, which selectively label spinal afferent, parasympathetic and sympathetic axons, respectively, were detected immunohistochemically in 1 ± 0.5% (n = 7), 15 ± 4.7% (n = 6) and 24 ± 4% (n = 7) of biotinamide-labeled extrinsic axons in myenteric ganglia. Immunoreactivity for vasoactive intestinal polypeptide, nitric oxide synthase, somatostatin, vesicular glutamate transporters 1 and 2 accounted for a combined maximum of 14% of biotinamide-labeled axons in myenteric ganglia. Thus, a maximum of 53% of biotinamide-labeled extrinsic axons in myenteric ganglia were labeled by antisera to one of these eight markers. Viscerofugal neurons were also labeled by biotinamide, and shown to have distinct morphologies and spatial distributions that correlated closely with their immunoreactivity for nitric oxide synthase and choline acetyltransferase. As reported for the rectum, nearly half of all extrinsic nerve fibers to the distal colon lack the key immunohistochemical markers commonly used for their identification. Their abundance may therefore have been significantly underestimated in previous immunohistochemical studies

Nuclear DNA content variation in life history phases of the Bonnemaisoniaceae (Rhodophyta)

Nuclear DNA content in gametophytes and sporophytes or the prostrate phases of the following species of Bonnemaisoniaceae (Asparagopsis armata, Asparagopsis taxiformis, Bonnemaisonia asparagoides, Bonnemaisonia clavata and Bonnemaisonia hamifera) were estimated by image analysis and static microspectrophotometry using the DNA-localizing fluorochrome DAPI (4′, 6-diamidino-2-phenylindole, dilactate) and the chicken erythrocytes standard. These estimates expand on the Kew database of DNA nuclear content. DNA content values for 1C nuclei in the gametophytes (spermatia and vegetative cells) range from 0.5 pg to 0.8 pg, and for 2C nuclei in the sporophytes or the prostrate phases range from 1.15-1.7 pg. Although only the 2C and 4C values were observed in the sporophyte or the prostrate phase, in the vegetative cells of the gametophyte the values oscillated from 1C to 4C, showing the possible start of endopolyploidy. The results confirm the alternation of nuclear phases in these Bonnemaisoniaceae species, in those that have tetrasporogenesis, as well as those that have somatic meiosis. The availability of a consensus phylogenetic tree for Bonnemaisoniaceae has opened the way to determine evolutionary trends in DNA contents. Both the estimated genome sizes and the published chromosome numbers for Bonnemaisoniaceae suggest a narrow range of values consistent with the conservation of an ancestral genome