Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis - acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.      

Increased spontaneous immunoglobulin secretion associated with cyclophosphamide-induced immune suppression

Spontaneous immunoglobulin (Ig) secretion by cells from multiple sclerosis (MS) patients (in the progressive phase) treated with monthly pulse doses of cyclophosphamide (CY) (1000–1600 mg/M²) was measured using the protein A plaque assay, to evaluate the effect of CY treatment on B-cell function. Surprisingly, an increase, rather than a decrease, in Ig-secreting cells was seen following CY treatment. CY-treated MS patients averaged 1380±535 spontaneous total (IgM+G+A) Ig plaque-forming cells (PFC) per 1×10⁶ peripheral blood mononuclear cells (MNC), measured at 15–22 days after monthly CY administration, while healthy adults had 280±47 Ig PFC/10⁶ MNC, and MS patients not treated with CY had 300±43 Ig PFC/10⁶ MNC. The observed increase was due to an increase in IgG and IgA PFC. PFC levels remained elevated for 4 weeks following CY treatment, decreasing to control levels by 7–8 weeks post-CY. A small increase in serum IgG level was noted after >12 months of pulse CY therapy; no increase was seen in CSF IgG levels. A preferential decrease in the number of CD4+ T cells was also seen in the CY-treated MS patients. We propose that the observed increase in the number of spontaneous Ig PFC was due to the CY-induced disruption of the CD4+ T cell-mediated control ofin vivo activated B cells.     

Novel Method for Processing Respiratory Specimens for Detection of Mycobacteria by Using C18-Carboxypropylbetaine: Blinded Study

A novel method for processing respiratory specimens to improve culture and acid-fast staining of mycobacteria is introduced. This new method utilized N,N-dimethyl-N-(n-octadecyl)-N-(3-carboxypropyl)ammonium inner salt (Chemical Abstract Service no. 78195-27-4), also known as C₁₈-carboxypropylbetaine (CB-18). In a blinded, five-center study, CB-18-based processing was compared to the standard method combining NALC and NaOH (NALC/NaOH). A total of 573 respiratory specimens were tested. Individual specimens were split approximately equally; the host institutions processed half of each specimen by the NALC/NaOH method, while the other half was processed with CB-18 at Quest Diagnostics—Baltimore. A total of 106 specimens were culture positive for acid-fast bacilli (AFB). Replacement of the primary decontamination agent with CB-18 caused changes in all diagnostic parameters. Aggregate culture sensitivity improved by approximately 43% (P < 0.01), and smear sensitivity improved by approximately 58% (P < 0.01). The sensitivity of smear relative to that of M. tuberculosis isolates exceeded 93% (P < 0.01) when specimens were processed with CB-18. The average times to a positive result were reduced by 7.3 days in liquid culture (P < 0.01) and 5.3 days on solid media (P < 0.05); however, the CB-18 method had a 20.8% contamination rate in liquid culture versus a rate of approximately 7.5% with NALC/NaOH processing. There were also unusual reductions in liquid culture sensitivity and smear specificity among CB-18-processed specimens. The characteristics of the latter parameters suggested that refinement of the CB-18 processing method should allow further improvements in culture sensitivity. This study showed that the CB-18 method has the potential to improve both smear and culture detection for these important human pathogens.     

Detection and identification of gastrointestinal microsporidia using non-invasive techniques.

AIMS--To detect enteric microsporidia in faecal specimens from patients with the acquired immunodeficiency syndrome (AIDS), and to identify the spores to species level without using invasive procedures. METHODS--Formalised faecal preparations were examined using a modification of the strong trichrome staining method to demonstrate microsporidian spores. Six positive specimens were prepared for electron microscopy by emulsification and separation using a 9% Ficoll gradient. RESULTS--The modified staining technique readily identified microsporidian spores. Spores of different species showed variation in size. Identification using electron microscopy was successful for five of the six positive specimens examined. It was unsuccessful for one specimen in which spores were less abundant on initial staining. CONCLUSIONS--The modified strong trichrome staining method is a useful way of detecting spores of intestinal microsporidia in faecal specimens. Variation in spore size may permit provisional identification by light microscopy. Electron microscopic examination of faecal preparations is useful for identifying spores to species level.     

Evaluation of new blood culture processing systems

The Antimicrobial Removal Device (ARD; Marion Scientific) was evaluated in vitro with simulated blood culture samples in fresh blood and clinically with samples from potentially septic patients to test its ability to remove antimicrobial agents and recover bacteria from blood culture specimens containing these drugs. In simulated specimens, the ARD was evaluated for adverse affects on microorganisms as well as compared with lysis-centrifugation (Isolator; Du Pont Co.), biphasic brain heart infusion bottles, and tryptic soy broth bottles for antimicrobial inactivation and organism recovery. There was no adverse effect of the ARD on organisms during a 4-h test period. The ARD was the only system to actually inactivate antimicrobial agents and removed greater than 99.2% of all antimicrobial agents tested from spiked and clinical specimens. Overall, with simulated blood culture specimens, the ARD recovered 90% of bacteria spiked into fresh blood containing antimicrobial agents, Isolator recovered 73%, biphasic brain heart infusion bottles recovered 31%, and tryptic soy broth bottles recovered 24%. In the clinical study, 43 of 86 clinically significant isolates were recovered only by ARD-assisted processing, 6 were recovered only by conventional processing, and 37 were recovered by both methods (the advantage of ARD processing over conventional processing in the clinical study was significant at P less than 0.001). Both clinical and simulated specimens demonstrated the ARD-associated blood culture processing to be the most efficient method for the isolation of microorganisms from specimens containing antimicrobial agents.     

Simultaneous reorganization in thalamocortical ensembles evolves over several hours after perioral capsaicin injections.

