Reduced collagen VI causes Bethlem myopathy: a heterozygous COL6A1 nonsense mutation results in mRNA decay and functional haploinsufficiency

We have identified a new pathogenic mechanism for an inherited muscular dystrophy in which functional haploinsufficiency of the extracellular matrix protein collagen VI causes Bethlem myopathy. The heterozygous COL6A1 mutation results in a single base deletion from the mRNA and a premature stop codon. The mutant mRNA is unstable, subject to nonsense- mediated mRNA decay, and is almost completely absent both from patient fibroblasts and skeletal muscle, resulting in haploinsufficiency of the alpha1(VI) subunit and reduced production of structurally normal collagen VI. This is the first example of a muscular dystrophy caused by haploinsufficiency of a structural protein or member of the dystrophin-glycoprotein complex, and identifies collagen VI as a critical contributor to cell-matrix adhesion in skeletal muscle.      

Rapid enzymatic characterization of clinically encountered anaerobic bacteria with the API ZYM system

The purpose of this study was to determine if enzyme profiles of anaerobic bacteria, obtained with the API ZYM system, are sufficiently distinctive and reproducible to merit future development of the system and an expanded data base for the four major groups of clinically encountered anaerobes. Of the total 155 clinical isolates and reference strains that were tested, 88 % had distinctive patterns. It was possible to differentiate between 89 % of the anaerobic gram-negative bacilli, 64 % of anaerobic cocci, 100 % of anaerobic gram-positive non-sporeforming bacilli and 100 % of theClostridium spp. using only the API ZYM plus Gram reaction, morphology and relation to oxygen. Overall reproducibility for 1,330 enzyme-substrate reactions was 97 %. It was concluded that the API ZYM could be a practical system for rapid identification of clinical anaerobe isolates provided that an expanded data base is developed and that certain additional recommended tests are used.     

Evaluation of Three Immunoassays Used for Detection of Anti-Rubella Virus Immunoglobulin M Antibodies

Three automated assays (Abbott AxSYM, Bayer ADVIA Centaur, and bioMerieux VIDAS) used for the detection of rubella virus-specific immunoglobulin M were evaluated. A total of 57 samples from individuals with evidence of infection with rubella virus were used to estimate sensitivity, and 220 samples from blood donors and individuals attending an antenatal clinic who had no evidence of recent infection were used to estimate specificity. Seroconversion panels comprising an additional 31 samples from four individuals were used to determine clinical sensitivity. Samples containing potentially cross-reacting substances were also tested. The sensitivities of the three assays ranged from 84.2 to 96.5%, and the specificities ranged from 96.8 to 99.9%. The Abbott AxSYM assay detected more reactive samples than the other two assays when a panel of 57 positive samples was tested. Bayer ADVIA Centaur detected more reactive samples in the seroconversion panels than the other two assays. All three assays evaluated reported a reactive result in 1 or more of the 48 samples containing potentially cross-reacting analytes. The assays demonstrated comparable performance in testing of a well-characterized panel of samples.     

Studies of percutaneous epididymal sperm aspiration (PESA) and intracytoplasmic sperm injection

Four distinct studies were carried out using two data sets ofpercutaneous epididymal sperm aspiration (PESA) and intracytoplasmicsperm injection (ICSI) procedures performed from March 1993to January 1997. In study A, an analysis of 181 ICSI treatmentcycles following PESA revealed a successful epididymal spermretrieval rate of 83%. It confirmed that PESA is an effectivesperm retrieval method and the associated ICSI pregnancy rate(35% per embryo transfer) compared favourably with that of othersperm retrieval methods. In study B, the relevance of a priordiagnostic PESA procedure was ascertained by comparing the spermretrieval rates in two groups of patients having their firstICSI treatment cycle with spermatozoa retrieved through PESA.Group B1 (n=50) had diagnostic PESA prior to the ICSI treatmentcycle PESA procedure, unlike patients in group B2 (n=64) whodid not. The sperm retrieval rate in the treatment cycle procedurewas not different at 90 and 82.8% for groups B1 and B2 respectively.However, the discontinuation of diagnostic PESA is fraught withproblems including liability to medico-legal sanctions. In studyC, analysis of 177 treatment cycles involving PESA and ICSIrevealed a successful sperm retrieval rate by PESA of 82% inthe first cycle, 93% in the second, 96% in the third and 100%in the fourth cycle. The same trend was evident when sperm retrievalwas examined in relation to each of the epididymides. Retrievedspermatozoa were found to be motile in 67-100% of cases andthe frequency of samples containing motile spermatozoa did notdecrease with increase in the number of PESA attempts. Theseresults show that PESA does not jeopardize future epididymalsperm retrieval. In study D, the outcome of treatment with ICSIusing ejaculated spermatozoa (305 cycles) (group D1) was comparedwith that of ICSI using spermatozoa obtained through PESA (54cycles) (group D2). The median age of women in the two groupsof couples was similar (34 years). In group D1, 70% of metaphaseII oocytes were fertilized compared with 61% in group D2 (P<0.01).The cleavage rate and the median numbers of transferred andcryopreserved embryos were similar in both groups. There wasno significant difference between the clinical pregnancy rates(33 and 42% in groups D1 and D2 respectively). Our results showthat the outcome of PESA-ICSI treatment compared favourablywith that of ICSI using ejaculated spermatozoa.     