Reorganization of the somatosensory system was quantified by simultaneously recording from single-unit neural ensembles in the whisker regions of the ventral posterior medial (VPM) nucleus of the thalamus and the primary somatosensory (SI) cortex in anesthetized rats before, during, and after injecting capsaicin under the skin of the lip. Capsaicin, a compound that excites and then inactivates a subset of peripheral C and Adelta fibers, triggered increases in spontaneous firing of thalamocortical neurons (10-15 min after injection), as well as rapid reorganization of the whisker representations in both the VPM and SI. During the first hour after capsaicin injection, 57% of the 139 recorded neurons either gained or lost at least one whisker response in their receptive fields (RFs). Capsaicin-related changes continued to emerge for >/=6 h after the injection: Fifty percent of the single-neuron RFs changed between 1-2 and 5-6 h after capsaicin injection. Most (79%) of these late changes represented neural responses that had remained unchanged in the first postcapsaicin mapping; just under 20% of these late changes appeared in neurons that had previously shown no plasticity of response. The majority of the changes (55% immediately after injection, 66% 6 h later) involved "unmasking" of new tactile responses. RF change rates were comparable in SI and VPM (57-49%). Population analysis indicated that the reorganization was associated with a lessening of the "spatial coupling" between cortical neurons-a significant reduction in firing covariance that could be related to distances between neurons. This general loss of spatial coupling, in conjunction with increases in spontaneous firing, may create a situation that is favorable for the induction of synaptic plasticity. Our results indicate that the selective inactivation of a peripheral nociceptor subpopulation can induce rapid and long-evolving (>/=6 h) shifts in the balance of inhibition and excitation in the somatosensory system. The time course of these processes suggest that thalamic and cortical plasticity is not a linear reflection of spinal and brainstem changes that occur following the application of capsaicin.     

Glucagon and glucagon-like immunoreactive cells in mid- and hindgut carcinoids

The occurrence of glucagon/glucagon-like immunoreactivity in 31 small intestinal, 34 rectal and 18 appendiceal carcinoids were investigated immunocytochemically using, sequence specific antisera. Glucagon/GLI immunoreactive cells were found in five small-intestinal and five rectal carcinoids, but none were observed in any of the appendiceal carcinoids examined. Glucagon/GLI immunoreactive cells constituted a minor cell population, except in one rectal carcinoid, where most of the tumour cells were of this type. Glucagon/GLI immunoreactive cells were detected with only some sequence-specific antisera, and not with antisera directed against the rest of the glucagon/glicentin molecule. This might indicate that these cells contain a molecule which shares some antigenic binding sites with glucagon/glicentin rather than genuine glucagon/glicentin. It is concluded that this finding contributes to explain why hindgut carcinoids rarely give rise to symptoms related to neuro-endocrine product(s).     

Long-term ST database: A reference for the development and evaluation of automated ischaemia detectors and for the study of the dynamics of myocardial ischaemia

The long-term ST database is the result of a multinational research effort. The goal was to develop a challenging and realistic research resource for development and evaluation of automated systems to detect transient ST segment changes in electrocardiograms and for supporting basic research into the mechanisms and dynamics of transient myocardial ischaemia. Twenty-four hour ambulatory ECG records were selected from routine clinical practice settings in the USA and Europe, between 1994 and 2000, on the basic of occurrence of ischaemic and non-ischaemic ST segment changes. Human expert annotators used newly developed annotation protocols and a specially developed interactive graphic editor tool (Semia) that supported paperless editing of annotations and facilitated international co-operation via the Internet. The database contains 86 two- and three-channel 24h annotated ambulatory records from 80 patients and is stored on DVD-ROMs. The database annotation files contain ST segment annotations of transient ischaemic (1155) and heart-rate related ST episodes and annotations of non-ischaemic ST segment events related to postural changes and conduction abnormalities. The database is intended to complement the European Society of Cardiology ST-T database and the MIT-BIH and AHA arrhythmia databases. It provides a comprehensive representation of ‘real-world’ data, with numerous examples of transient ischaemic and non-ischaemic ST segment changes, arrhythmias, conduction abnormalities, axis shifts, noise and artifacts.     

Muscle atrophy continues after early cast removal following tendon repair

We studied soleus (SOL), plantaris (PLN), and gastrocnemius (GST) muscles to determine whether early cast removal minimizes muscle atrophy or permits recovery from atrophy after tendon repair. After right tendocalcaneus (Achilles tendon) was transected and repaired, rabbit right hindlimbs were immobilized with the ankle plantar flexed and the knee flexed to 90°, Rabbits were maintained in the cast and sacrificed at 5, 15, or 21 days postoperatively or the cast was removed on day 5 and the animals sacrificed at day 15 or 21. SOL, PLN, and GST muscles of both limbs were removed and weights, mean fiber cross-sectional area, and percentage of Type I fibers and increased the percentage of Type IIc fibers. Ten days after cast removal (i. e., postoperative day 15), SOL muscle atrophy and fiber composition did not differ significantly from continuously immobilized controls. However, 16 days after cast removal (i. e., postoperative day 21), SOL muscle fiber cross-sectional area and fiber composition were near normal, differing significantly from continuously casted controls. At each of the time intervals studied, PLN (containing many glycolytic fibers) did not atrophy as much as SOL (containing mainly oxidative fibers). Our results indicate that (1) early cast removal prevents atrophy of PLN glycolytic fibers, but not oxidative fibers in either PLN or SOL, and (2) early cast removal promotes recovery from atrophy of both oxidative and glycolytic fibers. In spite of the many differences between rabbits and humans, these findings suggest that, although early cast removal may not prevent oxidative muscle fiber atrophy after postoperative immobilization, it may facilitate recovery from atrophy. © 1992 Wiley-Liss, Inc.