Increased light exposure consolidates sleep and strengthens circadian rhythms in severe Alzheimer's disease patients

Sleep in the nursing home environment is extremely fragmented, possibly in part as a result of decreased light exposure. This study examined the effect of light on sleep and circadian activity rhythms in patients with probable or possible Alzheimer's disease. Results showed that both morning and evening bright light resulted in more consolidated sleep at night, as measured with wrist actigraphy. Evening light also increased the quality of the circadian activity rhythm, as measured by a 5-parameter extended cosine model (amplitude, acrophase, nadir, slope of the curve, and relative width of the peak and trough). Increasing light exposure throughout the day and evening is likely to have the most beneficial effect on sleep and on circadian rhythms in patients with dementia. It would behoove nursing homes to consider increasing ambient light in multipurpose rooms where patients often spend much of their days.     

B7-CD28 interaction is a late acting co-stimulatory signal for human T cell responses

The interaction of CD28 with one of the B7 molecules (CD80 and CD86) on professional antigen-presenting cells (APC) is generally considered as the most important co-stimulatory signal for T cell activation. APC in a resting condition express either no or only low levels of B7 molecules. These are up-regulated as a result of interactions with activated T cells, thus suggesting that B7-CD28 interaction is not required at initiation of T cell activation. To study this issue, we blocked B7-CD28 interaction at various time points after in vitro stimulation of peripheral blood T cells with allogeneic monocytes. Epstein-Barr virus-transformed B cells or soluble antigens. We observed that T cell proliferation and IL-2 production were inhibited by B7- blocking agents (CTLA-4-Ig or anti-B7 mAb) almost to the same degree when added either at initiation of culture or 24 h later. B7-blocking agents still resulted in significant inhibition of allogeneic T cell activation when added after 48 h. Furthermore, when CTLA-4-Ig was added at the start of an allogeneic T cell stimulation, addition of anti-CD28 mAb after 24 h of culture nearly fully restored T cell proliferation to control levels. Finally, we demonstrate that delayed addition of B7- blocking agents together with cyclosporin A 1 day after the onset of culture of T cells with allogeneic B cells is highly efficient to induce energy as evaluated by lack of proliferation, cytotoxic T lymphocyte reactivity and IFN-gamma or IL-5 production upon alloantigen rechallenge. Taken together, our data can explain why B7 expression on APC is not required at the time of initial APC-T cell contact, and suggest that the effect of the CD28 signal indeed consists in prolonging IL-2 production and amplifying T cell responses, rather than in providing a critical co-stimulatory signal at the time of initial TCR triggering.      

Analysis of murine CD22 during B cell development: CD22 is expressed on B cell progenitors prior to IgM

CD22 is a B cell-restricted glycoprotein involved in cell adhesion and signaling. Since CD22 is likely to play an important role in interactions between B cells and other cells, and in regulating signaling thresholds, we characterized the expression of murine CD22 during different stages of B cell development. In contrast to previous reports, we show that CD22 is expressed on B cell progenitors prior to expression of IgM. IL-7-responsive B cell precursors from the fetal liver and early B lineage cells (B220+IgM-) from the bone marrow both express a low density of surface CD22. The majority of the earliest B cell progenitors (B220+IgM-CD43+) in the bone marrow, however, do not express CD22. As B cells mature, the density of CD22 molecules on the cell surface increases. B220brightIgM+ bone marrow cells express high levels of CD22, as do splenic B cells. The correlation of CD22 levels with B cell maturation is replicated in an in vitro culture system, which distinguishes stages of B cell development based on function. Following activation of mature resting splenic B cells with anti-mu mAb or lipopolysaccharide (LPS), levels of CD22 decrease. Finally, we show that the addition of anti-CD22 mAb augments the proliferative response of both anti-mu- and LPS-stimulated B cells, suggesting a role for CD22 in diverse signaling pathways.      

Illumination levels in nursing home patients: effects on sleep and activity rhythms

Studies examining levels of illumination in adult populations have demonstrated that the level and amount of light exposure are lower in the elderly compared with younger adults, particularly in institutionalized patients with dementia. Although insufficient light exposure has been implied as a cause of sleep fragmentation, evidence for such a relationship is scant. Sixty-six institutionalized elderly had their activity and light exposure monitored for a 3-day period. Mean and median light levels, minutes spent over 1000 and over 2000 lux, percent sleep and wake, and number of naps were computed for daytime intervals, defined as 07.00-18.59. Percentages of sleep and wake, number of awakenings and mean duration of wake periods were computed for night-time intervals, defined as 22.00-05.59. Mesor, amplitude and acrophase of activity and of light were determined by cosinor analysis. A mixed linear model was used to assess the effects of daytime Actillume measures on subsequent night-time measures, and vice versa. Spearman correlations were computed, and multiple regression analyses were carried out with light variables and dementia level as predictors and sleep-wake and activity measures as dependent variables. The median light level was 54 lux and a median of only 10.5 min were spent over 1000 lux. Higher light levels predicted fewer night-time awakenings, and severe dementia predicted more daytime sleep and lower mean activity. Increased bright light exposure predicted later activity acrophase. There was an association between the acrophases of light and of activity, with maximum illumination preceding peak activity. These results suggest that daytime light exposure has an impact on both night-time sleep consolidation and timing of peak activity level.